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Chronic Deficiency of Nitric Oxide Affects Hypoxia Inducible Factor-1α (HIF-1α) Stability and Migration in Human Endothelial Cells
Background: Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated.Results: Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by N G -Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1a (HIF-1a). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O 2 consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1a accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells.Conclusion: Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1a levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction.
# Introduction
Integrity of endothelial cells is crucial for the maintenance of vascular homeostasis. The endothelium explicates its physiological functions by producing active molecules, among which nitric oxide (NO) is particularly important. By diffusing into neighboring smooth muscle cells, endothelial-produced NO induces vasorelaxation, thereby controlling blood pressure levels [bib_ref] Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Palmer [/bib_ref] [bib_ref] Molecular control of blood flow and angiogenesis: role of nitric oxide, Sessa [/bib_ref]. NO generated in the endothelium also has antiaggregant activity that protects the cardiovascular system from thrombosis and acute events [bib_ref] Molecular control of blood flow and angiogenesis: role of nitric oxide, Sessa [/bib_ref].
Consistent with the key role of this gaseous messenger in cardiovascular physiology, NO loss is a dangerous event that is associated with endothelial dysfunction typical of diffuse pathological conditions like atherosclerosis and senescence [bib_ref] Therapeutic potential of nitric oxide donors in the prevention and treatment of..., Herman [/bib_ref] [bib_ref] Prevention of atherosclerosis by interference with the vascular nitric oxide system, Li [/bib_ref] [bib_ref] Nitric oxide activates telomerase and delays endothelial cell senescence, Vasa [/bib_ref]. Moreover, the deficiency of NO and endothelial nitric oxide synthase (eNOS) activity is thought to be crucial for the development and/or acceleration of the important vascular complications associated with diabetes [bib_ref] A review of endothelial dysfunction in diabetes: a focus on the contribution..., Triggle [/bib_ref].
In addition to its effect on smooth muscle cells and platelets, NO generated by the endothelium has important functions in the endothelial cells (ECs) themselves. Indeed, the gaseous messenger plays a key role in the process of angiogenesis, stimulating proliferation, migration and differentiation of ECs to form new blood vessels [bib_ref] The role of nitric oxide in tumour progression, Fukumura [/bib_ref]. In particular, NO acutely produced by angiogenic factors, such as Vascular Endothelial Growth Factor (VEGF) [bib_ref] Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for..., Dimmeler [/bib_ref] [bib_ref] Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is..., Morales-Ruiz [/bib_ref] [bib_ref] Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth..., Papapetropoulos [/bib_ref] , endothelin [bib_ref] Permissive role of nitric oxide in endothelin-induced migration of endothelial cells, Noiri [/bib_ref] , substance P [bib_ref] Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration..., Ziche [/bib_ref] and oxytocin [bib_ref] Oxytocin stimulates migration and invasion in human endothelial cells, Cattaneo [/bib_ref] is crucial for stimulation of EC migration.
Together with the stimulatory effect of acute NO on EC chemotaxis, also the concentration and timing of NO release appear to be of crucial importance in determining the final outcome on EC physiology. In particular, recent work from our laboratory has demonstrated that long term inhibition of eNOS in Human Umbilical Vein ECs (HUVECs) by exposure to the NOS inhibitor N G -Nitro-L-arginine methyl ester (L-NAME), increases the migratory behaviour of these cells in Boyden chambers assays carried out immediately after removal of the drug [bib_ref] Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells, Bulotta [/bib_ref]. These results suggest that basal NO, at variance with the gas released acutely in response to motogenic factors, diminishes the migratory ability of ECs. The tonic inhibitory effect of basal NO on migration, by acting as a brake on inappropriate migration, could prevent exaggerated angiogenic responses and thus be an important homeostatic factor in EC physiology.
In the present study, we have further investigated the effects of chronic NO deprivation on EC physiology, and attempted to unravel the pathway linking basal NO to migratory ability. Results obtained both by long term pharmacological inhibition and by genetic silencing of eNOS indicate that NO loss induces profound modifications in EC physiology, leading to a general decrease of mitochondrial mass and metabolic activity, to an accumulation of Hypoxia Inducible Factor-1a (HIF-1a) in normoxia and to enhanced chemotactic migration as a consequence of the increased HIF-1a levels. These results have important implication for our understanding of the consequences of NO deprivation in cardiovascular pathology.
# Results
HUVECs chronically treated with L-NAME are not apoptotic, but have decreased mitochondrial mass and function
To characterize the effects of long term NO deprivation on human ECs, we first analyzed possible changes in cell viability. As shown in [fig_ref] Figure 1: Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and... [/fig_ref] , treatment with L-NAME for 48 h did not induce caspase-3 cleavage, which instead occurred when HU-VECs were exposed to high glucose (30 mM for 48 h), a condition known to be apoptotic for these cells [bib_ref] High-glucose-triggered apoptosis in cultured endothelial cells, Baumgartner-Parzer [/bib_ref]. Moreover, quantification of apoptosis/necrosis by annexin V-conjugated FITC and PI staining followed by FACS analysis did not show any difference in the apoptotic index between control and L-NAME treated HUVECs (0.1660.03 and 0.1560.05 in control and L-NAME treated cells, respectively). Also the percentage of necrotic cells was unaffected by the treatment, ranging from 8.360.26% in control cells to 4.160.21% in cells treated with L-NAME. Finally, we checked the levels of Bcl-2 and Bax, well-known proteins involved in the regulation of apoptosis endowed with anti-apoptotic and pro-apoptotic activity respectively, and found that their expression was unchanged by L-NAME treatment [fig_ref] Figure 1: Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and... [/fig_ref].
In HeLa cells and adipocytes, basal NO enhances mitochondrial mass [bib_ref] Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide, Nisoli [/bib_ref] [bib_ref] Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals, Nisoli [/bib_ref] , however the effect of the gas on mitochondrial biogenesis in ECs had not been investigated. As shown in [fig_ref] Figure 1: Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and... [/fig_ref] , HUVECs treated with L-NAME for 48 h showed a decreased amount of mitochondrial DNA (mtDNA) as compared to untreated cells (by 3860.05%). Moreover, incorporation of the metabolic indicator MTS was lower in L-NAME treated cells (with a mean reduction of 2665%) [fig_ref] Figure 1: Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and... [/fig_ref] indicating a decreased mitochondrial activity in NO deficient cells as compared to control cells. In agreement, oxygen consumption as well as ATP levels were reduced in the L-NAME treated cells [fig_ref] Figure 1: Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and... [/fig_ref].
The NO donor DETA-NO completely reverts the increased migratory behavior induced in HUVECs by long-term L-NAME treatment
Our previous results demonstrated that long-term treatment of HUVECs with L-NAME induced a strong increase of the cell migratory capacity assayed in Boyden chambers [bib_ref] Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells, Bulotta [/bib_ref]. These results suggested that chronic, constitutive NO production exerts a tonic inhibition on HUVEC migratory behavior. To validate this hypothesis, we investigated whether long-term administration of the slow NO donor DETA-NO would reverse the L-NAME-enhanced migration of HUVECs. As shown in [fig_ref] Figure 2: The enhancement in HUVEC migration induced by L-NAME is reverted by the... [/fig_ref] , chronic treatment of HUVECs with L-NAME for 48 h was confirmed to increase the cell migratory response to VEGF by about 45%. Notably, also basal migration was augmented in a similar manner. No significant increase in cell migratory behavior was observed after 24 h treatment with L-NAME (data not shown). A dose as low as 500 nM of DETA-NO, supplied to the L-NAME treated cells for the last 24 h of the treatment, completely abolished the increased migration observed in both basal and VEGF-stimulated NO deficient HUVECs, thus confirming that enhanced HUVEC migration caused by L-NAME was due to NO deprivation. Notably, cells treated with the combination of L-NAME and DETA-NO showed diminished migration compared to cells treated with the donor alone. This result can probably be explained by a biphasic effect of chronic NO, as demonstrated for HeLa cells in our previous work [bib_ref] Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells, Bulotta [/bib_ref].
To investigate whether the increased migratory behavior caused by chronic NOS inhibition was due to a deficiency in cyclic GMP (cGMP) consequent to NO deprivation, we evaluated the effect of long-term treatment with the guanylate cyclase inhibitor ODQ on HUVEC migration in Boyden chambers. After treatment of the cells for 48 h either with L-NAME or with ODQ, migration assays were performed in the absence of the drugs. As shown in [fig_ref] Figure 2: The enhancement in HUVEC migration induced by L-NAME is reverted by the... [/fig_ref] , chronic treatment with ODQ did not induce any change either in basal or in VEGF-stimulated HUVEC motility. The measurement of cGMP accumulation in HUVECs chronically treated with L-NAME or ODQ confirmed the ability of both drugs to significantly reduce cGMP levels [fig_ref] Figure 2: The enhancement in HUVEC migration induced by L-NAME is reverted by the... [/fig_ref]. These results indicated that blunting of the cGMP signaling pathway is not involved in the stimulatory effect induced by NO depletion.
## Effects of long term l-name treatment on enos, vegf, kdr expression and vegf signaling in huvecs
We previously considered the hypothesis that the improved migratory ability of HUVECs chronically treated with L-NAME could be caused by a rebound effect: expression of eNOS in response to its chronic inhibition could have been increased, thus stimulating migration via enhanced NO release upon exposure of the cells to motogenic factors in the absence of the inhibitor. Our analysis, however, suggested that eNOS was not increased by chronic L-NAME treatment [bib_ref] Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells, Bulotta [/bib_ref]. To investigate this phenomenon further, we quantitatively analyzed both eNOS protein and mRNA levels in treated and untreated cells. As shown in [fig_ref] Figure 3: Effects of L-NAME treatment on eNOS, VEGF and KDR expression [/fig_ref] , a significant decrease in eNOS protein levels (by 4865%) was detected in treated cells. In contrast, no variation in the eNOS mRNA level, measured by RT-qPCR, was observed (1.0460.3 fold in comparison to untreated cells) [fig_ref] Figure 3: Effects of L-NAME treatment on eNOS, VEGF and KDR expression [/fig_ref] , suggesting that chronic inhibition of eNOS causes an increased degradation of the enzyme and/or impairment of the translation of its mRNA.
In an attempt to explain the mechanism through which NO deprivation enhances migration, we investigated how chronic L-NAME treatment affects the expression levels of the VEGF receptor-2 (kinase insert domain receptor, KDR). We also analyzed VEGF itself, as endogenous production of the growth factor could potentiate migration by an autocrine loop. RT-qPCR analysis demonstrated that both VEGF and KDR mRNA levels increased, 1.9160.2 and 1.7960.2 fold respectively, in treated compared to untreated cells [fig_ref] Figure 3: Effects of L-NAME treatment on eNOS, VEGF and KDR expression [/fig_ref]. In addition, increased VEGF production and KDR protein expression was demonstrated by ELISA measurement and biochemical analysis of HUVEC lysates, respectively. As shown in [fig_ref] Figure 3: Effects of L-NAME treatment on eNOS, VEGF and KDR expression [/fig_ref] , quantitative measurements of the secreted protein revealed a 1.7-fold increase of VEGF in conditioned media from L-NAME treated cells in comparison to untreated cells. Similarly, KDR protein level in cells chronically treated with L-NAME is 1.8-fold increased as compared to untreated cells [fig_ref] Figure 3: Effects of L-NAME treatment on eNOS, VEGF and KDR expression [/fig_ref] , in good agreement with the RT-qPCR data.
A major VEGF signaling pathway involved in endothelial cell migration includes the activation of phosphatidylinositol-3-kinase (PI-3-K) and the subsequent phosphorylation of the protein kinase AKT, which in turn activates eNOS by phosphorylating it on Ser1177. To determine if L-NAME treatment modified the ability of VEGF to activate this pathway, the phosphorylation state of eNOS and AKT after a 5-min VEGF stimulation in control and chronically L-NAME treated cells was measured. As shown in [fig_ref] Figure 3: Effects of L-NAME treatment on eNOS, VEGF and KDR expression [/fig_ref] , in control cells VEGF (25 ng/ml) increased eNOS and AKT phosphorylation by about 3 times, as expected (lane 2). In L-NAME treated cells, the basal levels of eNOS and AKT phosphorylation were already increased (see lane 3 vs lane 1), and VEGF was not able to induce any further phosphorylation (lane 4). A densitometric analysis performed on 4 independent experiments revealed that in L-NAME treated cells the basal level of phosphorylated eNOS was 3.4360.94 times greater than in control cells. The increase was less pronounced when the basal level of phosphorylated AKT was compared in treated and control cells (1.5760.24 times).
The results presented in Figs. 3 C-F are consistent with an activated VEGF/KDR system in L-NAME-treated HUVECs, and could explain the enhancement of both basal and VEGFstimulated chemotactic response in these cells.
## Inhibition of basal no production induces nuclear hif-1a accumulation in huvecs
Increased VEGF production and cell motility are typical events occurring in hypoxic cancer cells, due to the accumulation of L-NAME or 30 mM glucose (high glucose, h-Glc) were separated by 12% SDS-PAGE and immunoblotted with an anti-caspase 3 antibody which recognized full length caspase-3 (35 kDa) and its large fragment resulting from cleavage (17 kDa). b-actin was used as a loading control. Shown is a representative blot of 2 comparable experiments. (B) Total cell lysates prepared as described in (A) were separated by SDS-PAGE and immunoblotted with anti Bcl-2 or anti Bax antibodies. b-actin was used as a loading control. Shown is a representative blot of 2 comparable experiments. (C) Mitochondrial DNA (mtDNA) was quantified by RT-qPCR from control cells (CTRL) or from cells treated with L-NAME for 48 h, and normalized to the level of the housekeeping gene 18S. **p,0.01; t test; n = 3. (D) Mitochondrial activity of control and L-NAME treated cells was evaluated by means of MTS. In parallel samples, the total cell number was measured by crystal violet staining. ***p,0.001 vs control cells (CTRL, set at 100%); t test; n = 7. (E) After L-NAME treatment, oxygen consumption was reduced by 2566% in comparison to control cells. The values were normalized to the cell protein content. *p,0.05; t test, n = 3. (F) Total cellular ATP levels were reduced by 2567% after 48 h of 5 = mM L-NAME treatment. **p,0.01 vs control cells (CTRL, set at 100%); t test; n = 8. doi:10.1371/journal.pone.0029680.g001 hypoxia-inducible factor-1a (HIF-1a), which plays a major role in the transcriptional activation of genes encoding angiogenic factors [bib_ref] HIF-1: upstream and downstream of cancer metabolism, Semenza [/bib_ref] [bib_ref] Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling, Rey [/bib_ref]. Similarly, induction of VEGF expression during hypoxia has been described also in endothelial cells [bib_ref] Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells, Namiki [/bib_ref]. We therefore analysed the effect of long term L-NAME treatment on HIF-1a levels in HUVECs. Most interestingly, we observed that, after 48 h of treatment, L-NAME induced nuclear accumulation of HIF-1a in HUVECs (5.561.6 fold over basal) [fig_ref] Figure 4: L-NAME treatment induces HIF-1a nuclear accumulation [/fig_ref]. RT-qPCR analysis revealed no significant change in HIF-1a mRNA levels after L-NAME treatment (1.2160.1 fold in comparison to untreated cells) [fig_ref] Figure 4: L-NAME treatment induces HIF-1a nuclear accumulation [/fig_ref] , suggesting that HIF-1a accumulation in L-NAME-treated cells was mainly due to its stabilization, as occurs under hypoxic conditions. Taken together, these data suggest that prolonged L-NAME treatment induces in endothelial cells a pseudohypoxic state, with the consequent stabilization of HIF-1a under normoxic conditions.
In addition to HIF-1a, other factors could contribute to the upregulation of VEGF expression [bib_ref] Transcriptional regulation of the Vascular Endothelial Growth Factor gene-a concert of activating..., Pagès [/bib_ref]. To directly correlate the increase in VEGF expression induced in HUVECs by L-NAME treatment to the transcriptional activity of HIF-1, we performed experiments in which L-NAME treated cells and control cells were transfected with a plasmid expressing a dominant negative form of the HIF-1b subunit (DARNT), which maintains the capacity of forming an heterodimer but cannot bind DNA [bib_ref] Hepatocyte growth factor-activated NF-kappaB regulates HIF-1 activity and ODC expression, implicated in..., Tacchini [/bib_ref] [bib_ref] Role of HIF-1 and NF-kappaB transcription factors in the modulation of transferrin..., Tacchini [/bib_ref]. As shown in [fig_ref] Figure 4: L-NAME treatment induces HIF-1a nuclear accumulation [/fig_ref] , in HUVECs, the increase in VEGF protein induced by L-NAME treatment was totally blunted, confirming the central role of HIF-1a as regulator of VEGF expression in NO-deprived endothelial cells. Moreover, as shown in [fig_ref] Figure 4: L-NAME treatment induces HIF-1a nuclear accumulation [/fig_ref] , transfection with DARNT inhibited also the increased migratory capacity induced in HUVECs by L-NAME treatment.
The NO donor DETA-NO reverts the effects of L-NAME treatment on HIF-1a stabilization, and on VEGF and eNOS expression
To investigate whether the observed effects of L-NAME treatment on HUVEC gene expression were due to the chronic depletion of NO, we reconstituted NO levels by utilizing the long lasting NO donor DETA-NO. The addition of 500 nM DETA-NO completely reverted the stabilization of nuclear HIF-1a, the reduced expression of total eNOS protein and the increase in VEGF mRNA levels , which are all characteristic features detected in the long-term L-NAME treated cells.
## Silencing of enos mimics the effects of long-term l-name treatment of huvecs
To further investigate if the observed effects of L-NAME treatment were due to the specific inhibitory effect on eNOS activity, expression of the enzyme was silenced in HUVECs by using RNA interference methodology. HUVEC transfection with eNOS siRNA caused a mean reduction in eNOS protein levels of 7060.1% [fig_ref] Figure 6: Effect of eNOS silencing on HIF-1a accumulation, VEGF secretion, mtDNA and ATP... [/fig_ref]. In agreement with the results obtained after long-term exposure to L-NAME, HIF-1a accumulated in the nucleus of eNOS knock-down cells, whereas cells transfected with control siRNA were unaffected [fig_ref] Figure 6: Effect of eNOS silencing on HIF-1a accumulation, VEGF secretion, mtDNA and ATP... [/fig_ref]. Moreover, in eNOS silenced HUVECs, again similarly to long term L-NAME treated cells, VEGF production was increased [fig_ref] Figure 6: Effect of eNOS silencing on HIF-1a accumulation, VEGF secretion, mtDNA and ATP... [/fig_ref] , while the amount of mtDNA as well as ATP levels were decreased [fig_ref] Figure 6: Effect of eNOS silencing on HIF-1a accumulation, VEGF secretion, mtDNA and ATP... [/fig_ref].
# Discussion
Our results demonstrate that long-term pharmacologic and genetic down-regulation of eNOS induce relevant effects in HUVECs. The long-term treatment with L-NAME induced a significant decrease in mtDNA amount, accompanied by de-creased mitochondrial activity and ATP production. This observation is in agreement with previous results, which showed the important role of the gas in promoting mitochondrial biogenesis in different cell types and tissues [bib_ref] Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide, Nisoli [/bib_ref] [bib_ref] Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals, Nisoli [/bib_ref]. Similar results (i.e. decreased mitochondrial mass) were obtained in the microvasculature of mice treated with N G -monomethyl-L-arginine (L-NMMA) for a long period of time (1-2 months) [bib_ref] The Krebs cycle and mitochondrial mass are early victims of endothelial dysfunction:..., Addabbo [/bib_ref] ; however, given the length of the treatment, these results are not directly comparable to ours. To our knowledge, the effect of NO deprivation for shorter times (days) on mitochondrial biogenesis in ECs had not previously investigated. Our work demonstrated that HUVECs respond to NO deprivation already after 48 h of treatment, making them a useful model for the investigation of the effects of the gas on mitochondrial biogenesis and function.
An important consequence of the decrease in NO production observed by us is the accumulation of HIF-1a in the nucleus, with a resulting adaptation of gene expression to a pseudohypoxic state, i.e. a state of hypoxia in the presence of normal levels of oxygen. The increased expression of VEGF found in our experiments can be considered a consequence of this pseudohypoxic state since the dominant negative form of the HIF1-b subunit DARNT totally blunted the VEGF protein increase induced by L-NAME treatment. Accordingly, DARNT transfection inhibited also the increased motility in L-NAME treated HUVECs, thus indicating that the migratory behavior we observed in the treated cells can be considered the consequence of the transcriptional activity of HIF-1a. Of note, the increased HUVEC motility is observed despite a reduction in eNOS levels, and is not induced by chronic treatment with the guanylate cyclase inhibitor ODQ, indicating that cGMP and protein kinase G (PKG) are not involved in the observed effects.
Our results open the interesting question of the mechanism by which NO deficiency induces HIF1astabilization in HUVECs.
HIF-1a degradation depends on prolyl hydroxylases-catalysed proline hydroxylation, which induces binding of the factor to an ubiquitin ligase (the Von-Hippel Lindau protein) and targets it for proteasomal degradation. The activity of prolyl hydroxylases (PHDs) is dependent on the availability of oxygen and 2oxoglutarate (a Krebs cycle intermediate) as substrates and on Fe 2+ and ascorbate as cofactors. In addition to these mechanisms controlling HIF levels, HIF-1a transcriptional activity can also be negatively controlled via hydroxylation of an asparaginyl residue operated by an O 2 dependent enzyme called Factor Inhibiting HIF (FIH-1) [bib_ref] FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate..., Mahon [/bib_ref]. Under hypoxic conditions, PHD and FIH-1 activity are inhibited and HIF-1a accumulates in the nucleus to function as a transcription factor and to evoke adaptive responses to changes in tissue oxygenation. Under our experimental conditions, chronic eNOS inhibition induces HIF-1a nuclear accumulation, apparently as a result of a decreased degradation, (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs transfected with the empty vector (pcDNA3) or with the expression vector DARNT, and treated with L-NAME for the 48 h following transfection. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p,0.01 vs untreated cells transfected with pcDNA3; ***p,0.001 vs L-NAME treated cells transfected with pcDNA3 (One-way ANOVA with Bonferroni's test; n = 3). (E) HUVECs were transfected with pcDNA3 or DARNT, and treated with L-NAME for the 48 h following transfection when indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractant. Results are expressed as the number of migrating cells. #p,0.001 vs basal migration in untreated pcDNA3 cells; 1p,0.001 vs VEGF-induced migration in untreated pcDNA3 cells; ***p,0.001 vs basal migration in pcDNA3 cells treated with L-NAME; uuup,0.001 vs VEGF-induced migration in pcDNA3 cells treated with L-NAME; no significant differences between untreated pcDNA3 and DARNT transfected cells and between untreated and L-NAME treated DARNT tranfected cells (One-way ANOVA with Bonferroni's test, n = 10). doi:10.1371/journal.pone.0029680.g004 supporting the hypothesis of an impaired PHD activity. The question is then shifted to the mechanism through which NO may affect PHD activity in HUVECs.
It is well known that NO competes with O 2 for the binding to the heme moiety of cytochrome C oxidase [bib_ref] Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial..., Cleeter [/bib_ref] [bib_ref] Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with..., Brown [/bib_ref] [bib_ref] Nitric oxide, cytochrome C oxidase, and the cellular response to hypoxia, Taylor [/bib_ref]. Therefore, a possibility that we considered was that NO deficiency, by activating mitochondrial respiration, could cause an intracellular O 2 redistribution from the cytoplasm to the mitochondria [bib_ref] Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1alpha, Hagen [/bib_ref] , thus inducing oxygen depletion and PHD inactivation in the cytosol. However, it is unlikely that cytoplasmic hypoxia could have occurred in our experimental conditions where cells are exposed to an atmospheric O 2 concentration (21% O 2 ) [bib_ref] Does nitric oxide modulate mitochondrial energy generation and apoptosis?, Moncada [/bib_ref]. In addition, in NO deficient cells we observed a lower oxygen consumption, which can be explained by the reduced mitochondrial mass, thus making the hypothesis of an O 2 redistribution from the cytoplasm to the mitochondria difficult to support. We conclude therefore that NO deficiency affects PHD activity under normoxic conditions.
The relationship between NO and HIF-1a is complex and matter of intense debate [bib_ref] Oxygen-sensing under the influence of nitric oxide, Berchner-Pfannschmidt [/bib_ref]. Recent studies have suggested a dual role for NO in regulating HIF-1a function. By using NO donors or the controlled expression of an inducible NOS, it has been found that high concentrations of NO induced HIF-1a nuclear accumulation, even under normoxic conditions, whereas under hypoxic conditions, when HIF-1a levels are already high, low physiological concentrations of NO appear to have the opposite effect [bib_ref] Hypoxia response element of the human vascular endothelial growth factor gene mediates..., Kimura [/bib_ref] [bib_ref] Normoxic stabilization of hypoxiainducible factor-1 expression and activity: redox-dependent effect of nitrogen..., Palmer [/bib_ref] [bib_ref] Regulation of the hypoxiainducible factor 1alpha by the inflammatory mediators nitric oxide..., Sandau [/bib_ref] [bib_ref] Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial..., Liu [/bib_ref] [bib_ref] Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia, Sogawa [/bib_ref] [bib_ref] Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways, Mateo [/bib_ref]. The effects of the gas under the latter conditions are similar to those observed here. Further underscoring the similarity, the involvement of soluble guanylate cyclase and cGMP in the low NO-induced reduction of HIF-1a was, like in our migration experiments, excluded [bib_ref] Hypoxia response element of the human vascular endothelial growth factor gene mediates..., Kimura [/bib_ref] [bib_ref] Normoxic stabilization of hypoxiainducible factor-1 expression and activity: redox-dependent effect of nitrogen..., Palmer [/bib_ref] [bib_ref] Regulation of the hypoxiainducible factor 1alpha by the inflammatory mediators nitric oxide..., Sandau [/bib_ref] [bib_ref] The inhibitory effect of sodium nitroprusside on HIF-1 activation is not dependent..., Takabuchi [/bib_ref]. However, differently from the previously reported results, in our experiments, the ECs were not subjected to hypoxic conditions. It is possible that in ECs, the O 2 threshold for HIF-1a stabilization is higher than in other cells, and that under normoxic conditions, basal NO production counteracts the stabilization that would otherwise occur.
It should be also considered that PHD activity is affected not only by O 2 , but also by the availability of iron, ascorbate and the Krebs cycle intermediate 2-oxoglutarate [bib_ref] Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor..., Pan [/bib_ref]. The deficiency of NO may influence the intracellular availability of one or more of these factors, thus decreasing the enzymatic activity and causing HIF-1a accumulation. We cannot however exclude that other stabilizing mechanisms, not directly linked to PHD, are involved [bib_ref] The subtle side to hypoxia inducible factor (HIFalpha) regulation, Bilton [/bib_ref]. Furthermore, the possibility that reactive oxygen species may play a role is now under investigation in our laboratory. Certainly, more work is needed to clarify these important points.
In our experiments very low concentrations of DETA-NO abrogated the HIF-1a accumulation, the VEGF increased expression and the increased motility found in L-NAME treated cells. On the basis of this result one can hypothesize that low levels of the gas constantly produced by ECs under basal conditions contribute to keep HIF-1a levels under control. In other words, under physiological conditions, the ECs, by producing constitutive NO, could control all of the events set in motion by HIF-1a, including cell motility and VEGF production. In case of NO deficiency, HIF-1a would be released from this brake, would escape degradation and consequently accumulate, as shown in our experiments.
In conclusion, our results show that lack of NO in human endothelial cells induces pseudohypoxia and mitochondrial dysfunction with consequent decreased energy production. These events may very well occur in all of those pathological conditions where eNOS expression and/or activity are impaired, as in hypertension, type 2 diabetes, hypercholesterolemia, thus contributing to the endothelial dysfunction typical of such disorders. Our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study the effect of NO deficiency on vascular endothelial cells, and to find new therapeutic target (HIF-1a protein for example) possibly useful for treating endothelial dysfunction.
# Materials and methods
## Cell cultures
Human umbilical vein endothelial cells (HUVECs) were isolated from freshly derived umbilical cords by digestion with collagenase as described by Jaffe et al. [bib_ref] Culture of human endothelial cells derived from umbilical veins. Identification by morphologic..., Jaffe [/bib_ref]. Umbilical cords were donated anonymously after informed consent according to national ethical legislation. Cells were routinely grown in 199 medium, supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 mg/ml endothelial cell growth supplement (ECGS) and 50 mg/ml heparin, and used at passages 2-7. Where indicated, HUVECs were treated with 5 mM N G -Nitro-L-arginine . The NO donor DETA-NO reverts the effects of L-NAME treatment on HIF-1a stabilization, and on VEGF and eNOS expression. (A) HIF-1a protein levels were detected by western blotting of nuclear extracts from HUVECs treated with L-NAME and/or DETA/NO as described in [fig_ref] Figure 2: The enhancement in HUVEC migration induced by L-NAME is reverted by the... [/fig_ref]. An aliquot of total cell lysates was immunoblotted with anti eNOS antibodies, and with anti b-actin antibodies as loading control. A representative blot of 3 comparable experiments is shown. (B) VEGF RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. *p,0.05 vs control cells (CTRL); **p,0.01 vs L-NAME treated cells; no significant differences between control and DETA/NO treated cells (One-way ANOVA with Bonferroni's test; n = 3). doi:10.1371/journal.pone.0029680.g005 methyl ester (L-NAME) in 199 medium containing 10% FBS for 48 h preceding the experiments. The concentration of L-NAME was chosen according to Papapetropoulos et al. [bib_ref] Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth..., Papapetropoulos [/bib_ref].
## Apoptosis assays
Quantification of apoptosis/necrosis was performed by Annexin V-FITC conjugate and propidium iodide (PI) staining (Abcam, Cambridge, UK) followed by fluorescence activated cell sorting (FACS) performed with a FACScalibur flow cytometer equipped with a 488 nm argon laser (Becton Dickinson, San José, CA, USA). The collected data were evaluated by Cell Quest software. The degree of apoptosis was calculated as apoptotic index considering cells both in early and late apoptosis. In addition, active caspase-3, Bcl-2 and Bax proteins were detected by western blot on total cell lysates.
## Evaluation of mitochondrial dna (mtdna)
Total DNA was extracted with QIAamp DNA extraction kit (Qiagen, Hilden, Germany), and mtDNA levels were amplified with primers specific for the mitochondrial cytochrome b (CytB) gene and normalized to genomic DNA by amplification of the rRNA 18S nuclear gene. Primers used were: for CytB, F, 59-CTTCGCTTTCCACTTCATCTTACC-39 and R, 59-TTGGGTTGTTTGATCCT-GTTTCG-39, and for 18S, F, 59-CTGCCCTATCAACTTTCGATGGTAG-39 and R, 59-CCGTTTCTCAGGCTCCCTCTC-39. Quantitative real time PCR (RT-qPCR) reactions were run with the iQ SybrGreenI SuperMix (Bio-Rad, Segrate, Italy) on an iCycler iQ Real-Time PCR detection system (Bio-Rad) using 50 ng of total DNA. Calculations were performed with the 2 2DDCt methods using 18S rRNA as an internal control.
## Oxygen consumption
Cellular oxygen consumption was measured as previously described [bib_ref] TNF-alpha downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of..., Valerio [/bib_ref]. Briefly, HUVECs were re-suspended in respiration buffer (0.3 M mannitol, 10 mM KCl, 5 mM MgCl 2 , 10 mM K 2 PO 4 , pH 7.4) [bib_ref] Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography..., Barrientos [/bib_ref] at a density of 3.0610 6 /ml, and analyzed at 37uC in a gas-tight vessel equipped with a Clark-type oxygen electrode (Rank Brothers Ltd., Cambridge, UK) connected to a chart recorder. Protein content in cell samples was determined by the bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA).
## Cell metabolism assays
Cell metabolism was assessed by means of a Cell Titer 96H Aqueous ONE Solution Reagent colorimetric assay (MTS, Promega, Madison, WI, USA), and the total cellular ATP content using a CellTiter-GloH Luminescent Assay (Promega). Both the assays were performed according to the manufacturer's instructions on HUVECs plated at a density of 2610 4 cells/well in 96well microplates. Optical density at 490 nm (for MTS) and luminescence (for ATP) were measured using a multiplate spectrophotometer (Victor TM , PerkinElmer, Waltham, MA, USA). On parallel wells, cell proliferation was evaluated by crystal violet staining. Briefly, cells were fixed with 100% methanol, and then stained with a 0.1% crystal violet solution. After extensive washes with deionized water, the dye was solubilized in 10% acetic acid solution, and the absorbance was measured at 595 nm.
## Cell migration assays
HUVEC migration was evaluated by means of chemotaxis experiments in a 48-well modified Boyden chamber, as previously described [bib_ref] Oxytocin stimulates migration and invasion in human endothelial cells, Cattaneo [/bib_ref] [bib_ref] Basal nitric oxide release attenuates cell migration of HeLa and endothelial cells, Bulotta [/bib_ref]. Briefly, the filters coated with 10 mg/ml of type IV collagen were placed over a bottom chamber containing 25 ng/ml VEGF as attractant factor. The cells, suspended in 199 media containing 2% FBS, were added to the upper chamber at a density of 5.0610 4 cells/well. After 6 h of incubation at 37uC, the cells that had migrated to the lower side of the filter were stained with Diff-Quick stain (VWR Scientific Products, Bridgeport, NJ, USA), and 5 unit fields per filter were counted by a scorer blind to the experimental conditions using a Zeiss microscope. The assays were run in triplicate.
## Determination of cgmp accumulation
HUVECs were cultured in 60-mm Petri dishes and treated for 48 h with L-NAME or ODQ. During the last 30 min of treatment, 1 mM isobutylmethylxanthine (IBMX) was added to inhibit phosphodiesterases. cGMP was extracted in 500 ml of 0.1 N HCl, and its quantification was performed by an enzyme immunoassay (EIA) kit (Enzo Life Sciences, Vinci-Biochem, Vinci, Firenze, Italy) following manufacturer's instructions for the acetylated assay procedure.
## Immunoblot and immunoprecipitation analyses
For immunoblot analysis, HUVECs, plated in 35-mm diameter Petri dishes, were washed with phosphate-buffered saline (PBS), and then directly lysed in SDS-PAGE sample buffer (62 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.04% bromophenol blue). After SDS-PAGE electrophoresis, proteins were transferred onto nitrocellulose membranes that were blocked with 5% (w/v) non fat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), and probed overnight with the indicated primary antibodies. After incubation with the appropriate peroxidase-conjugated secondary antibody (DAKO, Denmark), the immunoreactive bands were visualized by chemioluminescence (LiteAblot Plus, EuroClone, Italy). For KDR immunoprecipitation, HUVECs were washed with PBS and lysed for 10 min on ice with RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM b-glycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate) supplemented with protease inhibitors. Aliquots of cleared cell lysates containing the same amount of protein (250 mg/sample) were incubated with anti KDR antibodies, followed by incubation with Protein A Sepharose. The immune complexes were washed with lysis buffer, eluted by boiling in sample buffer, and analyzed by SDS-PAGE. Densitometric analyses of the immunoblots were performed using the National Institute of Health (NIH) Image J program.
## Elisa determination of vegf levels
Cell supernatants were collected from HUVECs plated in 35mm Petri dishes, and VEGF measurements were performed using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) following manufacturer's instructions. VEGF levels were expressed relative to total cell protein (pg/mg of total protein) evaluated by BCA protein assay.
## Preparation of nuclear extracts
HUVECs, plated in 100-mm diameter Petri dishes, were washed with PBS and collected by scraping. The cells were then lysed for 10 min at 4uC in buffer A (10 mM HEPES pH 8.0, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.05% Nonidet P-40, 1 mM sodium orthovanadate) supplemented with protease inhibitors. After a 10 min centrifugation at 2,500 g at 4uC, the crude nuclei were washed with buffer A prior to lysis in buffer C (20 mM HEPES pH 8.0, 1.5 mM MgCl 2 , 420 mM NaCl, 1.0 mM DTT, 0.2 mM EDTA, 1 mM sodium orthovanadate, supplemented with protease inhibitors) for 30 min at 4uC. The nuclear extracts were clarified by centrifugation, and loaded on a 10% SDS-PAGE. The immunoreactive bands were visualized by chemioluminescence after overnight incubation with the anti HIF-1a antibody at a 1:500 dilution in 5% BSA in TBS-T.
Total RNA extraction for reverse transcription and quantitative real time PCR Total RNA was extracted using the RNeasyH Mini Kit and accompanying QIAshredder TM (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To avoid DNA contamination of samples, a 15 min on column incubation was carried out with DNase I (Qiagen). Reverse transcription was performed using the SuperScript TM III First-Strand Synthesis System for RT-PCR (Invitrogen), again following the manufacturer's instructions. For quantitative analysis of gene expression we used the ABI PrismH 7000 Sequence Detection System, SDS software version 1.2.3 (Applied Biosystems, CA, USA). Target sequences were amplified from 50 ng of cDNA. TaqManH Primer and Probe assays used were: human VEGF-A (Hs99999070_m1), KDR (Hs00176676_m1), eNOS (NOS3, Hs00167166_m1), HIF-1A (Hs00153153_m1) and the endogenous control 18S (Hs99999901_s1). For calculation of results, the 2 2DDCt method was used allowing normalization to 18S and to the calibrator which is set to a value of 1.
## Transient transfection
HUVECs, plated in 35-mm Petri dishes, were transfected with the expression vector pcDNA3ARNTdelta_b (DARNT) (kindly provided by Dr. Tacchini, Department of Human Morphology and Biomedical Sciences 'Città Studi', University of Milan, Milano, Italy), coding for a dominant negative mutant form of the HIF-1b ARNT subunit, and the void vector pcDNA3 using TransITTM LT1 (Mirus, Bologna, Italy). Six hours after transfection, the culture medium was replaced by fresh medium, and the cells were exposed to L-NAME for the following 48 h.
## Small interfering rna (sirna) transfection
Validated Stealth TM RNAi duplexes against human eNOS (GC content 48%) were provided from Invitrogen. As control RNA, we utilized a Stealth TM RNAi negative control duplex (Medium GC Duplex, Invitrogen) with a 48% GC content, suitable for use as a control with Stealth TM RNAi duplexes containing 45-55% of GC. All sets of RNAi molecules were transfected individually into HUVECs at a 30 nM concentration using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). The ability of the RNAi molecules to knockdown eNOS expression was analyzed 48 h after transfection by western blot analysis.
## Statistical procedures
All data were expressed as mean 6 s.e.m. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed
[fig] Figure 1: Effect of chronic NO deprivation on HUVEC vitality and mitochondrial mass and function. (A) Lysates of HUVECs treated for 48 h with 5 mM [/fig]
[fig] Figure 2: The enhancement in HUVEC migration induced by L-NAME is reverted by the NO donor DETA-NO and is independent of the cGMP pathway. (A) HUVECs were treated for 48 h with 5 mM L-NAME in the absence or in the presence of 500 nM DETA/NO for the last 24 h, as indicated. Chemotaxis experiments were then performed using 25 ng/ml VEGF as attractants. Results are expressed as the number of migrating cells. #p,0.001 vs basal migration in control cells (CTRL); 1p,0.01 vs VEGF-induced migration in control cells; ***p,0.001 vs basal migration in L-NAME treated cells; uuup,0.001 vs VEGF-induced migration in L-NAME treated cells; no significant differences between control and DETA/NO treated cells (One-way ANOVA with Bonferroni's test, n = 15). (B) HUVECs were treated for 48 h with 5 mM L-NAME or 1 mM ODQ, and chemotaxis experiments were performed as described in (A). Results are expressed as the number of migrating cells in the different experimental conditions. #p,0.001 vs basal migration in control cells (CTRL); 1p,0.001 vs VEGF-induced migration in control cells; no significant differences between control and ODQ treated cells (One-way ANOVA with Bonferroni's test, n = 3). (C) cGMP accumulation in HUVECs treated for 48 h with L-NAME or ODQ was evaluated by EIA and expressed as pmol of cGMP normalized to the cell protein content (pmol/mg protein). ***p,0.001; One-way ANOVA with Bonferroni's test; n = 3. doi:10.1371/journal.pone.0029680.g002 [/fig]
[fig] Figure 3: Effects of L-NAME treatment on eNOS, VEGF and KDR expression. (A) Densitometric analysis of eNOS protein expression. ***p,0.001; t test; n = 11. Inset: a representative blot out of eleven is shown. Total eNOS protein was evaluated by western blotting on lysates prepared from control cells (lane 1) or from 48 h L-NAME treated cells (lane 2). b-actin was used as a loading control. (B) eNOS RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (t test, n = 3). (C) VEGF and KDR RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. **p,0.01 vs control cells (CTRL); t test; n = 624 for VEGF and KDR, respectively. (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from control or 48 h L-NAME treated cells. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p,0.01; t test; n = 3. (E) KDR protein was visualized by western blot after immunoprecipitation with KDR antibodies of HUVEC lysates obtained from control (lane 1) or from 48 h L-NAME treated cells (lane 2). An aliquot of total cell lysates was immunoblotted with b-actin antibodies as a control (input). Shown is a representative blot of 2 comparable experiments. (F) Control cells (lanes 1 and 2) or 48 h L-NAME treated cells (lanes 3 and 4) were stimulated for 5 min with 25 ng/ml VEGF. Aliquots of cell lysates were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies. Actin was used as a loading control. Shown is a representative blot of 4 comparable experiments. doi:10.1371/journal.pone.0029680.g003 [/fig]
[fig] Figure 4: L-NAME treatment induces HIF-1a nuclear accumulation. (A) HIF-1a protein levels were detected by western blotting of nuclear extracts from control HUVECs (lane 1) or from HUVECs treated with L-NAME for 48 h (lane 2). Shown is a representative blot of 4 comparable experiments. HIF-1a migrates as a doublet with apparent molecular weight of 118 and 120 kDa. (B) Densitometric analysis of nuclear HIF-1a protein levels. *p,0.05; t test; n = 4. (C) HIF-1a RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (t test; n = 3). [/fig]
[fig] Figure 6: Effect of eNOS silencing on HIF-1a accumulation, VEGF secretion, mtDNA and ATP levels. (A) Characterization of HUVECs transfected with eNOS siRNA: densitometric analysis of eNOS protein expression where eNOS protein levels were normalized to b-actin protein. ***p,0.001; t test; n = 4. Inset: representative blots of eNOS protein in cells transfected with control (ctrl) or eNOS siRNA. (B) HUVECs were transfected with control (lane 2) or eNOS siRNA (lane 3), and HIF-1a protein was detected by western blotting on the corresponding nuclear extracts. In lane 1, nuclear extracts from untransfected cells. An aliquot of total cell lysates was immunoblotted with anti eNOS antibodies to check silencing, and with anti b-actin antibodies as loading control. A representative blot of 2 comparable experiments is shown. (C) VEGF protein levels were detected by ELISA measurement in conditioned media collected from HUVECs 48 h after transfection with control or eNOS siRNA. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). *p,0.05; t test; n = 3. (D) MtDNA (left axis) and total cellular ATP content (right axis) were measured in HUVECs transfected for 48 h with control or eNOS siRNA. In silenced cells, mtDNA and ATP were reduced by 3660.4 and 4569.7% respectively. **p,0.01 and ***p,0.001; t test; n = 3. doi:10.1371/journal.pone.0029680.g006 [/fig]
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Novel facile method for obtaining CdSe/polyaniline/C60 composite materials
This study presents a novel method for the oxidative polymerization of aniline (ANI) by employing fullerene C 60 /cadmium selenide (CdSe) quantum dots, as promoting agent of the polymerization system. The polymerization initiation mechanism is based on the difference between the HOMO-LUMO energy levels of the components which permits the formation of a continuous donor-acceptor exchange. Both the polymerization reaction evolution and the molecular weights of the obtained polymers have been characterized. The novelty of the paper consists in the synthesis of a novel nanocomposite material through a novel polymerization technique. The resulting material containing PANI, CdSe quantum dots and C 60 has been characterized by UV-Vis, NIR, fluorescence, TEM and GPC analyses.Polyaniline (PANI) is one of the oldest known conductive polymers 1 . Due to its low cost, good environmental stability and adequate electrical properties 2,3 , PANI has found application in the fabrication of bio/chemical sensors 4,5 , solar cells 6 , organic light emitting diodes 7 , supercapacitors 8 , field effect transistors 9 and electromagnetic interference shielding 10 . The synthesis and doping methods of PANI have a crucial role in determining the properties of the obtained materials 3,11 .Polyaniline (PANI) can be synthesized by electrochemical methods (potentiostatically or galvanostatically), by oxidation of the monomer using inert electrodes, such as stainless steel, platinum, gold, different types of carbon (vitreous or pyrolytic graphite), and glass covered with metal oxides. PANI can also be chemically synthesized in acidic media using an oxidizing agent, such as potassium dichromate, ammonium persulfate, hydrogen peroxide, cerium nitrate, etc.Over the past few decades hybrid nano-composites containing inorganic nanoparticles or carbon materials have become particularly attractive because of their promising applications in electronic and optoelectronic industries 3,12-15 .Thus, the aim of this study consist in a new synthesis route of ANI polymerization using strongly electrophile molecule, e.g. fullerene C 60 and quantum dots nanoparticles of CdSe (3 nm) as electron donating species. Considering the energy levels of the HOMO and LUMO for the two molecules, ANI and C 60 , the oxidative polymerization of the monomer is difficult to be achieved. In this case, a promotor for the polymerization is necessary, presenting electron donating properties towards ANI, making more accessible the electron transfer to C 60 . Quantum dots nanoparticles of CdSe (3 nm) were chosen as the promoting component.This straightforward synthesis approach allows the facile manufacturing of a novel hybrid material with improved properties. Therefore, both the materials as well as the synthesis strategy are original.MaterialsAniline (ANI) (Merck) has been purified through vacuum distillation and kept on molecular sieves 4 Å. Fullerenes (C 60 ) (Aldrich) has been used as received. Dimethylformamide (DMF) (Aldrich), toluene (T) (Fluka) and methanol (Aldrich) have been used without further purification.MethodsCdSe-quantum dots (3 nm) have been prepared according to our paper 16 .Polymerization procedure: in 5 mL of CdSe solution (0.04 mol/L in DMF), 2 mL ANI and 2 mL C 60 in T (1% weight) were added at room temperature under stirring for 2 hours. Methanol has been used as precipitation medium of the polymers. The polymers were filtered, washed several times with methanol and dried until a constant mass was attained.
Characterization. The UV-Vis-NIR spectra were recorded using a spectrofotometer Shimadzu UV-3600.
The fluorescence spectra were registered using a Jasco FP-6500 Able Jasco spectrofluorimeter.
The molecular weights of the resulted polymers and oligomers were analyzed using PL-GPC 50 Integrated GPC/SEC System (Agilent Technologies) using a 1 mL/min THF flow rate and a column oven temperature of 50 °C.
Infrared absorption spectra have been recorded at room temperature with a Nicolet 6700 FTIR spectrometer in the range of 4000-400 cm −1 .
The elemental analysis was performed with Perkin Elmer 2400 Series II CHNS/O Analyzer equipped with thermal conductivity detector (combustion temperature 975 °C, reduction temperature 500 °C).
The high resolution transmission electron microscopy (HRTEM) studies were performed on an atomic resolution analytical JEOL JEM-ARM 200F electron microscope, operating at 200 kV. Specimens for HRTEM were prepared in the following way: a drop of the solution was put on holey carbon TEM grid and dried at 100 °C for 5 min.
# Results and discussion
Literature presents the capacity of ANI to polymerize through an oxidative mechanism, respectively "pseudo-living cationic" polymerization process [bib_ref] Evolution of Polyaniline Nanotubes: The Oxidation of Aniline in Water, Trchová [/bib_ref] [bib_ref] Recent advances in polyaniline research: Polymerization mechanisms, structural aspects, properties and applications, Ćirić-Marjanović [/bib_ref]. The first step of polymerization consists in the monomer oxidation. Therefore, the monomer polymerization could be attained by an electron transfer process to a strongly electrophile molecule, e.g. fullerene C 60 18 , followed by chain growth process. Considering the energy levels of the HOMO and LUMO for the two molecules, ANI and C 60 , the oxidation of the monomer is difficult to be achieved. In this case, a promotor for the polymerization is necessary, presenting electron donating properties towards ANI, making more accessible the electron transfer to C 60. Quantum dots nanoparticles of CdSe [bib_ref] The effects of CdSe incorporation into bulk heterojunction solar cells, De Freitas [/bib_ref].
In order to highlight the ANI polymerization process, UV-Vis and fluorescence analyses were performed at different intervals.
Inis presented the absorption spectra of ANI/C 60 mixture which confirms the formation of a charge transfer complex [bib_ref] Photoinduced Intramolecular Electron Transfer in a Bridged C60 (Acceptor)-Aniline (Donor) System; Photophysical..., Williams [/bib_ref]. The analysis ofreveals the initial absorption specific for the CdSe quantum dots at 470 nm. There are no modifications in the visible range of the spectra after the addition of the ANI, until introduction of C 60 , which leads to the formation of the peaks at 620 nm and 890 nm. The first signal is specific for the PANI structure in basic medium [bib_ref] Polyaniline dispersions 2. UV-Vis absorption spectra, Stejskal [/bib_ref] , the absorbance declining with the reaction progress and pH decrease, evidence for monomer consumption. The signal at 890 nm is specific for the fullerene radical carbocation [bib_ref] On the Radical Cation Spectra of Fullerenes and Fulleranes, Cataldo [/bib_ref].
In order to highlight the formation of the intermediate species during the polymerization process, Vis-NIR spectroscopy was used to study the evolution of the system for a wavelength range between 700 nm and 1200 nm [fig_ref] Figure 2: NIR spectra at different intervals [/fig_ref]. The signals at 820 nm and 950 nm are specific to the carbocation resulting from C 60 23 , whereas the 1144 nm peak sustains the oxidized species of ANI. The signal at 1144 nm appears also for the CdSe-ANI mixture without C 60 , confirming this designation.
The fluorescence spectra [fig_ref] Figure 3: Emission spectra at an excitation wavelength of 370 nm [/fig_ref] reveal that the emission of CdSe (max around 460 nm) decreases on addition of ANI followed by a further decrease upon introduction of C 60 . This aspect sustains the donor activity of CdSe towards both components. The emission intensity registers only small decrease in time after initial quench caused by C 60 insertion in the system. This stabilization and the lack of emission increase indicate the hypothesis that the CdSe quantum dots act only as promotor for the polymerization.
Based on the presented experimental data, the schematic representation of the polymerization process (including the HOMO-LUMO levels for each component) is presented in [fig_ref] Figure 4: Schematic representation of the oxidative polymerization of ANI using CdSe-C 60 system [/fig_ref].
The polymerization process stages are: 1. excitation of the CdSe quantum-dots; 2. electron donating process from CdSe * to ANI molecules; 3. excitation of ANI molecules; 4. electron transfer from ANI * to C 60 , accompanied by the polymerization process; 5. C 60 excitation; 6. electron transfer from C 60 to ANI with the formation of fullerene carbocation (C 60 + - ); 7. electron transfer from ANI * to C 60 + - accompanied by the polymerization process. Following the polymerization experiments, we have determined that the CdSe quantum dots have a promoter role and do not actively participate in the polymerization process. Based on the steps 4 and 6 presented in [fig_ref] Figure 4: Schematic representation of the oxidative polymerization of ANI using CdSe-C 60 system [/fig_ref] , it is possible that the ratio between ANI and C 60 to influence the polymerization rate by modifying the concentration of the formed intermediate species. Thus, the process was followed by Vis-NIR spectroscopy, at different molar ratios of ANI/C 60 [fig_ref] Figure 5: NIR at different ANI/C 60 molar ratio [/fig_ref].
It is evident that with the acceptor concentration increase, the absorbance specific for the radical carbocation, as well as the absorbance of PANI formed also increases.
In order to better characterize the reaction mechanism two directions were followed: 1) polymerization experiments at different CdSe concentrations; 2) experiments at different ANI/C 60 ratio. In the first case, the values for the conversion are low, decreasing with the concentration increase of CdSe in the system; thus, confirming its promotor role in the polymerization process. Therefore, our next step was the study of the polymerization process kinetic at different concentrations of C 60 . In [fig_ref] Figure 6: The conversion versus ANI/C 60 ratio [/fig_ref] , it is presented the dependence of the conversion on the C 60 concentration. It can be observed that the conversion value decreases with the ANI/C 60 ratio increase.
In order to confirm a "living" polymerization mechanism GPC analyses were performed on the obtained polymers. The GPC results for lowest and highest conversion are summarized in .
From the GPC results it can be concluded that the conversion increase is accompanied by an increase of the molecular weight, respectively a decrease of the polydispersity index. In order to confirm the PANI structure, we have performed FTIR analysis [fig_ref] Figure 7: FTIR spectra for PANI synthesized using CdSe/C 60 system [/fig_ref]. The characteristics bands that can be noticed in the FTIR spectra are: 1598, 1439, 1220 and 1132 cm −1 due to quinoid ring C= C stretching, benzenoid C-C stretching, C-N stretching band and C-N +- 14,24 stretching vibration. The presence of C 60 can be confirmed by the presence of specific peaks at 1435 and 1186 cm −1 14 .
Elemental analysis of the PANI sample revealed the following values: 77.3% C, 7% H and 15.7% N. The results sustain the polymer structure of PANI.
For the characterization of the hybrid material in view of future applications, HRTEM analysis was performed [fig_ref] Figure 8: HRTEM images of the morphology of aniline layer covered with CdSe nanoparticles [/fig_ref].
The CdSe nanoparticles (dark contrast) is not uniformly spread over the PANI layer (gray contrast). White arrows indicate almost bare regions on the PANI layer where the density of CdSe nanoparticles is very small. Black arrows show holes in the aniline layer were the carbon grid is revealed (light gray contrast). The CdSe nanoparticles are in close contact with the PANI layer. No CdSe particles were observed outside PANI layer, this can be observed in the TEM image below taken at an even lower magnification [fig_ref] Figure 7: FTIR spectra for PANI synthesized using CdSe/C 60 system [/fig_ref]. C 60 crystallites were not observed.
# Conclusions
This study presents a novel method for the oxidative polymerization of ANI by employing C 60 /(CdSe) quantum dots, as promoting agent of the polymerization system. The polymerization initiation mechanism is based on the difference between the HOMO-LUMO energy levels of the components which permit the formation of a continuous donor-acceptor exchange. In order to highlight the formation of the intermediate species during the polymerization process, UV-Vis and Vis-NIR spectroscopy was used. Thus, the fullerene carbocation radical has been put into evidence.
Polymerization experiments performed at different ANI/C 60 ratio and CdSe concentrations revealed a decreasing conversion with the increase of CdSe. Further, GPC analyses confirmed a ''living" mechanism of the polymerization.
The HRTEM investigation of the morphology revealed CdSe nanoparticles non-uniformly distributed inside the PANI layer, however no free quantum dots were observed.
[fig] Figure 1: (A) UV-Vis spectra for ANI-C 60 charge transfer complex formation (B) UV-Vis spectra at 50 °C and reaction intervals. Scientific RepoRts | 6:32237 | DOI: 10.1038/srep32237 [/fig]
[fig] Figure 2: NIR spectra at different intervals. [/fig]
[fig] Figure 3: Emission spectra at an excitation wavelength of 370 nm. Scientific RepoRts | 6:32237 | DOI: 10.1038/srep32237 [/fig]
[fig] Figure 4: Schematic representation of the oxidative polymerization of ANI using CdSe-C 60 system. [/fig]
[fig] Figure 5: NIR at different ANI/C 60 molar ratio. [/fig]
[fig] Figure 6: The conversion versus ANI/C 60 ratio. [/fig]
[fig] Figure 7: FTIR spectra for PANI synthesized using CdSe/C 60 system. [/fig]
[fig] Figure 8: HRTEM images of the morphology of aniline layer covered with CdSe nanoparticles. White arrows indicate almost bare aniline. Scientific RepoRts | 6:32237 | DOI: 10.1038/srep32237 [/fig]
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Testing for HCV Infection: An Update of Guidance for Clinicians and Laboratorians
## Testing for hcv infection: an update of guidance for clinicians and laboratorians
In the United States, an estimated 4.1 million persons have been infected with hepatitis C virus (HCV), of whom an estimated 3.2 (95% confidence interval [CI] = 2.7-3.9) million are living with the infection. New infections continue to be reported particularly among persons who inject drugs and persons exposed to HCV-contaminated blood in health-care settings with inadequate infection control.
Since 1998, CDC has recommended HCV testing for persons with risks for HCV infection. In 2003, CDC published guidelines for the laboratory testing and result reporting of antibody to HCV. In 2012, CDC amended testing recommendations to include one-time HCV testing for all persons born during 1945-1965 regardless of other risk factors.
CDC is issuing this update in guidance because of 1) changes in the availability of certain commercial HCV antibody tests, 2) evidence that many persons who are identified as reactive by an HCV antibody test might not subsequently be evaluated to determine if they have current HCV infection, and 3) significant advances in the development of antiviral agents with improved efficacy against HCV. Although previous guidance has focused on strategies to detect and confirm HCV antibody, reactive results from HCV antibody testing cannot distinguish between persons whose past HCV infection has resolved and those who are currently HCV infected. Persons with current infection who are not identified as currently infected will not receive appropriate preventive services, clinical evaluation, and medical treatment. Testing strategies must ensure the identification of those persons with current HCV infection. This guidance was written by a workgroup convened by CDC and the Association of Public Health Laboratories (APHL), comprising experts from CDC, APHL, state and local public health departments, and academic and independent diagnostic testing laboratories, in consultation with experts from the Veterans Health Administration and the Food and Drug Administration (FDA). The workgroup reviewed laboratory capacities and practices relating to HCV testing, data presented at the CDC 2011 symposium on identification, screening and surveillance of HCV infection, and data from published scientific literature on HCV testing. Unpublished data from the American Red Cross on validation of HCV antibody testing also were reviewed.
## Changes in hcv testing technologies
Since the 2003 guidance was published (4), there have been two developments with important implications for HCV testing:
1. Availability of a rapid test for HCV antibody. The OraQuick HCV Rapid Antibody Test (OraSure Technologies) is a rapid assay for the presumptive detection of HCV antibody in fingerstick capillary blood and venipuncture whole blood. Its sensitivity and specificity are similar to those of FDA-approved, laboratory-conducted HCV antibody assays. In 2011, a Clinical Laboratory Improvements Amendments waiver was granted to the test by FDA. The waiver provides wider testing access to persons at risk for HCV infection, permitting use of the assay in nontraditional settings such as physician offices, hospital emergency departments, health department clinics, and other freestanding counseling and testing sites. 2. Discontinuation of RIBA HCV. The Chiron RIBA HCV 3.0 Strip Immunoblot Assay (Novartis Vaccines and Diagnostics) that was recommended (4) for supplemental testing of blood samples after initial HCV antibody testing is no longer available. As a result, the only other FDA-approved supplemental tests for HCV infection are those that detect HCV viremia.
## Identifying current hcv infections
In 2011, FDA approved boceprevir (Victrelis, Merck & Co.) and telaprevir (Incivek, Vertex Pharmaceuticals) for treatment of chronic hepatitis C genotype 1 infection, in combination with pegylated interferon and ribavirin, in adult patients with compensated liver disease. Boceprevir and telaprevir interfere directly with HCV replication. Persons who complete treatment using either of these drugs combined with pegylated interferon and ribavirin are more likely to clear virus (i.e., have virologic cure), compared to those given standard therapy based on pegylated interferon and ribavirin. Viral clearance, when sustained, stops further spread of HCV and is associated with reduced risk for hepatocellular carcinoma (10) and all-cause mortality. Other compounds under study in clinical trials hold promise for even more effective therapies.
Because antiviral treatment is intended for persons with current HCV infection, these persons need to be distinguished from persons whose infection has resolved. HCV RNA in blood, by nucleic acid testing (NAT), is a marker for HCV viremia and is detected only in persons who are currently infected. Persons with reactive results after HCV antibody testing should be evaluated for the presence of HCV RNA in their blood.
On , this report was posted as an MMWR Early Release on the MMWR website (http://www.cdc.gov/mmwr).
## Benefits of testing for current hcv infection
Accurate testing to identify current infection is important to 1) help clinicians and other providers correctly identify persons infected with HCV, so that preventive services, care and treatment can be offered; 2) notify tested persons of their infection status, enabling them to make informed decisions about medical care and options for HCV treatment, take measures to limit HCV-associated disease progression (e.g., avoidance or reduction of alcohol intake, and vaccination against hepatitis A and B), and minimize risk for transmitting HCV to others; and 3) inform persons who are not currently infected of their status and the fact that they are not infectious.
## Recommended testing sequence
The testing sequence in this guidance is intended for use by primary care and public health providers seeking to implement CDC recommendations for HCV testing. In most cases, persons identified with HCV viremia have chronic HCV infection. This testing sequence is not intended for diagnosis of acute hepatitis C or clinical evaluation of persons receiving specialist medical care, for which specific guidance is available.
Testing for HCV infection begins with either a rapid or a laboratory-conducted assay for HCV antibody in blood . A nonreactive HCV antibody result indicates no HCV antibody detected. A reactive result indicates one of the following: 1) current HCV infection, 2) past HCV infection that has resolved, or 3) false positivity. A reactive result should be followed by NAT for HCV RNA. If HCV RNA is detected, that indicates current HCV infection. If HCV RNA is not detected, that indicates either past, resolved HCV infection, or false HCV antibody positivity.
Initial Testing for HCV Antibody. An FDA-approved test for HCV antibody should be used. If the OraQuick HCV Rapid Antibody Test is used, the outcome is reported as reactive or nonreactive. If a laboratorybased assay is used, the outcome is reported as reactive or nonreactive without necessarily specifying signal-to-cutoff ratios.
Testing for HCV RNA. An FDA-approved NAT assay intended for detection of HCV RNA in serum or plasma from blood of at-risk patients who test reactive for HCV antibody should be used. There are several possible operational steps toward NAT after initial testing for HCV antibody:
1. Blood from a subsequent venipuncture is submitted for HCV NAT if the blood sample collected is reactive for HCV antibody during initial testing. 2. From a single venipuncture, two specimens are collected in separate tubes: one tube for initial HCV antibody testing; and a second tube for HCV NAT if the HCV antibody test is reactive.
## Figure. recommended testing sequence for identifying current hepatitis c virus (hcv) infection
* For persons who might have been exposed to HCV within the past 6 months, testing for HCV RNA or follow-up testing for HCV antibody is recommended. For persons who are immunocompromised, testing for HCV RNA can be considered. † To differentiate past, resolved HCV infection from biologic false positivity for HCV antibody, testing with another HCV antibody assay can be considered. Repeat HCV RNA testing if the person tested is suspected to have had HCV exposure within the past 6 months or has clinical evidence of HCV disease, or if there is concern regarding the handling or storage of the test specimen. 3. The same sample of venipuncture blood used for initial HCV antibody testing, if reactive, is reflexed to HCV NAT without another blood draw for NAT (13).
## A separate venipuncture blood sample is submitted for hcv
NAT if the OraQuick HCV Rapid Antibody Test for initial testing of HCV antibody has used fingerstick blood.
## Supplemental testing for hcv antibody
If testing is desired to distinguish between true positivity and biologic false positivity for HCV antibody, then, testing may be done with a second HCV antibody assay approved by FDA for diagnosis of HCV infection that is different from the assay used for initial antibody testing. HCV antibody assays vary according to their antigens, test platforms, and performance characteristics, so biologic false positivity is unlikely to be exhibited by more than one test when multiple tests are used on a single specimen.
## Test interpretation and further action
SeeLaboratory Reporting "Acute hepatitis C" and "hepatitis C (past or present)" are nationally notifiable conditions, and are subject to mandated reporting to health departments by clinicians and laboratorians, as determined by local, state or territorial law and regulation. Surveillance case definitions are developed by the Council of State and Territorial Epidemiologists in collaboration with CDC. In all but a few jurisdictions, positive results from HCV antibody and HCV RNA testing that are indicative of acute, or past or present HCV infection, are reportable. Specific policies for laboratory reporting are found at health department websites.
## Future studies
Research, development, validation, and cost-effectiveness studies are ongoing to inform the best practices for detecting HCV viremia and for distinguishing between resolved HCV infection and biologic false positivity for HCV antibody in persons in whom HCV RNA is not detected. Outcomes of these studies will provide comprehensive guidance on testing, reporting, and clinical management, and will improve case definitions for disease notification and surveillance. If distinction between true positivity and biologic false positivity for HCV antibody is desired, and if sample is repeatedly reactive in the initial test, test with another HCV antibody assay. In certain situations § follow up with HCV RNA testing and appropriate counseling.
* If HCV RNA testing is not feasible and person tested is not immunocompromised, do follow-up testing for HCV antibody to demonstrate seroconversion. If the person tested is immunocompromised, consider testing for HCV RNA. † It is recommended before initiating antiviral therapy to retest for HCV RNA in a subsequent blood sample to confirm HCV RNA positivity. § If the person tested is suspected of having HCV exposure within the past 6 months, or has clinical evidence of HCV disease, or if there is concern regarding the handling or storage of the test specimen. |
Diagnosis, consultation, treatment, and impact of migraine in the US: Results of the OVERCOME (US) study
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.AbstractObjective: The ObserVational survey of the Epidemiology, tReatment and Care of MigrainE (OVERCOME; United States) study is a multicohort, longitudinal web survey that assesses symptomatology, consulting, diagnosis, treatment, and impact of migraine in the United States.Background: Regularly updating population-based views of migraine in the UnitedStates provides a method for assessing the quality of ongoing migraine care and identifying unmet needs. Methods: The OVERCOME (US) 2018 migraine cohort involved: (I) creating a demographically representative sample of US adults using quota sampling (n = 97,478), (II) identifying people with active migraine in the past year via a validated migraine diagnostic questionnaire and/or self-reported medical diagnosis of migraine (n = 24,272), and (III) assessing consultation, diagnosis, and treatment of migraine (n = 21,143). The current manuscript evaluated whether those with low frequency episodic migraine (LFEM; 0-3 monthly headache days) differed from other categories on outcomes of interest. Results: Among the migraine cohort (n = 21,143), 19,888 (94.1%) met our International Classification of Headache Disorders, 3rd edition-based case definition of migraine and 12,905 (61.0%) self-reported a medical diagnosis of migraine. Respondents' mean (SD) age was 42.2 (15.0) years; 15,697 (74.2%) were women. Having at least moderate disability was common (n = 8965; 42.4%) and around half (n = 10,783; 51.0%) had consulted a medical professional for migraine care in the past year. Only 4792 (22.7%) of respondents were currently using a triptan. Overall, 8539 (40.4%) were eligible for migraine preventive medication and 3555 (16.8%) were currently using migraine preventive medication. | 123 HEADACHE
## Introduc ti on
Migraine is a chronic neurological disease that affects ~15% of individuals in the United States 1,2 and causes substantial personal and economic costs. [bib_ref] The prevalence and impact of migraine and severe headache in the United..., Burch [/bib_ref] [bib_ref] Indirect cost burden of migraine in the United States, Hawkins [/bib_ref] [bib_ref] Direct cost burden among insured US employees with migraine, Hawkins [/bib_ref] [bib_ref] Cost of health care among patients with chronic and episodic migraine in..., Stokes [/bib_ref] [bib_ref] National trends in direct health care expenditures among US adults with migraine:..., Raval [/bib_ref] [bib_ref] A narrative review on the burden of migraine: when the burden is..., Leonardi [/bib_ref] The World Health Organization ranks migraine as the second leading cause of years lived with disability and the leading cause of disability in women age 15-49 years. [bib_ref] Migraine remains second among the world's causes of disability, and first among..., Steiner [/bib_ref] Monitoring patterns of consultation, diagnosis, and treatment provides a method for assessing the quality of ongoing medical care and identifying barriers to better outcomes. The US population-based studies of migraine over the past 30 years have provided ongoing snapshots of migraine 9-13 that reflect evolving consulting, diagnostic, medication, and impact/burden patterns . These studies have shown that the percentage of those responding to population-based surveys whose symptoms identify them as having migraine has gone up over time and the impact of migraine may be increasing. [bib_ref] Prevalence of migraine headache in the United States. Relation to age, income,..., Stewart [/bib_ref] [bib_ref] Prevalence and burden of migraine in the United States: data from the..., Lipton [/bib_ref] [bib_ref] Migraine prevalence, disease burden, and the need for preventive therapy, Lipton [/bib_ref] [bib_ref] The impact of chronic migraine: the Chronic Migraine Epidemiology and Outcomes (CaMEO)..., Adams [/bib_ref] [bib_ref] Migraine in America Symptoms and Treatment (MAST) Study: baseline study methods, treatment..., Lipton [/bib_ref] Emerging developments in migraine treatment may herald a new era of migraine care. 14 In particular, novel monoclonal antibodies that target calcitonin gene-related peptide (CGRP), small molecule CGRP receptor antagonists, serotonin 5-HT 1F agonists, devices, and biobehavioral approaches offer healthcare providers (HCPs) a broader range of treatment options. [bib_ref] Non-invasive neuromodulation for migraine and cluster headache: a systematic review of clinical..., Reuter [/bib_ref] The ObserVational survey of the Epidemiology, tReatment and Care of MigrainE (OVERCOME; United States) study is a longitudinal, multicohort, web-based study annually recruited in a demographically representative sample of the US population. The primary objective of OVERCOME (US) is to monitor changes in patterns of consultation, diagnosis, acute and preventive treatment for migraine, and the impact of migraine over time as novel classes of treatment come into wider use. The current manuscript is the primary analyses of the initial OVERCOME (US) 2018 cohort and establishes the baseline of consulting, treatment, and impact of migraine while also considering potentially relevant covariates, such as sociodemographic characteristics and having a self-reported medical diagnosis (SR-MD) of migraine overall and across monthly headache day categories.
We hypothesized that those with low frequency episodic migraine
## Overcome (us) is a closed survey and requires panel mem-
bers to log-in to participate. Following consent, cookies were created to identify unique users, however, identifiable data were not collected in the study data. Prior to fielding, the survey underwent qualitative testing. During and following fielding, quality control measures included verifying programmed response ranges, performing consistency checks, evaluating length of interview, and ensuring an answer for each question was entered before moving to the next. Where appropriate, response options of "prefer not to answer," "don't know," "does not apply to me," and "don't remember" were included to accommodate those unable or unwilling to provide a definitive answer to a specific question. When appropriate, survey response option randomization and adaptive question logic were applied. provides full details regarding the programming, recruitment, enrollment, and survey administration of OVERCOME (US).
Establishing the migraine cohort involved three phases: [bib_ref] Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and..., Vos [/bib_ref] creating a demographically representative sample of US adults, [bib_ref] The prevalence and impact of migraine and severe headache in the United..., Burch [/bib_ref] identifying those with active migraine, and (3) characterizing Those with LFEM differed from moderate and high frequency episodic migraine and chronic migraine on nearly all measures of consulting, diagnosis, and treatment.
# Conclusion:
The OVERCOME (US) 2018 cohort revealed slow but steady progress in diagnosis and preventive treatment of migraine. However, despite significant impact among the population, many with migraine have unmet needs related to consulting for migraine, migraine diagnosis, and getting potentially beneficial migraine treatment.
Moreover, it demonstrated the heterogeneity and varying unmet needs within episodic migraine. Studies excluded for not using ICHD-based diagnoses were recently reviewed by Burch et al.symptomatology, consultation, treatment, and impact of migraine. The sample of respondents (n = 23,832) who met the above criteria for migraine were invited to complete the Migraine Assessment Survey in order to achieve our sample aim of at least 20,000 with migraine (to analyze smaller subgroups and account for potential loss at follow-up surveys).
Phase III: Establishing the migraine cohort Those with migraine were invited to complete the phase III survey, which assessed consultation, treatment, and impact of migraine.
To be included in the migraine cohort, individuals were required to answer all questions assessing the consultation, treatment, and impact of migraine. Among the 23,832 with migraine, 2689 (11.3%) did not complete the entire assessment and were not included in the migraine cohort. OVERCOME (US) aimed to have at least 20,000 people with migraine in each cohort in order to provide 90% power for detecting statistically significant differences within the cohort longitudinal analyses at 1 year and allow for evaluation of smaller subgroups that would not be feasible with a smaller baseline sample.
The 21,143 people who made up the 2018 migraine cohort met the cohort sample size aim. photophobia, and phonophobia) were experienced (response options included: "never," "rarely," "less than half the time," "half the time or more," and "all or nearly all the time"). [bib_ref] Migraine in America Symptoms and Treatment (MAST) Study: baseline study methods, treatment..., Lipton [/bib_ref] Respondents with migraine were asked about two aspects of health care consulting for headache: (1) lifetime consultation by specialty (primary care, neurology, headache specialist, pain specialist, emergency department, urgent care, retail clinic, and other) for headache/migraine (marking "yes" for all that applied) and then reporting the number of visits by specialty in the last 12 months
## Measures
and (2) reporting the number of HCP visits by specialty for any reason in the last 12 months. Within the survey, the term "specialist" (e.g., "headache specialist") did not imply a formal designation Respondents identified lifetime and current use of acute and preventive medications for migraine available at the time of the survey. Acute medication use was defined as "currently using or typically keeping on hand"; preventive medication use was defined by having "taken or used in the last 3 months" for migraine prevention. Eligibility for migraine prevention could be met in one of three ways: 3 monthly headache days with severe disability (MIDAS ≥21), 4-5 monthly headache days with at least some disability (MIDAS ≥6), or ≥6 monthly headache days. [bib_ref] Prevalence and burden of migraine in the United States: data from the..., Lipton [/bib_ref] [bib_ref] Migraine prevalence, disease burden, and the need for preventive therapy, Lipton [/bib_ref] Individuals also identified lifetime and current use of nonsurgical neurostimulation or biobehavioral treatments for migraine prevention. Both brand and generic name(s) for acute and preventive medications were provided in the survey.
# Statistical analysis
Mean and standard deviations (SDs) or median and range were reported for continuous variables and frequencies; percentages were reported for categorical variables. Sensitivity and positive predictive values of an SR-MD of migraine were calculated. Given that those who did not have a headache in the last 12 months not due to injury/illness were not asked any further questions regarding migraine, specificity the negative predictive value was not calculable.
Results were calculated for the overall cohort and then stratified by and RR, or 0.00 for beta, indicated a significant difference among the groups. The null hypothesis was that there was no difference between LFEM and other headache categories on these variables. For certain variables, the "prefer not to answer" response was excluded from analysis (see [fig_ref] TABLE 2: Continued) [/fig_ref]. Statistical analyses were performed using SAS Software version 9.4 (SAS Institute Inc., Cary, NC).
[formula] monthly [/formula]
## Re sults
## Respondent flow
Email invitations were sent to 1,888,290 individuals [fig_ref] Figure 1: provides a diagram of respondent flow [/fig_ref]
## Sociodemographics and migraine-related characteristics
As shown in [fig_ref] TABLE 2: Continued) [/fig_ref].
## Monthly headache day category consultation and diagnosis
The proportion with at least one lifetime medical consultation for headache/migraine was high (78.9%; [fig_ref] TABLE 2: Continued) [/fig_ref]. Consultation in primary care was most common (70. Preventive medications used for the preventive treatment of migraine included specific names (brand/generic) of antidepressants, antiseizure drugs, antihypertensive drugs, botulinum neurotoxins, and calcitonin gene-related peptide (CGRP) monoclonal antibodies available at the time of the survey.
n Neurostimulation involved non-surgical external nerve stimulator devices.
o Biobehavioral treatments involved those treatments used specifically for migraine prevention.
p "Current" defined as "currently using or typically keep on hand" for acute medications and "last 3 months" for preventive medications.
q Models assessed the effect of each monthly headache day group 4-7, 8-14, and ≥15 versus 0-3 monthly headache days on each dichotomized variable (predicted event italicized). Logistic regression models were used to assess odds ratios (ORs) for binary outcomes, linear regression models were used and the beta was reported for continuous outcomes with a normal distribution, and Poisson regression models were used and rate ratios (RRs) were approximated and presented for continuous variables with a Poisson distribution. All models were unadjusted, and 95% CIs reported. Parameter estimates (OR, beta, or RR) were bolded to indicate statistical significance. [fig_ref] TABLE 2: Continued) [/fig_ref]. shows that 40.4% of respondents met eligibility criteria for migraine prevention; and 16.8%
## Ta b l e 2 (continued)
were currently using a migraine preventive medication and this increased with higher monthly headache day frequency (LFEM = 13.2%; MFEM = 18.4%; HFEM = 20.4%; and CM = 28.9%; . [fig_ref] TABLE 2: Continued) [/fig_ref] shows that lifetime use of nonsurgical neurostimulation was 2.
## Discuss ion
The OVERCOME (US) study was designed to longitudinally monitor and characterize changes in healthcare consulting for migraine, acute and preventive migraine medication use, and impact on people with migraine in a large representative sample of people in the United States. The current manuscript focuses on cross-sectional data for the first cohort in this multicohort longitudinal study. This is the most recent in a series of US studies conducted over the past 30 years [bib_ref] Prevalence and burden of migraine in the United States: data from the..., Lipton [/bib_ref] [bib_ref] Migraine prevalence, disease burden, and the need for preventive therapy, Lipton [/bib_ref] Eligibility was defined three ways: ≥6 monthly headache days, 4-5 monthly headache days with at least some disability (MIDAS ≥6), or 3 monthly headache days with severe disability (MIDAS ≥21). b Currently taking was defined as "taken or used in the last 3 months." Migraine preventive medication eligibility considered disability and monthly headache day frequency as specified by the American Headache Society.Currently taking migraine preventive medication use was defined as use within the last 3 months for migraine and is reflective of the percentage among the overall total population within that monthly headache day frequency (regardless of current eligibility for migraine preventive medication) [bib_ref] The prevalence and burden of migraine and severe headache in the United..., Burch [/bib_ref] Lifetime consultation for migraine at an emergency department was 24.0%; this is higher than the 5%-6% in AMS-I and AMPP. [bib_ref] Use of the emergency department for severe headache. A population-based study, Lipton [/bib_ref] Overall, 31.0% had consulted at an emergency department or urgent care center and 12.3% had consulted at a retail clinic. The high rate of utilization outside primary and specialty care may be reflective of broader consulting trends in the United States related to the growth of ambulatory clinics. [bib_ref] Convenient ambulatory care-promise, pitfalls, and policy, Chang [/bib_ref] However, the emergency department/urgent care setting is not ideal for delivering ongoing migraine care due to its environment, the prioritization migraine presentation may receive, the increased likelihood of unnecessary neuroimaging, and the use of opioids as a first-line treatment for migraine. [bib_ref] American Headache Society Choosing Wisely Task Force. Choosing wisely in headache medicine:..., Loder [/bib_ref] [bib_ref] Trends in opioid analgesic use for headaches in US emergency departments, Mazer-Amirshahi [/bib_ref] [bib_ref] Use of narcotic analgesics in the emergency department treatment of migraine headache, Colman [/bib_ref] [bib_ref] The prevalence and burden of migraine and severe headache in the United..., Burch [/bib_ref] SR-MD for migraine (58.6% among those screening positive for migraine and 61.0% overall) is numerically higher than previous for those with LFEM, which likely reflects both those who do not need triptans as well as failures to deliver guideline-based care.
Rates of current triptan use are modest among those with MFEM (18.4%) and HFEM (28.7%). Although triptans are contraindicated for certain comorbid conditions, this alone cannot fully account for their lack of use. 46,47 Current opioid use for migraine (19.1%) was lower than lifetime use (47.7%) and may reflect favorable shifts in treatment away from opioids and toward triptans. [bib_ref] Characterizing opioid use in a US population with migraine: results from the..., Lipton [/bib_ref] [bib_ref] Vital signs: changes in opioid prescribing in the United States, Guy [/bib_ref] Regular use of opioids is well documented to increase risk of headache worsening and the onset of CM. [bib_ref] Opioid use and dependence among persons with migraine: results of the AMPP..., Buse [/bib_ref] [bib_ref] Acute migraine medications and evolution from episodic to chronic migraine: a longitudinal..., Bigal [/bib_ref] Overall, 40.4% of respondents were deemed prevention eligible and 16.8% were currently using a migraine prevention medication.
The proportion of use among those with at least one headache day a week is higher, yet only 18.4% of those with MFEM, 20.4% with HFEM, and 28.9% with CM were currently taking migraine preventive medication. Although low, the overall rate of 16.8% is higher than the 12%-13% rate reported in AMPP 11 and suggests potentially modest improvement in preventive medication use. The proportion of people with migraine is similar to the findings from the AMPP study but the overall rates of use have increased by 25%.
The reported current use of nonsurgical neurostimulation (1.2%) and biobehavioral treatments (14.2%) is novel. Biobehavioral use was higher than expected given how infrequently healthcare professionals specializing in biobehavioral treatments (e.g., psychologists and licensed clinical social workers) are utilized. However, individuals can access biobehavioral treatments (e.g., mindfulness, meditation, relaxation, biofeedback, and cognitive-behavioral therapy) through written/auditory/visual online materials, books, and mobile apps.
This may account for the reported utilization rate.
Overall, the percentage of current preventive treatment use is concerning given the high rates of at least moderate disability (57.5% within LFEM, 68.8% within HFEM, and 79.7% within CM). These increasing rates of disability as monthly headache day frequency increases is consistent with other population-based findings. [bib_ref] The effect of psychiatric comorbidities on headache-related disability in migraine: results from..., Lipton [/bib_ref] This study had several strengths. The OVERCOME (US) 2018 cohort drew from a representative US sample that allowed invitees an equal opportunity to participate in the phase I screening and included respondents with varying levels of monthly headache day frequency and burden. Data were collected at a time (2018) when novel preventive and acute treatments designed specifically for migraine were becoming available and prior to the introduction of novel medications for the acute treatment of migraine.
Respondents were identified as having migraine using the validated AMS/AMPP migraine diagnostic questionnaire and/or SR-MD. The latter, although not typical of other studies in this area, allows for a more complete picture of the spectrum of people with migraine.
The study captured respondents (not consulting, not diagnosed, and not treated with prescription medication for migraine) and patient-reported outcomes not commonly found in other large-scale real-world evidence studies that use claims or electronic health records data. Validated measures were used wherever possible (e.g., AMS/AMPP migraine diagnostic questionnaire, MIDAS). The inclusion of migraine frequency spanning from LFEM to CM and including those not consulting for migraine aimed to reduce selection bias.
Moreover, evaluating LFEM, MFEM, and HFEM separately provides a more nuanced understanding of those with EM so that the needs of those with EM garner appropriate attention.
Along with these strengths, this study had several important limitations. The participation rate of 7% created the potential for participation bias. The eligible sample approximated US Census through quota sampling rather than traditional random sampling methods.
Relative to US Census data, women, individuals over the age of 55 years, and people who were married were over-represented whereas people of Hispanic origin or with annual household incomes greater than or equal to $100,000 were under-represented (see [fig_ref] TABLE 2: Continued) [/fig_ref].
Requiring internet access may underestimate the needs of some of the most vulnerable and the requirement to read/write in English may account for the under-representation of those of Hispanic origin. However, these patterns are not unusual in internet-based population survey research. Requiring individuals to complete the entire survey to be included in the cohort and the analyses may have introduced another form of participation bias and loss of potentially relevant information/non-random missingness of data. Relative to another population survey (AMPP), the OVERCOME (US) 2018 migraine cohort includes individuals with higher monthly headache day frequency and higher reports of severe migraine-related disability (see . This may have been a result of different sampling methods, participation bias, evolving recognition of migraine, and its impact among the population, or increased impact from migraine. Finally, survey data were self-reported and were not validated by a medical professional, healthcare claims, or electronic health records.
As such, they may be susceptible to recall bias.
The current findings spur ideas about other questions that OVERCOME (US) or other population-based studies may address, including better understanding how sociodemographics, geography, clinical characteristics, individual's beliefs about migraine and migraine care, and migraine burden influence the likelihood of consulting; how many patients only use the emergency department, urgent care, or retail clinics for migraine; how prescribing patterns may differ across care settings; and the influence of including an SR-MD only group. OVERCOME (US) will be able to build on the current findings as new data emerges longitudinally from this cohort and baseline/longitudinal findings from other cohorts. Finally, there is a need for manuscripts that provide a more detailed analysis of the trajectory of migraine care as viewed from population-based surveys over the last three plus decades.
The OVERCOME (US) 2018 cohort provides the latest serial "snapshot" of the migraine landscape. It is timely as the snapshot is of a time just as newly available therapies were becoming available. The results show that, relatively to previous population-based findings, consultation may now be more likely to include ambulatory clinics, diagnosis rates have shown slow and consistent improvement over time, and use of preventive medication may be slowly improving. The current study also provides evidence that the needs of those with EM vary and should not be considered homogenous when it comes to treatment or research. Several opportunities for optimizing migraine care, including patients seeking care in primary care, more people getting diagnosed with migraine and prescribed potentially beneficial acute and preventive medication, were identified. This baseline OVERCOME (US) 2018 cohort study will be followed by other unique cohorts followed longitudinally to assess changes in the migraine care landscape concurrent with the introduction of novel therapeutic classes for preventing or treating migraine.
[fig] Figure 1: provides a diagram of respondent flow. [/fig]
[fig] Phase: II: Identifying respondents with migraine The purpose of phase II was to identify respondents with migraine in the demographically representative sample. This phase involved initially asking a series of questions surrounding the respondent's health and comorbidities, including a question about whether the respondent had at least one headache in the past 12 months not associated with head injury/illness/hangover. These potentially eligible individuals were assessed for migraine in two ways: (1) they completed the validated American Migraine Study (AMS)/American Migraine Prevalence and Prevention Study (AMPP) migraine diagnostic questionnaire to determine if they had migraine (algorithmic details can be found elsewhere) 9,10,11,17 and/or (2) they had an SR-MD of migraine defined by indicating that a health care provider had told them that they had migraine" and/or "chronic migraine or transformed migraine." We identified 23,832 individuals with migraine and also sampled a control group of 10,000 individuals without active migraine in the past year and no SR-MD of migraine. The control group was demographically matched to the US Census data. [/fig]
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The Prevalence of Adolescent Social Fears and Social Anxiety Disorder in School Contexts
# Introduction
Social fears refer to anxiety or discomfort in social situations in which one may be and/or anticipates being exposed to evaluation from others, which in turn may result in embarrassment or humiliation. From an evolutionary perspective, social fears are an adaptive experience-they provide a social barometer that contributes to the adjustment of oneself to peers, to the improvement and preservation of social desirability, and to careful awareness of one's presentation and/or behaviors [bib_ref] Evolution and social anxiety: The Role of Attraction, Social Competition, and Social..., Gilbert [/bib_ref]. However, difficulties may arise when normative social situations are perceived as posing disproportionate threats, and that perception causes a myriad of other symptoms associated with significant impairment. That is the case with social anxiety disorder (SAD), which is at the extreme end of the social anxiety continuum. SAD is characterized by a marked and persistent fear of everyday social and/or performance situations. Individuals with SAD often report concerns such as fear of one's anxiety symptoms (i.e., shaking, sweating, blushing, stuttering) being visible to others and/or fear of seeming anxious, strange, incompetent, or uninteresting [bib_ref] Social anxiety in young people: A prevalence study in seven countries, Jefferies [/bib_ref].
SAD has its most frequent onset during adolescence (i.e., before the age of 18) [bib_ref] The cross-national epidemiology of social anxiety disorder: Data from the World Mental..., Stein [/bib_ref] [bib_ref] Lifetime Prevalence and Age-of-Onset Distributions of DSM-IV Disorders in the National Comorbidity..., Kessler [/bib_ref]. Most of the social situations reported to be difficult by adolescents with SAD occur in school contexts and include talking to peers, starting or joining a conversation, writing on the board, and other social performances tasks (e.g., athletic, reading aloud in class) [bib_ref] Childhood Social Anxiety Disorder: From Understanding to Treatment, Chavira [/bib_ref]. When left untreated, difficulties associated with SAD seem to perpetuate into adulthood [bib_ref] Disability and Quality of Life in Social Phobia: Epidemiologic Findings, Stein [/bib_ref] [bib_ref] Influence of Psychiatric Comorbidity on Recovery and Recurrence in Generalized Anxiety Disorder,..., Bruce [/bib_ref] following a typically chronic and debilitating course [bib_ref] What do we know today about the prospective long-term course of social..., Steinert [/bib_ref]. SAD can lead to significant impairments and have impact in various domains of the life of adolescents (e.g., greater risk for school dropout, poorer qualifications, increased likelihood to be a victim of bullying, fewer friends, less gratifying interpersonal relationships) [bib_ref] Adolescent loneliness and social anxiety as predictors of bullying victimisation, Acquah [/bib_ref] [bib_ref] Linking Social Anxiety and Adolescent Romantic Relationship Functioning: Indirect Effects and the..., Hebert [/bib_ref]. Over time, these difficulties are associated with an overall reduction in quality of life [bib_ref] Linking Social Anxiety and Adolescent Romantic Relationship Functioning: Indirect Effects and the..., Hebert [/bib_ref]. Furthermore, SAD has been linked to increased vulnerability to other anxiety and mood disorders [bib_ref] Sociodemographic Correlates and Mental Health Comorbidities in Adolescents with Social Anxiety: The..., Jystad [/bib_ref] [bib_ref] Social anxiety disorder among children and adolescents: A nationwide survey of prevalence,..., Mohammadi [/bib_ref] , behavioral disorders [bib_ref] Social anxiety disorder among children and adolescents: A nationwide survey of prevalence,..., Mohammadi [/bib_ref] , and substance abuse [bib_ref] Sociodemographic Correlates and Mental Health Comorbidities in Adolescents with Social Anxiety: The..., Jystad [/bib_ref] [bib_ref] Social Phobia and Subtypes in the National Comorbidity Survey-Adolescent Supplement: Prevalence, Correlates,..., Burstein [/bib_ref].
Despite the growing body of research concerning the recognition of, evaluation of, and intervention with SAD, the disorder still seems to be neglected. Specifically, SAD presents one of the lowest rates of treatment seeking [bib_ref] Social anxiety disorder in childhood and adolescence: Current status and future directions, Kashdan [/bib_ref]. This may be associated with several factors: on the one hand, social anxiety seems to be underrecognized and elicit less concern and help-seeking recommendations by adolescents themselves [bib_ref] Adolescent Mental Health Literacy: Young People's Knowledge of Depression and Social Anxiety..., Coles [/bib_ref] ; additionally, social anxiety is perceived to be embarrassing to have, which in turn predicts (alongside other variables) how much others want to distance themselves from the socially anxious person [bib_ref] How people evaluate others with social anxiety disorder: A comparison to depression..., Anderson [/bib_ref] ; finally (and paradoxically), experiencing SAD symptoms and being aware of the stigma associated with mental health in general has also been associated with less intention to seek help, particularly from peers and informal adults [bib_ref] Do stigma and level of social anxiety predict adolescents' help-seeking intentions for..., Mcdonagh [/bib_ref]. This reluctance to ask for support might indicate that the real impact of this disorder is not known, especially in adolescents. Alongside this gap, evidence regarding the prevalence of SAD is scarce, particularly in adolescent samples. Existing research on the prevalence estimates of SAD can be found in [fig_ref] Table 1: SAD prevalence estimates from existing studies [/fig_ref]. It shows that only a minority of studies included referred to adolescent samples [bib_ref] Social Phobia and Subtypes in the National Comorbidity Survey-Adolescent Supplement: Prevalence, Correlates,..., Burstein [/bib_ref] [bib_ref] A pilot study on the prevalence of psychiatric disorders among Saudi children..., Al-Modayfer [/bib_ref] [bib_ref] Social phobia in Swedish adolescents, Gren-Landell [/bib_ref] [bib_ref] Adolescents: Results from the National Comorbidity Survey Replication-Adolescent Supplement (NCS-A), Merikangas [/bib_ref]. Prevalence rates greatly varied, with estimates ranging from 0.8% to 36%. Prevalence estimates may present discrepancies due to geographical and cultural factors [bib_ref] Measurement equivalence of the Social Interaction Anxiety Scale (SIAS) and Social Phobia..., Wong [/bib_ref]. As such, studies conducted in Eastern countries [bib_ref] Prevalence and correlates of social fears in Hong Kong, Lee [/bib_ref] and Western countries [bib_ref] Social fear and social phobia types among community youth: Differential clinical features..., Knappe [/bib_ref] showed disparate prevalence rates, concluding that Eastern societies tend to report lower estimated rates of SAD. Additionally, age ranges (i.e., increasing age can lead to higher SAD prevalence; e.g., [bib_ref] A profile of social, separation and generalized anxiety disorders in an Australian..., Spence [/bib_ref] , methodological differences and/or gender seem relevant variables. Prevalence rates of SAD were systematically found to be higher in females when compared to males, with women also reporting greater clinical severity and higher levels and number of social fears. This gender-sensitive prevalence of SAD seems to be more preponderant in adolescence and become more moderate along the course of life [bib_ref] Gender differences in social anxiety disorder: A review, Asher [/bib_ref]. However, some studies reported an equal gender distribution in adolescence (i.e., statistically non-significant differences between females and males regarding the prevalence of SAD) [bib_ref] Social anxiety disorder among children and adolescents: A nationwide survey of prevalence,..., Mohammadi [/bib_ref] [bib_ref] Prevalence of Psychiatric Disorders in Children and Adolescents of South Khorasan Province..., Farshidfar [/bib_ref].
Acknowledging the prevalence of impairing mental disorders such as SAD in a critical developmental phase such as adolescence is an important epidemiological responsibility [bib_ref] Interpreting the Prevalence of Mental Disorders in Children: Tribulation and Triangulatio, Holbrook [/bib_ref]. Advancing epidemiological understanding of SAD may help to promote preventive and interventional approaches targeted at early ages, who are usually more reluctant to look for specialized treatment [bib_ref] Do stigma and level of social anxiety predict adolescents' help-seeking intentions for..., Mcdonagh [/bib_ref]. Still, evidence on that prevalence has been scarce recently, particularly so considering adolescent samples only and considering the continuity of social anxiety, going from normative to high and then impacting intensity. As such, the current work will explore the prevalence of heightened levels of social anxiety and of SAD; those presenting with heightened levels of social anxiety may be at higher risk for SAD but are rarely considered in the literature. Because SAD is proposed to be more prevalent in females [bib_ref] Gender differences in social anxiety disorder: A review, Asher [/bib_ref] , we expect to replicate that finding in relation to adolescent SAD. Moreover, we expect to confirm previous indications that SAD presents one of the lowest rates of treatment seeking [bib_ref] Social anxiety disorder in childhood and adolescence: Current status and future directions, Kashdan [/bib_ref] , but we are also interested in exploring the prevalence of who would be willing to receive treatment if offered, which has not been considered before. Finally, we are interested in exploring the intensity of different core social fears associated with SAD (i.e., observation, social interaction, and social performance). The existence of such core fears is implicit in the diagnostic criteria for SAD, but adolescent SAD has not been explored in relation to those fears. Because these criteria consider a performance specifier for SAD, we would expect those fears to be particularly noteworthy. Findings taken from this work may help refine intervention guidelines for those presenting with SAD that are or not proactively seeking treatment.
# Materials and methods
## Participants
The initial sample consisted of 1495 participants aged between 15 and 18 years old (M = 15.71; SD = 0.82), of which 62.3% were girls (n = 932) and 37.7% were boys (n = 563).
## Instruments
## Screening for intense social fears
The Social Anxiety Scale for Adolescents (SAS-A) [bib_ref] Social Anxiety among Adolescents: Linkages with Peer Relations and Friendships, Greca [/bib_ref] was used for screening adolescents who might present with heightened social anxiety and could be further assessed for SAD. It is a self-report questionnaire that assesses adolescents' subjective experiences of social anxiety. It consists of 22 items (e.g., "I worry that others don't like me") answered on a 5-point Likert scale according to how much the item "is true for you" (1 = "not at all" to 5 = "all the time"). Scores may range from 22 to 110 and higher scores reflect higher levels of social anxiety. The scale comprises a total score and three subscales, namely the Fear of Negative Evaluation (FNE), the Social Avoidance and Distress of New Situations (SAD-New), and the Generalized Social Avoidance and Distress (SAD-General). In the original study, the FNE, SAD-New, and SAD-General subscales presented good internal consistencies with Cronbach's alpha values ranging from 0.76 to 0.91 [bib_ref] Social Anxiety among Adolescents: Linkages with Peer Relations and Friendships, Greca [/bib_ref]. In the SAS-A Portuguese version, Cronbach's alpha values reached 0.87 for FNE, 0.74 for SAD-New, 0.71 for SAD-General, and 0.88 for the total score, also representing a good internal consistency. For the screening phase of the current work only the total score was used, and it presented a good internal consistency with a Cronbach's alpha of 0.89.
## Diagnostic assessment
The Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID) [bib_ref] Reliability and Validity of the Mini International Neuropsychiatric Interview for Children and..., Sheehan [/bib_ref] [bib_ref] Mental health problems in male young offenders in custodial versus community based-programs:..., Rijo [/bib_ref] was used for defining the presence/absence of a diagnosis of SAD. It is a structured diagnostic interview for the assessment of DSM-V Axis I diagnoses in children and adolescents. The MINI-KID utilizes a branching tree logic, wherein two to four screening questions are presented for the evaluation of specific diagnostic criteria for each clinical diagnosis. All questions are answered in a yes/no format. If the screening questions are positively answered, additional symptom questions are presented. The MINI-KID includes items that allow the exclusion of medical, organic, and/or drug causes for disorders. Administration time ranges from 30 to 90 min. In its original version, the MINI-KID presented excellent interrater reliability across diagnoses except for dysthymia [bib_ref] Reliability and Validity of the Mini International Neuropsychiatric Interview for Children and..., Sheehan [/bib_ref]. The Portuguese version of MINI-KID resulted from a careful translation and backtranslation process and has been previously utilized as a method for diagnoses assessment [bib_ref] Mental health problems in male young offenders in custodial versus community based-programs:..., Rijo [/bib_ref]. Clinicians conducting the assessment phase of the present study received specific training, engaged in role-play training exercises, and observed experienced evaluators applying the interview before conducting individual evaluations.
## Self-reported core social fears
The Social Anxiety and Avoidance Scale for Adolescents (SAASA) [bib_ref] Social Fears in Adolescence-The social anxiety and avoidance scale for adolescents, Cunha [/bib_ref] was used to characterize the core social fears of adolescents presenting with SAD. It comprises 30 items in its adapted version for late adolescents [bib_ref] Evaluating social fears in late adolescence: Study with a Portuguese sample, Vagos [/bib_ref]. It includes two scales assessing the intensity of anxiety and the frequency of avoidance in social situations across the same six factors: interaction with the opposite sex, assertive interaction, observation by others, interaction in new social situations, performance in social situations, and eating and drinking in public. The items are rated on a five-point Likert-type scale (ranging from 1 = "none" to 5 = "very much" for anxiety, and from 1 = "never" to 5 = "almost always" for avoidance). Scores may range from 30 to 150 for each scale and higher scores reflect higher levels of social anxiety/avoidance. In its 30-item version tailored for late adolescents, the SAASA has shown adequate internal consistency values with Cronbach's alphas over 0.70 [bib_ref] Evaluating social fears in late adolescence: Study with a Portuguese sample, Vagos [/bib_ref]. For the present study, a version of the scale that groups the items into three factors related to the main social fears reported by adolescents (i.e., being observed in public, performance, and social interactionswas used for comparing core social fears experienced by adolescents with SAD. Using this sample and this version of the measurement model, good internal consistency was found with Cronbach's alphas ranging from 0.81 to 0.89.
## Procedures
The sample was collected from Portuguese high schools located in the north and center regions of Portugal. The procedures and goals of the study were shared with thirty-eight schools, following which consent for participating in this work was requested from their executive boards. Twenty-six schools (4 from the north region and 22 from the center region) accepted to collaborate in the implementation of the project. Written and informed consent from the guardian or legal representative was requested for all students attending the 10th and 11th grades. Potential participants were informed on the goals and procedures of the research and the confidentiality and anonymity of their responses were guaranteed. Students were asked to voluntarily participate in the study and informed verbal assent was requested.
The study then included three phases (i.e., screening for intense social fears, diagnostic assessment, and intervention referral). The screening phase consisted of requesting assenting students to fill in the Portuguese version of the Social Anxiety Scale for Adolescents (SAS-A;. Adolescents who participated in this screening composed the initial sample of this work (see above) that was used to analyze the prevalence of intense self-reported social fears. The following diagnostic assessment phase of this work invited adolescents scoring one standard deviation above the mean found for a large normative sample on the SAS-A (i.e., M = 46.97, SD = 11.16;for an individual structured clinical interview (i.e., the Portuguese version of the Mini International Neuropsychiatric Interview for Children and Adolescents; [bib_ref] Mental health problems in male young offenders in custodial versus community based-programs:..., Rijo [/bib_ref]. This allowed us to analyze the prevalence of SAD. Based on their diagnostic assessment using the MINI-KID, participants fulfilling inclusion (i.e., primary diagnosis of SAD) and exclusion criteria (i.e., primary diagnosis other than SAD, psychotic symptoms, suicidal risk, special education needs) segued to intervention referral. At this stage, adolescents who were referred to intervention within the research project from which these data were taken filled in a self-report protocol on social fears and other related variables; their scores on social fears were used to characterize diverse core social fears associated with adolescent SAD. Further information on the intervention implementation phase of the project will not be detailed in the present study-for more information contact the principal investigator of the research project and/or ClinicalTrials.gov Identifier: NCT04979676. [fig_ref] Figure 1: Flowchart of the prevalence estimates found for the screening, diagnostic assessment, and... [/fig_ref] summarizes prevalence estimates found for the screening, diagnostic assessment, and intervention referral phases of the current work.
# Results
## Prevalence of intense self-reported social fears
Of our initial sample (n = 1495), 26% (n = 388) presented with intense self-reported social fears (i.e., scoring one standard deviation above the mean found for a large adolescent normative sample; [bib_ref] Do stigma and level of social anxiety predict adolescents' help-seeking intentions for..., Mcdonagh [/bib_ref]. There were significant differences between participants reporting and not reporting intense self-reported fears concerning their scores on the complete
## Prevalence of intense self-reported social fears
Of our initial sample (n = 1495), 26% (n = 388) presented with intense self-reported social fears (i.e., scoring one standard deviation above the mean found for a large adolescent normative sample; [bib_ref] Do stigma and level of social anxiety predict adolescents' help-seeking intentions for..., Mcdonagh [/bib_ref]. There were significant differences between participants re-
## Prevalence of social anxiety disorder
Of adolescents participating in the diagnostic assessment phase of the study (n = 209), 140 (67%) presented a diagnosis of SAD, of which 118 (56.5%) had SAD as their primary diagnosis; alternatively, 33% (n = 69) did not fulfil diagnostic criteria for SAD. Those who fulfilled (M = 67.64, SD = 7.59) and did not fulfil (M = 67.12, SD = 737) those diagnostic criteria did not differ significantly on self-reported social anxiety based on the complete measure of the SAS-A (t(207) = 0.48, p = 0.63, d = 0.06). Relating to the initial sample (n = 1495), 9.4% of the participants had a SAD diagnosis, of which 7.9% had SAD as their primary diagnosis. Of the 140 adolescents diagnosed with SAD, 76.4% were girls (n = 107) and 23.6% were boys (n = 33). In relation to the complete sample (n = 1495), this signifies a prevalence point-estimate of SAD of 11.16% in girls and 5.86% for boys. Significant differences were found between boys and girls regarding the presence/absence of a SAD diagnosis (χ2(1) = 13.82, p < 0.001, d = 0.19). The standardized residual value (>|1.9|) shows that there are more girls than boys than statistically expected to have a SAD diagnosis. Alternatively, fewer boys than statistically expected had a SAD diagnosis. Still, though girls scored higher (M = 68.34, SD = 7.55) than boys (M = 65.39, SD = 7.39), the betweengender difference on the complete measure of the SAS-A was not statistically significant (t(138) = 1.97, p = 0.05, d = 0.39); it was, however, statistically significant for the fear of negative evaluation subscale only (t(138) = 3.04, p = 0.003, d = 0.56), with girls presenting significantly higher scores (M = 25.39, SD = 3.33) than boys (M = 23.21, SD = 4.30).
## Intervention referral
Of the adolescents diagnosed with SAD (n = 140), 92 (65.7%) were available for segueing to the intervention phase of the study. Alternatively, 17 (12.1%) refused intervention and 13 (9.3%) adolescents did not segue into the intervention phase due to other motives (e.g., special education needs, SAD not being the primary diagnosis). Only 18 (12.9%) were already receiving psychological intervention for SAD. Self-reported social fears, based on the complete SAS-A, did not differ significantly across these groups (F(3139) = 1.81, p = 0.15, f = 0.21).
The 92 participants who segued to the intervention phase of the study completed the SAASA (along with other measures) at pre-intervention. Using those data, a mixed ANOVA using gender as between-subject and measures as within-subject factors showed that only the main effect of measure was statistically significant (F(2, 180) = 42.11, p < 0.001, η2 = 0.32); the main effect of gender (F(1, 90) = 2.57, p = 0.11, η2 = 0.028) and the interaction effect (F(2, 180) = 1.99, p = 014, η2 = 0.022) were not statistically significant (see [fig_ref] Figure 2: Core social fears by gender [/fig_ref]. Post hoc pairwise comparisons using the Bonferroni correction showed that all three factors differ from each other, and this difference is statistically significant (p < 0.001).
# Discussion
SAD is a debilitating and highly prevalent condition in adolescence and is related to significant impairments in multiple domains of life [bib_ref] Linking Social Anxiety and Adolescent Romantic Relationship Functioning: Indirect Effects and the..., Hebert [/bib_ref]. Because difficulties associated with this disorder tend to persist throughout life, early detection and intervention are critical research concerns [bib_ref] Influence of Psychiatric Comorbidity on Recovery and Recurrence in Generalized Anxiety Disorder,..., Bruce [/bib_ref]. However, epidemiological information regarding SAD prevalence rates, particularly in adolescence, is still very limited. The present study aimed to bridge this gap by estimating the current prevalence rates of intense self-reported social fears and of SAD as per DSM-V diagnostic criteria, using a representative Portuguese sample.
About a quarter of adolescents recruited from school contexts reported intense social fears (i.e., 26%), though only about half of them was available to better explore their social fears by participating in a diagnostic interview. This unavailability may reflect the mental health stigma associated with social anxiety in adolescence. Previous evidence has
# Discussion
SAD is a debilitating and highly prevalent condition in adolescence and is related to significant impairments in multiple domains of life [bib_ref] Linking Social Anxiety and Adolescent Romantic Relationship Functioning: Indirect Effects and the..., Hebert [/bib_ref]. Because difficulties associated with this disorder tend to persist throughout life, early detection and intervention are critical research concerns [bib_ref] Influence of Psychiatric Comorbidity on Recovery and Recurrence in Generalized Anxiety Disorder,..., Bruce [/bib_ref]. However, epidemiological information regarding SAD prevalence rates, particularly in adolescence, is still very limited. The present study aimed to bridge this gap by estimating the current prevalence rates of intense self-reported social fears and of SAD as per DSM-V diagnostic criteria, using a representative Portuguese sample.
About a quarter of adolescents recruited from school contexts reported intense social fears (i.e., 26%), though only about half of them was available to better explore their social fears by participating in a diagnostic interview. This unavailability may reflect the mental health stigma associated with social anxiety in adolescence. Previous evidence has pointed to social anxiety being subjected to perceived/societal stigma [bib_ref] How people evaluate others with social anxiety disorder: A comparison to depression..., Anderson [/bib_ref] and that such perceived stigma had a demotivating effect in help-seeking behavior, even in the presence of SAD symptoms [bib_ref] Do stigma and level of social anxiety predict adolescents' help-seeking intentions for..., Mcdonagh [/bib_ref]. Because this subthreshold social anxiety has been linked with significant impairment [bib_ref] Social anxiety disorder: What are we losing with the current diagnostic criteria?, Filho [/bib_ref] , it seems important to be aware of and invest in prevention strategies towards participants that might present risk factors for SAD. For instance, following upon previous evidence on the efficacy of school-based intervention directed at improving general knowledge about mental illness and at promoting help-seeking and help behaviors (e.g., [bib_ref] Evaluation of a youth mental health literacy and action program: Protocol for..., Marinucci [/bib_ref] [bib_ref] A quasi-cluster randomized controlled trial of a classroom-based mental health literacy educational..., Yamaguchi [/bib_ref] , it would be important to specify these interventions to social anxiety in particular, which is particularly unrecognized by adolescents [bib_ref] Adolescent Mental Health Literacy: Young People's Knowledge of Depression and Social Anxiety..., Coles [/bib_ref].
Of those participants who reported intense self-reported social fears and were willing to be assessed, 33% did not fulfil criteria for a SAD diagnosis based on the DSM-V criteria. Thus, though those participants experienced subjective social anxiety, their experience did not (yet) convey a psychological disturbance. According to previous findings, the trajectory of these participants' social anxiety symptomatology may depend on several intrapersonal (e.g., behavioral inhibition, [bib_ref] Trajectories of socially anxious behavior from age 5 to 13: Temperamental and..., Poole [/bib_ref] ; biased interpretation of ambiguous social events, [bib_ref] Trajectories of Social Anxiety during Adolescence and Relations with Cognition, Social Competence,..., Miers [/bib_ref] and interpersonal (e.g., less successful social interactions, [bib_ref] Trajectories of Social Anxiety during Adolescence and Relations with Cognition, Social Competence,..., Miers [/bib_ref] variables. Hence, it would be relevant for school contexts to be aware of these vulnerability profiles, so that adolescents within them may be directed early on to appropriate interventions.
As for the presence of SAD, our results place the point-estimated prevalence of SAD in Portuguese adolescents at 9.4%; specifically, a ratio of approximately 1 to 3 was found regarding SAD prevalence. This value is slightly higher than data from previous studies that considered similar age groups (e.g., [bib_ref] Social Phobia and Subtypes in the National Comorbidity Survey-Adolescent Supplement: Prevalence, Correlates,..., Burstein [/bib_ref] [bib_ref] Social fear and social phobia types among community youth: Differential clinical features..., Knappe [/bib_ref] [bib_ref] Adolescents: Results from the National Comorbidity Survey Replication-Adolescent Supplement (NCS-A), Merikangas [/bib_ref] and it is much higher than a previous work using a Portuguese adolescent sample. This may reflect the post-confinement period the world is now facing. Specifically, while confined, adolescents might have temporarily felt comfortable and protected from demanding social situations [bib_ref] School Closures and Social Anxiety during the COVID-19 Pandemic, Morrissette [/bib_ref]. However, and because social isolation has been generally associated with increased social anxietyrelated symptomatology [bib_ref] The role of social isolation in social anxiety disorder: A systematic review..., Teo [/bib_ref] , these same adolescents may have felt more prominent social fears upon returning to school contexts [bib_ref] A Qualitative Study of Social Anxiety and Impairment amid the COVID-19 Pandemic..., Coyle [/bib_ref]. As such, SAD may be a more pressing concern nowadays. This point-estimated prevalence was much higher for girls then for boys. Additionally, girls were significantly more prone to experience pathological levels of social anxiety when compared with boys. This gender difference may have been highlighted by the age group under study, as some studies have shown that gender differences in the prevalence of SAD are greatest among adolescents and seem to diminish along the course of life (see [bib_ref] Gender differences in social anxiety disorder: A review, Asher [/bib_ref] for a review). It is also worth noting that the presentation of self-reported social anxiety was similar across boys and girls, except for fear of negative evaluation. The prospective association between fear of negative evaluation and social anxiety over time has been found for adolescence [bib_ref] Prospective Associations between Fears of Negative Evaluation, Fears of Positive Evaluation, and..., Fredrick [/bib_ref] , and so may be one of the mechanisms through which girls are more vulnerable to social anxiety than boys. It is also worth mentioning that adolescents with SAD reported most fearing performance in formal events (such as school presentations), followed by interaction with others and then being observed. On the one hand, this lends support to some youth presenting with a more evident (and perhaps circumscribed) fear of performance events, such as anticipated in the DSM-V specifier for performance SAD only. On the other hand, the prominence of this fear may be related to the age range we considered in this work, in which adolescents are likely starting to focus on school achievement as a necessity to progress to higher education. Unfortunately, this fear (and social anxiety in general) may be counterproductive to achieving desired academic goals (e.g., [bib_ref] Much more than just shyness: The impact of social anxiety disorder on..., Vilaplana-Pérez [/bib_ref].
Of the 140 diagnosed adolescents, only 18 (12.9%) were already receiving treatment. This is in accordance with previous literature stating that the prevalence of adolescents with SAD diagnosed in school contexts that are not under treatment is very high [bib_ref] School-Based Intervention for Adolescents with Social Anxiety Disorder: Results of a Controlled..., Masia-Warner [/bib_ref] , perhaps because difficulties associated with social anxiety have less impact on the school dynamics themselves (i.e., socially anxious students typically do not disturb classes) but rather impact mostly on how the individual internalizes their difficulties (i.e., subjective suffering) and achieves academic success.
Interestingly, when the possibility of receiving treatment was available, high rates of acceptance were found. Specifically, 65.7% of those diagnosed with SAD were willing to be provided with free psychological intervention for SAD. This result is, surprisingly, higher than what was found in previous studies that have showcased reluctance of adolescents with SAD in seeking treatment (e.g., only 24.1% of adolescents with SAD looked for specialized treatment;. It has been stated that doubts about where to obtain treatment and the fear of being negatively evaluated for seeking it are the main reasons why individuals with SAD are reluctant to seek treatment [bib_ref] Barriers to the Treatment of Social Anxiety, Olfson [/bib_ref]. In this case, however, adolescents did not have to proactively seek treatment; rather, it was offered to them. This suggests that if treatment is made available for adolescents to accept it (instead of waiting for them to seek it), adherence results will improve and progress can be achieved in closing the gap between the beginning of symptomatology and treatment seeking (previously stated to be of 15 years; [bib_ref] The impairments caused by social phobia in the general population: Implications for..., Kessler [/bib_ref]. Hopefully-because there are empirically validated interventions available to be implemented specifically in school contexts (e.g., [bib_ref] Treating adolescents with social anxiety disorder in school: An attention control trial, Warner [/bib_ref] -the trajectories of social fears and SAD of these adolescents can also be changed.
The findings of the present study further point to school settings as privileged contexts for identifying and treating mental health issues in young ages. Offering school-based prevention and/or intervention programs constitutes an important mental health responsibility that may help bring efficacious treatments to communities that have been systematically found to be under-diagnosed and lacking access to treatment. It may additionally contribute to increasing literacy in mental health (i.e., in school personnel, parents, caregivers) leading to more accurate and earlier identification and referral of mental health issues, including SAD [bib_ref] Adolescent Mental Health Literacy: Young People's Knowledge of Depression and Social Anxiety..., Coles [/bib_ref]. Considering the possible advantages that interventions in schools can have, future studies may investigate this from a community-based perspective on schools that foster parents, teachers, students and other school personnel as active ingredients of change [bib_ref] Treating Adolescents with Social Anxiety Disorder in Schools, Ryan [/bib_ref].
Despite the relevance of the present study, some limitations should be considered when interpreting its findings. For example, prevalence results/estimates may greatly vary due to different methodological aspects (e.g., age, period of reference). Following this perspective, results should be compared carefully. Additionally, gender was the only sociodemographic variable assessed in the current study. It might be relevant to explore differences between adolescents within different age groups, different years of education and/or different social economic status in future studies. Even though the school context proved to be a privileged setting for the identification of adolescents with SAD, this does not invalidate that other contexts may be important for a broader detection of the disorder. In addition, the present study considered only schools from the north and center regions of the country. Future studies should aim for a broader recruitment. The selection method can also be considered a limitation, since we did not evaluate the entire sample for diagnostic criteria (i.e., only those who scored high on the SAS-A;. As such, we may have lost teenagers who did not self-report social fears.
# Conclusions
The results of this study corroborate that SAD is a highly prevalent mental health disorder among adolescents and that, though being particularly prevalent in girls, boys' and girls' social fears seem to present similarly. Though we were not able to explore other demographic correlates and social backgrounds in relation to prevalence of intense social fears or SAD, their potential impact should not be overlooked and that should be considered in future research. Given the major impact SAD holds not only when difficulties emerge but also throughout life when left untreated [bib_ref] What do we know today about the prospective long-term course of social..., Steinert [/bib_ref] , current findings emphasize the importance of early diagnosis, prevention, and intervention for adolescent social anxiety, which may be particularly useful if adopting a broadened perspective. Specifically, on the one hand, it may be useful to consider not only those adolescents presenting with SAD but also those presenting with intense social fears, and how they may benefit from similar or specific intervention approaches. Cognitive therapy was found to be effective in adult SAD [bib_ref] Psychological and pharmacological interventions for social anxiety disorder in adults: A systematic..., Mayo-Wilson [/bib_ref] as well as promising in adolescent SAD, whether when delivered in person [bib_ref] Cognitive Therapy for Social Anxiety Disorder in Adolescents: A Development Case Series, Leigh [/bib_ref] [bib_ref] Delivering cognitive therapy for adolescent social anxiety disorder in NHS CAMHS: A..., Leigh [/bib_ref] or remotely [bib_ref] Online Cognitive Therapy for Social Anxiety Disorder in Adolescence: A Clinical Case..., Ganho-Ávila [/bib_ref]. Other approaches are also showing potential and should be further explored (e.g., acceptance and commitment therapy. In addition to studying their therapeutic efficacy, it might be important to consider the optimal length of treatment for different intensities of social anxiety symptoms and by that means have different preventive and adequate cost-benefit approaches to adolescent social anxiety. It may be the case that adolescents with intense social fears, compared to those with SAD, are less reluctant to seek help and/or may require less intervention sessions to achieve relevant therapeutic gains; there is no previous evidence on this at this time. On the other hand, schools were an evident context to identify and scrutinize social anxiety and thus should be seen as holistic contexts to be made more aware of how they can help identify vulnerable students and help them cope, in addition to directing them to available interventions. Previous findings have shown that the most frequent recommendation made by adolescents to someone experiencing social anxiety is to seek help from friends [bib_ref] Do stigma and level of social anxiety predict adolescents' help-seeking intentions for..., Mcdonagh [/bib_ref]. Friends may, then, have a dual role towards their socially anxious peers: they can help their peers appropriately cope with social events if they have sufficient literacy about the difficulties associated with social anxiety, and/or they can be an optimal (albeit indirect) route for socially anxious adolescents seeking formal help. Moreover, because our findings indicate that adolescents with SAD seem to be receptive to intervention being offered to them, school personnel should also be made more aware of the symptoms and suffering associated with social anxiety, so that they can make help-seeking options openly available to those who may benefit from them. Overall, the impact of schools' mental health literacy on help-seeking, helping and/or being open to treatment could be the focus of future work. Regardless, this inclusive and community-based perspective may overall be the route to prompting higher levels of social functioning, better interpersonal relationships, increased school satisfaction and success on the part of vulnerable adolescents, and consequently, better overall mental health. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Because the research project from which data for this manuscript were taken is still ongoing, data were not, at this time, made publicly available. Data that supported the results reported in the manuscript may be obtained, upon reasonable request, from the corresponding author.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Figure 1: Flowchart of the prevalence estimates found for the screening, diagnostic assessment, and intervention referral phases of the current work. [/fig]
[fig] Figure 2: Core social fears by gender. [/fig]
[fig] Author: Contributions: Conceptualization, F.A., D.V.F. and P.V.; methodology, F.A. and D.V.F.; software, F.A. and D.V.F.; validation, F.A. and D.V.F.; formal analysis, P.V.; investigation, F.A., D.V.F. and P.V.; resources, F.A., D.V.F. and P.V.; data curation, F.A. and D.V.F.; writing-original draft preparation, F.A.; writing-review and editing, P.V.; visualization, F.A., D.V.F. and P.V.; supervision, P.V.; project administration, P.V.; funding acquisition, P.V. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research was supported by FEDER-European Social Fund-through the COMPETE 2020-Operational Program for Competitiveness and Internationalization (POCI), and by Portuguese funds through the Portuguese Foundation for Science and Technology (FCT) in the framework of the project POCI-01-0145-FEDER-029445. Institutional Review Board Statement: The Study's procedures were approved by the ethics committee of the hosting institution (15 January 2018). [/fig]
[table] Table 1: SAD prevalence estimates from existing studies.Table 1. Cont. [/table]
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Chaperones as Suppressors of Protein Misfolded Oligomer Toxicity
Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates.
# Introduction
The various proteins that constitute the human proteome are functional if they fold correctly, remain soluble, can be trafficked properly, form functional complexes and perform their task correctly. These abilities rely on the existence of a proteostasis network and on its proper running. The proteostasis network is constituted by the translational machinery, a large body of molecular chaperones and co-chaperones, the autophagy/lysosome system (ALS) and the ubiquitin/proteasome system (UPS). Molecular chaperones and co-chaperones can be grouped in distinct families, including ribosome-binding chaperones, Hsp40s, Hsp70s, chaperonins, Hsp90s, Hsp100, prefoldins, small heat shock proteins (sHsps) and TPR-domain containing co-chaperones. It has been estimated that this large body of proteins amounts to ca. 330 distinct polypeptide chains in the human proteome [bib_ref] A chaperome subnetwork safeguards proteostasis in aging and neurodegenerative disease, Brehme [/bib_ref].
Molecular chaperones have long been recognized to: (i) assist protein folding from unfolded or partially folded states and promote formation and maintenance of multisubunit complexes;
(ii) mediate protein degradation via the UPS or ALS systems; (iii) inhibit protein aggregation by binding to fully or partially unfolded states; and (iv) promote disassembly of undesired aberrant protein aggregates [bib_ref] Chaperones in autophagy, Kaushik [/bib_ref] [bib_ref] The biology of proteostasis in aging and disease, Labbadia [/bib_ref] [bib_ref] Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation, Mogk [/bib_ref] [bib_ref] In vivo aspects of protein folding and quality control, Balchin [/bib_ref]. Although this last function has long been known to involve only chaperones of the ClpB and Hsp104 families present in bacteria, protozoa, plants and fungi , it has recently been discovered in higher eukaryotes as well [bib_ref] Metazoan Hsp70-based protein disaggregases: emergence and mechanisms. Front, Nillegoda [/bib_ref].
However, in addition to these four well established functions, evidence is mounting that molecular chaperones can also interact with protein aggregates (macromolecular species resulting from the aberrant self-assembly of proteins) and inhibit their toxicity without promoting their disassembly. In this review we will show the evidence in this direction, showing how molecular chaperones can interact with both protein oligomers and amyloid fibrils, that represent the small species forming at the beginning of the aggregation process and the fibrillar end-product of this phenomenon, respectively. We will describe the mechanism through which molecular chaperones suppress the detrimental effects of such aggregates in the absence of their disaggregation or clearance.
## Chaperones bind to protein oligomers
An increasing number of reports has shown the ability of molecular chaperones to interact with oligomeric species that form early during the aggregation of proteins. The extracellular chaperone clusterin has been observed, through Thioflavin T (ThT) kinetics and dot blot assays, to bind to prefibrillar species formed by six different amyloidogenic proteins or peptides at a substoichiometric 1:10 molar ratio [bib_ref] The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with..., Yerbury [/bib_ref]. Using transmission electron microscopy (TEM), ThT kinetics and size exclusion chromatography (SEC), Hsp104 was found to bind to Aβ 42 oligomers and protofibrils, but also to small fibrils, and abolish their ability to convert into amyloid fibrils through their further addition to preformed fibrils, as well as to abrogate their capacity to seed the addition of monomers to the fibril surface, up to a Hsp104:Aβ 42 stoichiometric ratio of 1:1000 [bib_ref] Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding..., Arimon [/bib_ref]. By means of single-molecule fluorescence methods, clusterin [bib_ref] The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β 1-40 peptide, Narayan [/bib_ref] and the sHsp αB-crystallin or HSPB5 [bib_ref] Amyloid-β oligomers are sequestered by both intracellular and extracellular chaperones, Narayan [/bib_ref] were observed to form longlived, stable complexes with Aβ 40 oligomers at equimolar ratios. Such sequestration was found also in the case of αB-crystallin and SOD1 aggregates, using ThT kinetics and SEC coupled with SDS-PAGE, with αB-crystallin:SOD1 molar ratios of 1:100 and 1:1, respectively [bib_ref] The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in..., Yerbury [/bib_ref]. A quantitative kinetic analysis and immunochemistry studies revealed that the chaperone DNAJB6, from the Hsp40 family, preferentially binds to oligomeric species of Aβ 42 at low substoichiometric molar ratios (up to 1:200), preventing their growth into longer fibrils as well as the formation of new fibril nuclei [bib_ref] Interaction of the molecular chaperone DNAJB6 with growing amyloid-β 42 (Aβ42) aggregates..., Månsson [/bib_ref].
This ability of chaperones to bind to protein oligomers is not just aimed at interfering with the process of amyloid fibril formation, but has also the important effect to inhibit directly the toxicity of these aberrant species. In fact, oligomers are characterized by physicochemical properties that make them harmful to cells [bib_ref] A causative link between the structure of aberrant protein oligomers and their..., Campioni [/bib_ref] [bib_ref] Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions, Olzscha [/bib_ref] [bib_ref] Protein misfolded oligomers: experimental approaches, mechanism of formation, and structure-toxicity relationships, Bemporad [/bib_ref]. Among the structural determinants of toxicity identified so far, the small size and the high extent of hydrophobic surface of the oligomers have been recognized to play an important role in causing cellular dysfunction [bib_ref] ANS binding reveals common features of cytotoxic amyloid species, Bolognesi [/bib_ref] [bib_ref] Size-dependent neurotoxicity of β-amyloid oligomers, Cizas [/bib_ref] [bib_ref] Toxicity of protein oligomers is rationalized by a function combining size and..., Mannini [/bib_ref]. It is just on these structural determinants that molecular chaperones seem to act with the goal of counteracting the damages mediated by the oligomers: in fact chaperones increase the size of the oligomers and mask hydrophobic patches exposed on their surface. Such evidence will be described in the next two sections.
## Chaperones induce clustering of the oligomers and inhibit their toxicity
In an early report, the ability of clusterin to inhibit the toxicity of preformed oligomers of Aβ 42 and of the SH3 domain of phosphatidylinositol 3-kinase (PI3-SH3) was observed on human neuroblastoma SH-SY5Y cells at 1:10 clusterin:substrate ratio [bib_ref] The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with..., Yerbury [/bib_ref]. Although the mechanism of action of the chaperone against the aggregates was not investigated in detail, the authors reported a sedimentation assay in which the formation of high molecular weight Aβ 42 /clusterin complexes were observed [bib_ref] The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with..., Yerbury [/bib_ref].
Later on, Hsp27 (HSPB1) was found to increase the size of preformed Aβ 42 oligomers, making them unable to exert their toxicity on N2a mouse neuroblastoma cell cultures [bib_ref] Sequestration of toxic oligomers by HspB1 as a cytoprotective mechanism, Ojha [/bib_ref]. In particular, atomic force microscopy (AFM), TEM and light scattering showed that the incubation of a substoichiometric concentration of Hsp27 with preformed Aβ 42 oligomers (1:5) in vitro generates larger aggregates. In these larger species Hsp27 co-precipitated with the Aβ 42 oligomers, without affecting significantly the structure of the oligomers, as shown by unaltered ThT binding and far-UV circular dichroism (CD) spectra. Interestingly, even the ability to bind to 8-anilinonaphthalene-1-sulfonic acid (ANS) was unalterd, indicating that the extent of the hydrophobic surface exposure does not change after the incubation with the chaperone.
In another study, five different chaperones, namely, αB-crystallin, Hsp70 (HSPA1A), clusterin, α 2 -macroglobulin and haptoglobin were able to suppress the toxicity of oligomers formed by three different proteins, the Aβ 42 peptide, the islet amyloid polypeptide (IAPP), and the model protein HypF-N at a substoichiometric concentration (up to 1:1000) on SH-SY5Y cells [bib_ref] Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein..., Mannini [/bib_ref]. Several methods of investigation applied to the HypF-N system, such as AFM, confocal microscopy coupled to immunostaining, centrifugation assays and intrinsic fluorescence, showed that large clusters of HypF-N oligomers are formed following the incubation with substoichiometric concentrations of chaperones. In the resulting large aggregates, chaperone molecules are trapped inside the large aggregates. Again, the chaperone-mediated conversion of the small oligomers into bigger nontoxic aggregates occurs without a remodeling of the structure of the oligomers, as assessed by Fourier-transform infrared spectroscopy and pyrene labeling [bib_ref] Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein..., Mannini [/bib_ref].
Recently, it was found that high amounts of insoluble aggregates containing chaperones, in particular the sHsps, accumulate in the long-lived daf-2 mutants of the nematode Caenorhabditis elegans during aging, and that such amounts are higher compared to the wild-type controls that had a normal lifespan [bib_ref] Widespread proteome remodeling and aggregation in aging C. elegans, Walther [/bib_ref]. It was suggested that the chaperones neutralize aberrant, potentially toxic, proteins and soluble oligomers by driving them into insoluble large aggregates and that this strategy enables to slow down the decline of the proteostasis network during normal aging and extend the lifespan of the mutant nematodes [bib_ref] Widespread proteome remodeling and aggregation in aging C. elegans, Walther [/bib_ref].
A similar mechanism was observed on various cell culture systems, such as CHO, N2a, NIH-3T3, PC12 and MEF, treated with NT219, an inhibitor of the insulin/IGF-1 signaling pathway [bib_ref] The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and..., Moll [/bib_ref]. NT219 was found to enhance the aggregation of misfolded prion proteins and promote its deposition in intracellular inclusions such as the aggresomes. Although NT219 was also found to increase the concentrations of certain molecular chaperones, it also reduces proteasome activity and impairs autophagy, indicating that conversion of proteins into large aggresomes is a protective mechanism even in the absence of their immediate clearance [bib_ref] The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and..., Moll [/bib_ref].
## Chaperones shield the hydrophobic moieties of the oligomers and inhibit their toxicity
The chaperone-induced clustering of aberrant protein oligomers is not the only mechanism through which these important protein molecules protect against oligomer toxicity. Evidence has been shown, using surface plasmon resonance (SPR), on the ability of clusterin to bind to toxic Aβ 42 oligomers at substoichiometric ratio (1:1000) and shield their reactive hydrophobic patches on their surface [bib_ref] Clusterin binds to Aβ1-42 oligomers with high affinity and interferes with peptide..., Beeg [/bib_ref]. The pre-incubation of these aggregates with clusterin also reverted their ability to reduce the pharyngeal mobility in C. elegans nematodes [bib_ref] Clusterin binds to Aβ1-42 oligomers with high affinity and interferes with peptide..., Beeg [/bib_ref]. In another study, Hsp70 was modified to be released in the extracellular space in order to address its protective activity against Aβ 42 in Drosophila melanogaster models [bib_ref] Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila, Fernandez-Funez [/bib_ref]. The secreted form, called secHsp70, suppressed Aβ 42 toxicity, as deduced by decreased eye degeneration, reduced neuronal death, structural integrity of adult neurons, suppression of locomotor neuron dysfunction, and lifespan extension. An assay based on luciferase-derived luminescence showed that secHsp70 stabilizes Aβ 42 oligomeric species and masks their neurotoxic epitopes, thus promoting the accumulation of nontoxic aggregates [bib_ref] Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila, Fernandez-Funez [/bib_ref]. This activity was carried out in the absence of ATP, indicating therefore that secHsp70 exploits its holdase function to interact with the oligomers in the absence of detectable clustering [bib_ref] Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila, Fernandez-Funez [/bib_ref]. It is interesting to note that other Hsps that were found able to interact with aggregates, such as Hsp104 [bib_ref] Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding..., Arimon [/bib_ref] [bib_ref] Repurposing Hsp104 to antagonize seminal amyloid and counter HIV infection, Castellano [/bib_ref] , Hsp70 [bib_ref] Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein..., Mannini [/bib_ref] and Ssa1p [bib_ref] Influence of specific HSP70 domains on fibril formation of the yeast prion..., Xu [/bib_ref] , exert the capacity to bind to the aggregates without consumption of ATP.
Stabilization of oligomeric Aβ 42 has also been observed in the presence of human prefoldin (hPFD) at substoichiometric molar ratio (up to 1:500) using western blot and TEM. Viability assays on cultured PC12 cells or primary cortical neurons from embryonic mice show that the Aβ 42 /hPFD complexes are less toxic than complexes of similar size obtained by incubating Aβ 42 oligomers with archaeal prefoldin (PhPFD). The different biological activity was attributed to the higher hydrophobic exposure and β-sheet content of Aβ 42 /PhPFD complexes, as assessed by ANS and ThT binding [bib_ref] Human prefoldin inhibits amyloid-β (Aβ) fibrillation and contributes to formation of nontoxic..., Sörgjerd [/bib_ref].
These observations highlight the existence of different mechanisms of action mediated by the chaperones against the toxicity of the oligomers, that is ''binding followed by clustering'' and ''binding causing hydrophobic shielding in the absence of clustering''. Both mechanisms have been indeed observed on a recent report in which the effect of the chaperones αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin known to have a chaperone-like activity, was investigated over a wide range of concentrations, both superand sub-stoichiometric relative to HypF-N toxic oligomers, ranging from 4:1 to 1:16 [bib_ref] Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric..., Cappelli [/bib_ref]. AFM images and light scattering measurements showed that the chaperones increase the size of the aggregates to an extent that correlates with chaperone concentration, ranging from null to remarkable increase. Notably, the protective effect on N2a cells was observed at all chaperone concentrations, irrespective of the size increase. Measurements of ANS binding showed that in the large clusters the overall exposure of the hydrophobic surface does not change, whereas when the clustering promoted by the chaperones is negligible the ANS binding is reduced, indicating that the hydrophobicity on the surface is shielded by the chaperones.
## Chaperones bind to amyloid fibrils
Prefibrillar oligomers are not the only molecular target of molecular chaperones. An increasing body of reports also describe the ability of these guardian proteins to bind to mature fibrils. In this regard, the sHsp αB-crystallin has been extensively studied because it colocalizes with Aβ plaquesand Lewy bodies [bib_ref] The lewy body in Parkinson's disease: molecules implicated in the formation and..., Wakabayashi [/bib_ref] , that are the neuropathological hallmarks of Alzheimer's disease and Parkinson's disease, respectively. Indeed, this chaperone has been found to bind to fibrils from Aβ 40 [bib_ref] αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an..., Raman [/bib_ref] , Aβ 42 and the Arctic variant E22G Aβ 42 [bib_ref] Binding of the molecular chaperone αb-crystallin to Aβ amyloid fibrils inhibits fibril..., Shammas [/bib_ref] , and α-synuclein [bib_ref] The interaction of αB-crystallin with mature α-synuclein amyloid fibrils inhibits their elongation, Waudby [/bib_ref]. The binding prevents fibril growth of Aβ 40 , as revealed by ThT binding assays, total reflection fluorescence microscopy and CD measurements [bib_ref] αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an..., Raman [/bib_ref]. A strong inhibition of fibril growth was also demostrated for Aβ 42 [bib_ref] Binding of the molecular chaperone αb-crystallin to Aβ amyloid fibrils inhibits fibril..., Shammas [/bib_ref] and α-synuclein [bib_ref] The interaction of αB-crystallin with mature α-synuclein amyloid fibrils inhibits their elongation, Waudby [/bib_ref] , by measurements of seeded fibril elongation kinetics, both in solution and on the surface of a quartz crystal microbalance (QCM). Immunoelectron microscopy images showed that αB-crystallin binds along the entire lenght and ends of the Aβ 42 [bib_ref] Binding of the molecular chaperone αb-crystallin to Aβ amyloid fibrils inhibits fibril..., Shammas [/bib_ref] and α-synuclein fibrils [bib_ref] The interaction of αB-crystallin with mature α-synuclein amyloid fibrils inhibits their elongation, Waudby [/bib_ref]. Although cell toxicity measurements were not reported directly, it was hypothesized that the binding of αB-crystallin to fibrils may reduce their toxicity by shielding the exposed hydrophobic residues and by preventing the generation of new oligomers that occurs on the fibril surface due to secondary nucleation [bib_ref] The interaction of αB-crystallin with mature α-synuclein amyloid fibrils inhibits their elongation, Waudby [/bib_ref].
The ability of αB-crystallin to bind to fibrillar species and inhibit their growth has also been observed with many other non-neuronal amyloid fibril systems, such as insulin fibrils at low pH [bib_ref] Kinetics and thermodynamics of amyloid formation from direct measurements of fluctuations in..., Knowles [/bib_ref] , β 2 -microglobulin (β 2 -m) fibrils at low pH [bib_ref] αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an..., Raman [/bib_ref] and apolipoprotein C-II (apoC-II) fibrils at neutral pH [bib_ref] Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein..., Binger [/bib_ref]. Interestingly, by exploiting the property of β 2 -m fibrils to depolymerize when shifted to neutral pH, it was found that αB-crystallin retards fibril depolymerization [bib_ref] αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an..., Raman [/bib_ref]. A chaperone-induced stabilization effect was also observed in the case of apoC-II fibrils, when their fragmentation promoted by dilution was inhibited in the presence of the chaperone [bib_ref] Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein..., Binger [/bib_ref]. In the same report, αB-crystallin was also found, using TEM images and sedimentation assays, to induce the formation of large fibrillar tangles. Although the authors did not provide direct experimental evidence, they argued that these stabilized clumped inclusions represent a protective strategy because they are unable to release cytotoxic oligomers and to promote events of secondary nucleation [bib_ref] Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein..., Binger [/bib_ref].
A direct proof of the beneficial effect of the binding of chaperones to fibrils was obtained for the human Brichos domain [bib_ref] A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers, Cohen [/bib_ref]. This chaperone binds to the surface of Aβ 42 fibrils, where the formation of oligomeric intermediates is catalyzed, and therefore minimizes the formation of toxic species, as demonstrated by several techniques, such as ThT kinetic analysis, TEM coupled with immunogold labeling, SPR and SEC in cojunction with immunoblot. Electrophysiology experiments in living mouse brain tissue, as well as cell viability measurements based on the MTS assay and the capsase-3 activity quantification on SH-SY5Y cultured cell lines, verified that this mechanism effectively suppresses the oligomer-mediated damage.
## The ability of other non-chaperone proteins to bind to protein aggregates and inhibit their toxicity
Other proteins that are generally not classified as chaperones have been recognized to bind to aggregates and suppress their toxic effects, thus acting as officially recognized molecular chaperones. Soluble collagen VI was found to rescue mice neocortical/hippocampal neurons from the toxicity mediated by Aβ 42 oligomers by altering the interaction of the oligomers with neurons [bib_ref] Collagen VI protects neurons against Aβ toxicity, Cheng [/bib_ref]. Indeed, immunostained confocal microscopy images showed that collagen VI prevents the association of Aβ 42 oligomers with the surface of cultured neurons and was found to colocalize with Aβ 42 into large deposits in the extracellular space, with the latter finding being confirmed by AFM. This mechanism of sequestration was found to result in a lower amount of soluble toxic oligomers in the extracellular space, with lack of binding to the neuron surface and protection from the damage [bib_ref] Collagen VI protects neurons against Aβ toxicity, Cheng [/bib_ref].
This protective strategy has also been observed, by means of immunolabeled confocal microscopy images, for the complement protein C1q, shown to prevent the association of fibrillar Aβ 42 to cultured mouse primary cortical neurons and to increase the size of Aβ 42 oligomeric species [bib_ref] C1q-induced LRP1B and GPR6 proteins expressed early in Alzheimer disease mouse models,..., Benoit [/bib_ref]. In particular, the accumulation of the oligomers into large deposits in the extracellular space impedes their internalization in the neurons, as demonstrated by the lower colocalization between the oligomers with a lysosomial marker in samples treated with C1q [bib_ref] C1q-induced LRP1B and GPR6 proteins expressed early in Alzheimer disease mouse models,..., Benoit [/bib_ref].
Interesting is the case of transthyretin, which has been recently found to possess a generic ability to deal with protein misfolded oligomers [bib_ref] Neuronal production of transthyretin in human and murine Alzheimer's disease: is it..., Li [/bib_ref] [bib_ref] Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro, Cascella [/bib_ref]. When pre-incubated with two different oligomeric species formed by the Aβ 42 peptide and the HypF-N protein, human tetrameric transthyretin (hTTR) and its engineered monomeric variant (M-TTR) effectively suppress their toxicity on SH-SY5Y cell cultures, again promoting the clusterization of the oligomers into larger species, as shown by AFM and immunostained confocal microscopy, in the absence of their structural reorganization, as shown by ThT fluorescence and the patterns of the pyrene spectra [bib_ref] Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro, Cascella [/bib_ref].
The maltose binding protein (MBP) from Escherichia coli has been shown to induce the formation of bigger clusters of preformed Aβ 42 oligomers, visible in TEM images, which have a reduced perniciouness to SH-SY5Y cell cultures [bib_ref] The inhibitory effects of Escherichia coli maltose binding protein on β-amyloid aggregation..., Sharoar [/bib_ref]. These assamblies were found to display a lower level of ThT binding and β-sheet content in the CD spectra, indicating in this case a change in the secondary structure induced by MBP. Finally, the protein α s1 -casein from bovine milk was found to inhibit fibril generation of Aβ 40 by redirecting the process towards the formation of amorphous aggregates [bib_ref] Inhibiting effect of α s1 -casein on Aβ 1-40 fibrillogenesis, Carrotta [/bib_ref]. AFM images and data obtained with CD spectroscopy and ThT binding assays showed that α s1 -casein affects the formation of the oligomers and the growth of the protofibrils. The aggregates formed in its presence have a lower content in fibrillar structure and a large globular appearance [bib_ref] Inhibiting effect of α s1 -casein on Aβ 1-40 fibrillogenesis, Carrotta [/bib_ref].
Following all this experimental evidence it is clear that many proteins cooperate with molecular chaperones in defending the cells from the insults caused by aberrant protein oligomers and that a widespread proteostasis control exists, with a multiplicity of guardians in vivo.
FIGURE 1 | Mechanisms used by molecular chaperones to maintain proteins in their soluble states and reduce the toxicity of protein aggregates. Chaperones assist protein folding, promote formation and maintenance of multisubunit complexes, mediate protein degradation, inhibit protein aggregation and promote disassembly of undesired protein aggregates. In addition, several strategies are employed by molecular chaperones to reduce the toxicity of protein aggregates: they act on small soluble oligomers by shielding their hydrophobic patches or by sequestering them into larger aggregates; they also promote the clustering of the fibrils, inhibit their elongation, the generation of oligomers through secondary nucleation occurring on the fibril surface, their fragmentation/oligomer release, and mask the reactive hydrophobic residues exposed on the fibril surface.
Frontiers in Molecular Neuroscience | www.frontiersin.org
## Assembly of protein oligomers and fibrils into large aggregates in vivo
The neutralization of protein misfolded oligomers and fibrils following their binding to molecular chaperones and subsequent clustering into larger aggregates observed in vitro has also been found in vivo. It has been shown that in all living organisms, from bacteria to high eukaryotes, the aggregates formed intracellularly are assembled together in one or a limited number of inclusions [bib_ref] Protein aggregation in recombinant bacteria: biological role of inclusion bodies, Villaverde [/bib_ref] [bib_ref] Clearance of misfolded and aggregated proteins by aggrephagy and implications for aggregation..., Hyttinen [/bib_ref] [bib_ref] Spatially organized aggregation of misfolded proteins as cellular stress defense strategy, Miller [/bib_ref] , which are termed aggresomes in mammalian cells and are in close proximity to the centrosome [bib_ref] A cellular response to misfolded proteins aggresomes, Johnston [/bib_ref] [bib_ref] Clearance of misfolded and aggregated proteins by aggrephagy and implications for aggregation..., Hyttinen [/bib_ref].
The formation of the aggresome is thought to be a protective process able to sequester harmful aggregates and to act as a storage center for eventual degadation via authophagy [bib_ref] Protein aggregation in recombinant bacteria: biological role of inclusion bodies, Villaverde [/bib_ref] [bib_ref] Clearance of misfolded and aggregated proteins by aggrephagy and implications for aggregation..., Hyttinen [/bib_ref] [bib_ref] Spatially organized aggregation of misfolded proteins as cellular stress defense strategy, Miller [/bib_ref]. The assembly of small aggregates into the large aggresome is not mediated by a single chaperone, as observed in simplified experiments in vitro, but is a finely regulated process mediated by a complex machinery: misfolded aggregates are polyubiquitinated and associate with the microtbule-associated protein dynein, which transports them to the microtubule organizing center (MTOC) to merge them into the growing aggresome [bib_ref] Clearance of misfolded and aggregated proteins by aggrephagy and implications for aggregation..., Hyttinen [/bib_ref]. The association of the polyubiquitinated aggregate to the microtubule/dynein complex is mediated by chaperones of the Hsp70 family, the Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG-3) and the protein 14-3-3 which has binding sites for both BAG-3 and dynein [bib_ref] 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes, Xu [/bib_ref] [bib_ref] 14-3-3 and aggresome formation: implications in neurodegenerative diseases, Jia [/bib_ref]. It can also be mediated by histone deacetylase 6 (HDAC6), which has binding sites for both ubiquitin and dynein [bib_ref] The deacetylase HDAC6 regulates aggresome formation and cell viability in response to..., Kawaguchi [/bib_ref] [bib_ref] Protein aggregates are recruited to aggresome by histone deacetylase 6 via unanchored..., Ouyang [/bib_ref]. Importantly, however, the outcome is similar to that observed in vitro, that is neutralization of diffusible and potentially harmful oligomers into an innocuous and easily manageable large aggregate, which will later be degraded via autophagy.
Formation of large inclusion bodies has also been widely studied in yeasts, where three distinct inclusion bodies have been observed, namely the cytosolic quality control bodies (Q-bodies or cytoQ), the intranuclear quality control compartment (INQ, previously termed JUNQ) and the insoluble protein deposit (iPOD) forming close to the vacuole [bib_ref] Spatially organized aggregation of misfolded proteins as cellular stress defense strategy, Miller [/bib_ref]. Although the molecular mechanisms underlying formation of such inclusions are still largely unclear, it has been shown that cytoQ and INQ form from the fusion of smaller aggregates and that their formation is mediated by chaperones such as the small heat shock protein Hsp42 and the heat shock protein Btn2, respectively [bib_ref] Spatially organized aggregation of misfolded proteins as cellular stress defense strategy, Miller [/bib_ref].
All the results that have shown the ability of molecular chaperones to interact with protein oligomers and neutralize their deleterious effects have mainly been obtained in cultured cell models. As described above, there is evidence that chaperones also induce formation of large aggregates in vivo, but the molecular mechanism by which this occurs and how such a complex tissue as the human brain benefits from this possibly protective process awaits specific experimental studies.
# Conclusions
Overall, all the experimental evidence collected so far from both in vitro and in vivo studies indicate that chaperones do not just maintain proteins in their soluble native states, as thought until 5 years ago, but also directly bind to protein oligomers and fibrils and neutralize their deleterious effects. This may occur through: (i) the direct binding to the hydrophobic patches exposed on the oligomer/fibril surface, which are responsible for the aberrant interactions with a number of targets in the cell and on the cell membrane; or (ii) their conversion into large and more innocuous species, which appear to minimize their surface-tovolume ratio, their diffusibility and to be more easily manageable by clearance mechanisms, such as autophagy. A schematic representation of the mechanisms used by molecurar chaperones to maintain protein homeostasis is shown in . In the light of the fact that the formation of large aggregates in the cells has a protective role, it is conceivable that the large size and spatially circumscribed nature of the histopathological signatures of various neurodegenerative diseases, such as the amyloid plaques and neurofibrillarly tangles in Alzheimer's diseases, the Lewy bodies in Parkinson's disease, the round and skein inclusions in amyotrophic lateral sclerosis, represent an extrema ratio of the cells to limit the damages of these undesired oligomers, the choice by the cell of the lesser of two evils: the small reactive oligomers and the large inert deposits, that become unable to be cleared with aging or disease progression.
# Author contributions
FC and BM designed the work, performed literature search, wrote the manuscript and critically revised it.
April 2017 | Volume 10 | Article 98
Frontiers in Molecular Neuroscience | www.frontiersin.org
Conflict of Interest Statement:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Copyright © 2017 Mannini and Chiti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
Treatment of Painful Diabetic Neuropathy—A Narrative Review of Pharmacological and Interventional Approaches
Citation: Gupta, M.; Knezevic, N.N.; Abd-Elsayed, A.; Ray, M.; Patel, K.; Chowdhury, B. Treatment of Painful Diabetic Neuropathy-A Narrative Review of Pharmacological and Interventional Approaches.
# Introduction
The number of patients living with diabetes mellitus (DM) is growing in the United States, and the estimated prevalence of this condition rose from 9.5% in 1999-2000 to 12.0% in 2013-2016. Neuropathy, which can produce both painful and non-painful symptoms, is the most common complication of DM, and the management of neuropathic symptoms has been estimated to comprise 27% of the total annual cost of diabetes care or 9% of all healthcare costs for people with DM. Painful diabetic neuropathy (PDN) has been estimated to affect 20% to 24% of all patients with DM, and PDN has been reported in 19% of those with insulin-dependent DM and 49% of those with non-insulin-dependent DM. Pain has also been reported in twice as many patients with DM and neuropathic symptoms (60%) than those with DM but no neuropathy (30%).
Like other chronic pain conditions, PDN has been associated with substantial declines in quality of life measures including sleep, recreational activities, normal mobility, general activity, social activities, and mood. Like neuropathic pain from all etiologies, PDN is often refractory to treatment and challenging to treat, and a variety of strategies are currently employed to manage this condition. Because no single treatment modality is beneficial or appropriate for every patient with PDN, it is undoubtedly desirable to have multiple options for managing this condition; however, some of the evidence supporting different options varies substantially. The pivotal studies supporting the use of current treatments for PDN, including pharmacotherapies and neuromodulation, are presented here with a focus on results regarding their effectiveness and safety. These results reveal important differences in what is and is not known about these treatments and their adverse events (AEs), which can help guide clinicians in choosing the best option.
## Clinical presentation and general management
The most common clinical presentation of diabetic neuropathy is distal symmetric polyneuropathy (DSP), which affects the limbs symmetrically in a characteristic "glove and stocking" pattern. Common symptoms of DSP include numbness, tingling, and weakness, in addition to pain, and many patients experience sensations similar to bunched-up socks or ill-fitting shoes. Subjects with PDN have described the pain as 'burning', 'electric', 'sharp', and 'dull/ache', and the intensity of pain has been reported to worsen in about half of those with PDN at night, when tired, or when stressed.
PDN is a diagnosis of exclusion; therefore, a combination of a thorough medical history, clinical testing, and neurological examination is required to eliminate possible secondary causes of pain. It is important to closely monitor patients with DM for neuropathic symptoms, since over 12% of patients in one study did not report painful symptoms to their physicians. Glycemic control can help to help prevent or slow the progression of PDN, but there is currently no available treatment to reverse existing nerve damage. This means symptoms, including pain, will need to be managed chronically and a therapy that is both effective and safe in the long-term is required. In the following sections, different types of treatments, including pharmacological as well as non-pharmacological therapies that are currently available or under evaluation, are summarized.
## Criteria for selection of articles
Pivotal randomized studies that met the following criteria were identified through literature search and discussed in the review. The qualification criteria included:
(a) Studies that evaluated the safety and efficacy of the drug or device. (b) Studies that supported the new drug application (NDA) or the post market followup requirements.
Where indicated, additional information was obtained from the FDA-approved package insert and discussed in the review.
## Pharmacotherapy
The first treatment for pain, including chronic pain, is often pharmacotherapy, but neuropathic pain is not the same as musculoskeletal pain, and commonly used analgesics such as opioids are not appropriate or effective for managing chronic neuropathic pain such as PDN. The pharmacotherapies approved and used to manage PDN are mostly not traditional analgesics or opioids that can be taken "as needed" but rather agents such as anticonvulsants or antidepressants that must be taken regularly for a period of time to achieve full effect. There are now more pharmacotherapies available for treating PDN than in the recent past, which are summarized in, and clinicians should consider patient-specific factors such as age, quality-of-life goals, functional status, and comorbidities when determining appropriate management. Information included in the table is summarized from FDA-approved package inserts and literature. Side-effects profile listed in the table is not exhaustive and only includes commonly seen events. * Gabapentin approved for postherpetic neuralgia. # Not FDA approved for PDN.
## Anticonvulsants
Pregabalin and gabapentin are both gabapentanoids that act as anticonvulsive drugs by inhibiting α 2 -δ calcium channels in the dorsal horn, thereby inhibiting neurotransmitter release. Pregabalin has been approved by the U.S. Food and Drug Administration (FDA) for use in treating PDN, and is recommended by the American Diabetes Association (ADA) as a first-line treatment, and while gabapentin is not approved for this indication, it is also recommended by the American Academy of Neurology (AAN) and ADA for this use.
The efficacy of pregabalin was shown in a double-blind, parallel-group, randomized controlled trial (RCT) in subjects with PDN. A total of 146 subjects were randomized to treatment with 300 mg/day pregabalin or placebo for 8 weeks, and treatment with pregabalin was associated with a 38% decrease in pain scores from baseline, which was significantly more than the 13% reported in placebo-treated subjects. The most frequently reported AEs were reported more often in the treatment arm than placebo and included dizziness, somnolence, infection, and peripheral edema. More pregabalintreated subjects discontinued treatment due to AEs (11%) than those treated with placebo (3%), but more subjects in the placebo arm discontinued due to lack of efficacy (4%) than in the treatment arm (1%). The FDA approved dosage for treatment of PDN is 50 mg TID (150 mg/day) at initiation and can be titrated up to 100 mg TID (300 mg/day) to achieve adequate effect. AEs reported with pregabalin treatment across many clinical studies include somnolence, dizziness, blurred vision, difficulty with concentration or attention, dry mouth, edema, and weight gain. Gabapentin is another anticonvulsant frequently used to treat PDN, although it is characterized as a second-line alternative to pregabalin due to the lower quality of clinical data available for gabapentin, its less predictable pharmacokinetics, longer titration periods, less flexible dosing, and requirement for dosing adjustments in patients with renal impairments. Dosing for chronic pain starts at 300 mg/day and is titrated up until suitable pain relief is achieved with effective doses ranging from 1800 mg to 3600 mg per day. Gabapentin has been tested for the treatment of PDN in a multi-center, double-blind RCT that enrolled 165 subjects. Subjects were initiated at a dose of 900 mg/day gabapentin and were titrated to a maximum of 3600 mg/day. Mean pain relief in the treatment arm was 39%, significantly greater than the 22% decrease reported in the placebo arm after 8 weeks of treatment, and the most common AEs were sedation and dizziness. AEs reported across a range of clinical studies using gabapentin to treat neuropathic pain include dizziness, somnolence, peripheral edema, and gait disturbance.
## Antidepressants
Antidepressants including serotonin and norepinephrine reuptake inhibitors (SNRIs) like duloxetine and venlafaxine treat chronic neuropathic pain by increasing the activity of noradrenergic and serotonergic neurons in the descending pathways of the dorsal horn. These descending neurons inhibit the activity of dorsal horn neurons, suppressing excessive input, which is perceived as pain, from reaching the brain. Tricyclic antidepressants (TCAs), likewise, block monoamine reuptake, including serotonin and norepinephrine, and are also used to treat chronic pain, especially neuropathic pain.
Duloxetine became the first agent approved by the FDA for treating PDN in 2004, and is recommended as a first-line treatment for neuropathic pain by the AAN and the ADA. The first of two pivotal trials of this agent was a multicenter, parallel, double-blind RCT that tested duloxetine at 60 mg or 120 mg/day for 12 weeks against placebo treatment in 348 subjects and reported mean pain reductions of 64% to 68% in the treatment groups, which were both significantly higher than the 43% mean pain reduction reported in placebo-treated controls. Treatment-emergent AEs (TEAEs) including nausea, somnolence, hyperhidrosis, and anorexia were more common in both duloxetine-treated groups than among placebo-treated controls, and the most frequently cited reasons for treatment discontinuation were vomiting and nausea. The second pivotal trial was, likewise, a multicenter, double-blind RCT in 457 subjects with PDN that tested duloxetine doses of 20 mg, 60 mg, and 120 mg per day. Mean pain relief after 12 weeks of treatment was significantly greater than that observed in placebo-treated subjects (33%) in the 60 mg/day (48%) and 120 mg/day (54%) treatment groups. TEAEs that were significantly more common with 120 mg/day duloxetine treatment than placebo include constipation, dry mouth, hyperhidrosis, decreased appetite, anorexia, weakness, nausea, and severe somnolence. The recommended dosage of duloxetine for PDN is 60 mg/day, and lower initial doses may be used in cases with tolerability concerns or renal impairment, which is a common complication of diabetes. The duloxetine label includes a black box warning for the potential emergence or worsening of suicidal thinking or behavior in children and young adults, while frequently reported AEs include nausea, dry mouth, somnolence, constipation, decreased appetite, and hyperhidrosis.
In addition to duloxetine, the SNRI venlafaxine and TCAs have shown evidence of efficacy for PDN and may be considered for PDN according to recommendations by the ADA, although they have not received FDA approval for this use. Venlafaxine is mechanistically similar to duloxetine, but there are fewer published data for this drug in PDN. An extended release (ER) formulation of venlafaxine was tested in a 6-week, double-blind, RCT in 244 subjects with PDN, and the investigators found that doses from 150 mg to 225 mg/day resulted in 50% lower pain scores than the baseline, which was significantly greater than the 27% pain reduction in the placebo group. The reported AEs in this trial were nausea, somnolence, and electrocardiogram abnormalities, and there was no significant difference in the rate of serious AEs between subjects receiving placebo (10%) or 150-225 mg/day venlafaxine ER (12%).
Amitriptyline is the most commonly used TCA for treating PDN, but the high risk of AEs requires careful monitoring and this drug is best suited as a last resort. A metaanalysis of trials testing amitriptyline in subjects with PDN included four studies with doses ranging from 10 mg to 90 mg/day for 12 to 14 weeks. The authors concluded amitriptyline was 1.95-fold more effective than placebo at producing at least 50% pain relief, which was not significant, but the odds ratio (OR) for subject withdrawals due to AEs for amitriptyline was 10.24 relative to placebo and 7.03 relative to gabapentin. Common AEs encountered with TCAs include gastrointestinal issues, orthostatic hypotension, dry mouth, urinary retention, and QTc prolongation, and this safety profile reflects concurrent actions at histaminergic, adrenergic, and cholinergic receptors.
## Opioids
Opioids have been commonly used in the past for treating chronic non-cancer related pain; however, there is little evidence that opioids effectively reduce chronic pain, including neuropathic pain. In addition, the serious risks presented by long-term opioid use include respiratory depression and addiction, which have led to an emphasis on avoiding their long-term use, whenever possible.
Currently, tapentadol is the only opioid specifically approved by the FDA for use in treating PDN. Tapentadol is a strong analgesic that combines the mechanisms of a µ-opioid receptor agonist, like a typical opioid, and a norepinephrine reuptake inhibitor. The opioid effects inhibit ascending pain signals in the spine, while increased synaptic levels of norepinephrine potentiate descending inhibitory signaling. Tapentadol was approved for use with PDN in 2012, but the dangers of chronic opioid treatment make the use of this drug for PDN controversial.
The efficacy of tapentadol in treating neuropathic pain was documented in two nearly identical phase 3 trials, which randomized 713 subjects in total. Both studies started with a three-week open-label stage, during which subjects with PDN were titrated to an optimal dose of tapentadol (200 mg/day-500 mg/day) for pain management, and all subjects who responded to tapentadol were randomized to continue on tapentadol treatment or placebo in a blinded manner. Schwartz et al. reported a 37% increase in pain scores in subjects switched to placebo and no change in those continuing on tapentadol, a significant difference, while the RCT by Vinik et al. showed subjects who continued on tapentadol treatment in the double-blind part of the study also had significantly less pain (26%) than those who were withdrawn to placebo. TEAEs led to discontinuation of tapentadol in 17% and 20% of subjects during the open-label portions of these studies, and the primary causes of discontinuations were nausea, vomiting, and dizziness. Since both study populations were enriched for patients who responded to tapentadol, the applicability of these results to the general population is debated. This fact, combined with safety concerns about chronic opioid intake, prompted the ADA not to recommend this medication as a first-or second-line treatment of PDN. Due to the addictive nature of opioids, it is advised to avoid prescribing tapentadol for PDN and consider alternate options for the management of pain.
## Topical capsaicin
In 2020, the FDA approved a capsaicin 8% topical patch for treating PDN. Topical capsaicin has been used for treating pain with a variety of etiologies and works via agonism of the transient receptor potential vanilloid 1 receptor (TRPV1). Topical exposure is thought to reduce the TRPV1-expressing nociceptive nerve endings in the affected area, providing a period of pain relief lasting several months. The efficacy of topical capsaicin in reducing pain due to PDN was demonstrated in a 12-week, double-blind RCT study in 369 subjects, showing that mean pain was reduced by 28% in the treatment group and by 21% in the placebo group, which was statistically significant. Only three subjects in this study, all in the 8% capsaicin treatment group, had severe drug-related TEAEs, including two with severe burning sensations and one with severe application site pain, but no subjects discontinued due to drug-related TEAEs.
## Non-pharmacological treatments: neuromodulation
The International Neuromodulation Society defines neuromodulation as medical technologies that reversibly enhance or suppress nervous system activity with the goal of treating disease and includes both implantable and non-implantable devices that deliver electrical, chemical, or other agents. Types of neuromodulation tested in subjects include transcutaneous electrical nerve stimulation (TENS), intrathecal pain therapy, and spinal cord stimulation (SCS). Although such methods have been used with welldocumented success in conditions such as musculoskeletal pain or failed back surgery syndrome, few well-controlled studies have examined their use in PDN. However, there is increased interest in new, non-opioid methods for treating neuropathic pain, and the use of neuromodulation is expected to expand in the coming years. Several neuromodulation methods with positive published results are reviewed here and include varying levels of invasiveness and efficacy, and this information is summarized in. a-IT pain therapy tested in subjects with non-malignant pain, including neuropathic pain; b-Ongoing study; d-
## Transcutaneous electrical nerve stimulation
TENS is a non-invasive, inexpensive, and easy-to-use form of neuromodulation to treat both acute and chronic pain with few contraindications or AEs, and no known drug interactions. Patients treated with TENS have electrical stimulation applied to the skin via adhesive electrodes using a variety of waveforms that are broadly classified as high frequency (>50 Hz), low frequency (<10 Hz) or burst. The mechanism by which TENS produces its analgesic effects is currently unknown, but multiple complimentary hypotheses have been proposed including improved microcirculation, higher levels of beta endorphin and met-enkephalin, increased expression of proteins including calcitonin gene regulating protein and nerve growth factor, and reduced inflammation.
Despite its long history of clinical use, there is no consensus on the efficacy of TENS for treating pain, and trials of this treatment in neuropathic pain have tended to be small, short in duration, and with large placebo effects, making the utility of TENS for managing PDN uncertain. A pair of small trials in the 1990s showed improvements in both pain and other neuropathic symptoms in patients with PDN when receiving TENS compared to sham controls. In the first of these, TENS produced pain relief by 54% in 18 subjects and improvement of neuropathic symptoms in 15, significantly more than the 18% decline in pain scores and neurologic improvement in 5 of 13 sham-treated controls. The second prospective, randomized study tested TENS in 14 subjects with PDN who had not responded to four weeks of treatment with amitriptyline. In this trial, TENS plus amitriptyline produced 66% pain relief, which was significantly more than the 55% pain relief in subjects receiving amitriptyline plus sham stimulation. No AEs were reported in either trial among patients receiving TENS stimulation. An RCT that included 25 subjects with PDN compared three days of treatment with TENS (≤35 Hz) to treatment with high-frequency external muscle stimulation (>4 kHz), and the authors reported a significant reduction in "total symptom score" including pain with both treatments. However, fewer subjects with painful PDN responded to TENS treatment (25%) than high-frequency external muscle stimulation (69%), with a response defined as alleviation of at least 1 symptom by 3 or more points on an 11-point scale.
Low-frequency pulsed electromagnetic fields (PEMF) was tested in 225 subjects with PDN, but the reported mean pain relief of 28% was not significantly different from placebo after a three-month RCT. The only AE reported was allodynia leading to two subjects each from the PEMF and sham treatment groups to drop out of the study. Frequencymodulated electromagnetic neural stimulation (FREMS) was also tested in a long-term RCT in 110 patients with symptomatic diabetic neuropathy and produced a statistically significant effect, decreasing pain scores by about 50%, but this effect was transient, and undetectable three months after the last treatment.
## Intrathecal pain therapy
Intrathecal pain therapy is a targeted drug delivery strategy to bypass first pass metabolism and the blood-brain barrier by delivering analgesic medication directly into the intrathecal cerebrospinal fluid via a pump and catheter to treat refractory chronic pain when conventional medical treatments are ineffective. Intrathecal therapy using either ziconotide or morphine is recommended and FDA-approved for chronic neuropathic pain such as that associated with PDN. Ziconotide is recommended more strongly by the Polyanalgesic Consensus Conference (PACC) because it is supported by evidence from well-designed trials, unlike the use of morphine, and it is not associated with some of the serious AEs observed with opioids, especially respiratory depression.
Mechanistically, intrathecal morphine acts as a µ-opioid agonist to reduce pain, as in other delivery methods, but targeted delivery to the spine can increase efficacy, improve alertness, and reduce AEs compared to systemic opioid therapy. Intrathecal ziconotide, in contrast, is a nonopioid analgesic that binds selectively and reversibly to N-type voltage-sensitive calcium channels, thereby blocking the release of pro-nociceptive neurotransmitters in the spinal dorsal horn. Tolerance and withdrawal do not develop in response to intrathecal ziconotide, which is a significant advantage over intrathecal morphine, and serious AEs are rare, even in cases of overdose. However, this agent requires careful dose titration, is contraindicated for patients with psychosis, and carries a black box warning for potential severe psychiatric symptoms and neurological impairment.
The efficacy of ziconotide has been demonstrated for chronic nonmalignant pain in a double-blind RCT involving 255 total subjects randomized 2:1 to treatment with ziconotide or placebo. Over 75% of the subjects in the treated arm had chronic neuropathic pain, and the results showed significant reductions in pain scores among subjects treated with ziconotide relative to placebo during the six-day study period. Among patients receiving ziconotide, 95% experienced at least 1 AE, and AEs likely to be related to ziconotide included nausea, hypotension, dizziness, somnolence, urinary retention, asthenia, amblyopia, nystagmus, abnormal gait, and confusion. Far fewer data from prospective trials exist for the efficacy and safety of intrathecal morphine; however, a small RCT was reported in subjects with non-cancer pain who had received intrathecal morphine for at least 1 year. The investigators progressively reduced the morphine dose for subjects in the intervention arm, while maintaining the doses for those in the control group, and found increased pain scores and study discontinuations among those receiving reduced doses, demonstrating efficacy although the sample size was small. Serious AEs commonly reported in association with intrathecal morphine include respiratory depression that can lead to death, the formation of inflammatory masses (granulomas) around the catheter tip, and myoclonus.
## Conventional scs
Conventional, tonic SCS was first used to treat human pain in 1967, and it has been established as a standard treatment for chronic, refractory pain since the 1980s. Conventional SCS is administered with a variety of waveforms via electrode leads implanted in the epidural space. Typically, a frequency of 40 Hz with a pulse width of 400 µs is delivered at intensities high enough to produce paresthesia, which is necessary to produce analgesic effects and must overlap the painful area. The current understanding of the mechanism by which conventional SCS produces analgesia is based, in part, on the Gate Control Theory by Melzak and Wall. Stimulation through the epidural electrodes activates large diameter spinal Aβ fibers in the dorsal column of the spine, which is thought to produce both pain relief and paresthesia, and the intensity of stimulation is correlated with the inhibition of wide-dynamic range neurons in the dorsal horn. In addition, functional MRI imaging of patients undergoing tonic SCS at conventional frequencies has shown the activation of supraspinal areas that modulate pain transmission in the dorsal horn via descending serotonergic and noradrenergic projections.
The first clinical study of conventional SCS in patients with chronic, refractory PDN included 10 subjects who had a trial stimulation and 8 who proceeded to a permanently implanted system. The investigators reported significant relief of both background and peak neuropathic pain through 14 months of stimulation, and follow-up visits at 3.3 and 7.5 years found continued pain relief in these subjects. The investigators reported several stimulation-related AEs including loss of analgesia in 1 subject, superficial wound infections, hematoma, electrode migration and displacement, and electrode failure secondary to trauma. The positive efficacy results were supported by two more small open-label prospective studies of SCS for treating PDN that reported positive responses in 9 of 11 subjects after 6 months of treatment, and 10 of 15 subjects after 12 months of stimulation.
Based on this work, two RCTs were conducted to obtain better evidence regarding the safety and efficacy of SCS for treating PDN refractory to conventional medical treatment. In the first of these, 60 subjects were randomized 2:1 to treatment with best conventional medical practice with or without SCS therapy and followed for six months. The authors reported a significant reduction of 55% in pain scores in the group treated with SCS and no change in pain intensity among controls, and 60% of subjects treated with SCS (vs. 5% of controls) reported more than 50% pain relief, a common threshold used to define SCS responders. AEs included infections, pain at the implant site, and electrode lead migration. In the second RCT, 36 subjects were randomized to receive the best medical treatment with or without SCS, and the investigators reported pain relief of 44% during the day and 38% at night in those receiving SCS for 6 months, while controls reported 0% relief during the day and 10% at night. The proportion of subjects reporting at least 50% pain relief after SCS treatment was 41% during the day and 36% at night. Serious AEs reported in this study were one death due to subdural hematoma and one infection resulting in explant and autonomic neuropathy. After six months, 93% of subjects in the control arm crossed over to the SCS treatment arm and 15 subjects in total were evaluable after 24 months of treatment. The mean pain relief was 45% during the day and 48% at night, and the proportion of subjects experiencing at least 50% pain relief was 47% during the day and 35% at night. After 24 months of stimulation, 13% of subjects had undergone surgery to replace the implanted pulse generator (IPG), and 27% underwent lead revisions. Most recently, long-term results for subjects from this RCT plus those from the pilot study by Pluijms et al.have been published, showing that among 48 subjects treated for a median of 5 years, mean pain relief decreased to 36% during the day and 31% at night, while the proportion of subjects experiencing at least 50% pain relief decreased to 36% and 32% during the day and night, respectively.
Despite the benefits of conventional tonic SCS for some patients with PDN, many patients do not respond to this treatment, and physiological adaptation, or habituation, frequently results in a loss of therapeutic effect. Moreover, paresthesia itself is a limiting factor for a substantial number of patients, particularly paresthesia affecting areas outside of the painful region and carrying intensity variations resulting from postural changes. Alternative waveforms have been developed with the aim of addressing some of these limitations and have been studied in patients with PDN, including burst SCS and high-frequency SCS.
## Burst scs
Unlike conventional and high-frequency SCS, which deliver stimulation at a constant, or tonic, frequency, burst SCS is characterized by clusters of high frequency pulses separated by longer inter-pulse intervals and is intended to emulate naturally occurring neuronal firing patterns. Like low-frequency tonic SCS, burst SCS produces analgesia via GABAergic mechanisms, and intrathecal administration of GABA A and GABA B antagonists abolish the analgesic effects of both types of SCS. Brain imaging in humans has also shown supraspinal effects of burst SCS that activates areas involved with emotion and motivation to a greater extent than tonic, low-frequency SCS.
Limited evidence for the efficacy of burst SCS in PDN was shown by a prospective trial of subjects with at least six months of previous experience with conventional SCS that included 12 subjects with PDN. Subjects with PDN reported additional pain reduction averaging 44% after two weeks of burst stimulation, which was significant, and eight (67%) preferred burst SCS to conventional stimulation. AEs reported among all 48 subjects in this brief study were headaches, dizziness, and the sensation of "heavy legs", and several reported feeling paresthesia in the supine position.
## High frequency (or 10 khz) scs
In contrast to conventional and burst SCS, evidence from a multicenter RCT has shown that high-frequency SCS delivered at 10 kHz (10 kHz SCS) produces deep and durable paresthesia-free pain relief for chronic neuropathic pain. The specific benefits and unique physiological responses associated with 10 kHz SCS may be attributed to its unique mechanism of action, although the precise mechanism of action is not yet understood.
Research in rodents using in vivo and ex vivo electrophysiological methods has shown that sub-sensory threshold stimulation at 10 kHz selectively activated inhibitory interneu-rons in the spinal dorsal horn, unlike such stimulation delivered at 1 kHz or 5 kHz, suggesting that low-intensity 10 kHz SCS may produce paresthesia-free pain relief by activating inhibitory interneurons in the spine without activating dorsal column fibers. A study using a spared nerve injury-induced (SNI) neuropathic pain model in rats showed that 10 kHz SCS significantly reduced hyperalgesia compared to sham stimulation and was also associated with reduced levels of inflammatory mitogen-activated protein kinases (MAPKs) in the dorsal root ganglia. Alterations in glutamatergic signaling in the dorsal horn has been shown to be involved in the development of neuropathic pain in rodents, and results from a second study-a rat SNI pain model-demonstrated that 10 kHz SCS relieved pain and partially restored altered spinal glutamate uptake activity, spinal glutamate levels, and miniature excitatory postsynaptic currents.
Work in humans has produced other possible mechanisms of action. Investigators who recorded 10-channel electroencephalograms in nine patients during SCS surgery reported a shift in peak frequencies from theta at baseline or with tonic stimulation at 60 Hz to alpha rhythms with high frequency stimulation at 1 or 10 kHz. In addition, the authors reported a positive correlation between disability scores and the high-frequency stimulation-induced alpha/theta peak power ratio in patients' frontal and somatosensory brain regions. Investigators who examined human subjects using voxel-based morphometry reported that in subjects with pain due to failed back surgery syndrome who were treated with 10 kHz SCS, pain relief was correlated with bilateral decreases in hippocampal volume, demonstrating an effect of this therapy on structural brain architecture over time.
A prospective trial, SENZA-PPN, was the first to test 10 kHz SCS in 26 subjects with peripheral polyneuropathy (PPN) refractory to conventional management, including 18 who got a permanently implanted device. A sub-analysis of subjects in SENZA-PPN showed 6 of 7 subjects who had PDN were responders (≥50% pain relief) and pain remitters (VAS ≤ 3.0 cm) after 12 months of 10 kHz stimulation, and 5 subjects demonstrated improvements in sensory and/or reflex testing, suggesting 10 kHz SCS could be associated with beneficial neurological effects beyond simple analgesia. All study-related AEs were mild or moderate and resolved without sequalae.
More recently a prospective, multicenter RCT, SENZA-PDN, evaluated 10 kHz SCS in 216 subjects with refractive PDN who were randomized to two treatment groups, conventional medical management (CMM) and 10 kHz SCS plus CMM. All subjects had pain for at least one year that was refractive to treatment with at least two pharmacologic interventions including pregabalin or gabapentin, and their pain intensity was ≥5 cm on a 10 cm VAS. Moreover, this study includes a battery of neurological assessments to monitor motor, sensory, and reflex function, and subjects were required to have stable neurological status at baseline. Neurological assessments include lower limb motor function, L1-S1 sensation to light touch, pinprick and Semmes-Weinstein 10-g monofilament sensory testing of the feet, patellar and Achilles reflexes, and Babinski response. SENZA-PDN will also examine medication use, since previous studies have shown treating chronic pain with 10 kHz SCS is associated with decreased use of opioid analgesics. This study is the largest RCT of SCS undertaken in patients with PDN, with 5 times as many patients in the treatment group as the RCT conducted with conventional SCS, and subjects will ultimately be followed for 24 months, providing important long-term data on the efficacy and safety of 10 kHz SCS.
The six-month results of this study were presented at the 2021 meeting of the North American Neuromodulation Society (NANS) and have been published recently; the primary endpoint of this study was a composite of ≥50% pain relief and no deterioration in neurological status including motor, sensory, and reflex categories at three months. The six-month results showed 86% of subjects who received 10 kHz SCS plus CMM met this endpoint, while only 5% of subjects who received CMM alone did. Overall, the mean pain relief was 76% with 10 kHz SCS and −2% in control subjects. Like the SENZA-PPN subanalysis, the investigators reported neurologic improvements in motor, sensory, and/or reflex categories in 62% of subjects treated with 10 kHz SCS and only 3% of the control arm. Finally, 18 AEs were reported during the study in the 10 kHz SCS arm, including two explants due to infection (2.2%). The study is ongoing to determine the durability of pain relief after two years (24 months) of treatment.
# Discussion
It is obvious from the foregoing review that there are many possible options for treating PDN, but it is also clear that the evidence supporting each of these options is not of equivalent quality. Pharmacotherapies are often the first option for treating chronic pain conditions, including PDN and other neuropathies, and the pivotal trials for approved drugs showed significant, but modest, effects on pain by anticonvulsants and antidepressants. However, many patients do not achieve adequate pain relief with pharmacological treatments. Moreover, these trials tested the efficacy or safety of these agents for 12 weeks or less, but the chronic nature of PDN requires patients to take these drugs for years. Topical capsaicin was tested longer, but had a modest benefit over placebo (7%), while the clinical trials assessing tapentadol were enriched with responders before randomization, making it difficult to interpret the implications of these results for the wider population.
Among the neuromodulation treatments available, RCTs for TENS and intrathecal pain therapy are similarly limited by short follow-up intervals, small sample sizes, or both. Conventional SCS has been tested for up to five years in PDN, but the number of implanted subjects was small, less than two dozen, and neither the amount of pain relief nor the proportion of responders reached 50% at these extended follow-up times. In addition, the paresthesia necessary for analgesia with conventional SCS is not tolerable for all patients, reducing the number of people who can benefit from this modality.
In contrast, 10 kHz SCS is paresthesia-free and is currently being tested in PDN in an ongoing, multicenter RCT much larger than the trials in conventional SCS, with over 100 patients randomized to the 10 kHz SCS arm, and these subjects will be followed for a full two years to produce high-quality data regarding the durability of effects. As a chronic, incurable condition, any treatment for PDN must be durable over years. Loss of therapeutic effect due to tolerance or habituation is one of the most common reasons for failure of conventional SCS over timeand remains a significant challenge in its use for chronic pain. Although the reasons for tolerance are poorly understood, 10 kHz SCS has been demonstrated to produce sustained back pain relief for two years, and SENZA-PDN is, likewise, designed to probe long-term treatment efficacy in PDN over 24 months. The results over six months have been robust with 76% mean pain relief and a responder rate of 85%, while over 60% of subjects showed neurological improvement, which has not been observed with conventional SCS or any other treatment.
Finally, complete treatment of PDN involves more than clinical outcomes such as effective, durable, and safe management of pain. A more holistic view of PDN management includes outcomes such as changes in pain medication use, health-related quality of life measures, and costs of treatment. Most of these outcomes have not been included in most clinical trials of pharmacotherapies or neuromodulation, including SCS, but SENZA-PDN addresses these areas by examining outcomes including neurological functioning, analgesic and diabetic medication use, health-related quality of life measures including sleep quality, and the cost-effectiveness of 10 kHz SCS. However, further studies may be needed to quantitatively assess the neurological improvements following 10 kHz SCS treatment and the results also need to be reproduced in real-world setting.
It must also be noted that SCS is an invasive procedure and the adverse events associated with SCS need to be carefully weighed against the benefits before recommending the therapy. It is opined that a fraction of PDN patients may have improvement in pain symptoms within a year without any specific treatment. Therefore, it is important to carefully select the patients who are refractory to conventional medical management. In fact, the main inclusion criteria in SENZA-PDN study was pain for >1 year that is refractory to at least two pharmacologic treatments.
# Conclusions
PDN is a common complication of DM that is associated with a significant decline in quality of life. This review has summarized the primary evidence for these therapies including efficacy and AEs. Although all have been shown to adequately address pain in patients with PDN, 10 kHz SCS provides evidence in a large RCT for clinically significant pain relief in addition to improvement in neurologic symptoms. SCS therapies along with pharmacological interventions provide growing armamentarium for pain management in PDN patients, in conjunction with the current state of the art in clinical management of diabetic patients.
Funding: The authors did not receive funding for the study. |
Metabolic Differences in Glutamine Utilization Lead to Metabolic Vulnerabilities in Prostate Cancer
The new oncologic paradigm of precision medicine is focused on identifying metabolic, proteomic, transcriptomic and genomic variabilities in tumors that can be exploited to tailor treatments and improve patient outcomes. Metabolic changes are a hallmark of cancer, and inhibition of metabolic pathways is now a major strategy in medicinal chemistry for targeting cancers. However, non-invasive biomarkers to categorize metabolic subtypes are in short supply. The purpose of this study was to characterize the intracellular and extracellular metabolic profiles of four prostate cancer cell lines with varying degrees of aggressiveness. We observed metabolic differences between the aggressive prostate cancer cell line PC3 and the even more aggressive, metastatic subline PC3M assessed by hyperpolarized in vivo pyruvate studies, nuclear magnetic resonance spectroscopy, and carbon-13 feeding studies. On further examination of the differences between these two cell lines, we found increased glutamine utilization in the metastatic PC3M subline that led directly to sensitivity to glutaminase inhibitor CB-839. Our study supports the theory that metastatic progression increases glutamine utilization and the inhibition of glutaminolysis could have clinical implications.Prostate cancer (PCa) is the second leading cause of cancer death in men in the United States. Despite the approval of multiple new therapies for PCa 1-5 , metastatic disease remains incurable. The metabolism of normal prostate cells versus cancer cells has been revealed in several biochemistry assays to be significantly different 6 . For example, the concentration of citrate is higher in normal prostate than in PCa, reaching concentrations as high as 180 mM in prostatic fluid 7 . The significant reduction in citrate level in PCa 8-10 results from citrate utilization in other metabolic pathways 6,11 . Several recent publications have found differences between PCa and normal prostate in expression of metabolic enzymes such as glutaminase (GLS) 12,13 , acetyl-CoA synthetase 14 , and the monocarboxylic acid transporters MCT1/MCT4 15-17 (SeeFig. 1a). Our long-term goal is to identify the roles of these metabolites in prostate cancer growth and progression.The major changes that occur in PCa are similar to what is seen in other cancers including increased glycolysis 18 . The increases in glycolysis in PCa progression has been observed both in cell culture 19 and in vivo using a new metabolic imaging technique called hyperpolarized magnetic resonance 20 . Hyperpolarization can enable a >10,000 fold sensitivity enhancement of the magnetic resonance (MR) signal of contrast agents over Boltzmann polarization 20-23 . More importantly, this signal enhancement is retained on the metabolites of the hyperpolarized molecule allowing direct observation and measurement of metabolic flux and real-time metabolic imaging. The most widely used method for providing hyperpolarized contrast agents for metabolic MR imaging is dynamic nuclear polarization (DNP) 24,25 and the most studied hyperpolarized 13 C tracer with DNP is pyruvate. Recently, the first Phase I study with hyperpolarized 1-13 C pyruvate was completed 26 . Hyperpolarized 1-13 C pyruvate is taken up by glycolytic cancer cells quickly and is then converted to 1-13 C lactate through lactate dehydrogenase (LDHA) 20,26-30 . This conversion rate can be measured in real time and in vivo by measuring the integration of the
lactate signal over the overall hyperpolarized signal (nLac). Multiple laboratories have used hyperpolarized 1-13 C pyruvate to image the transgenic adenocarcinoma of mouse prostate (TRAMP) model [bib_ref] Hyperpolarized C-13 spectroscopic imaging of the TRAMP mouse at 3T-initial experience, Chen [/bib_ref]. In the TRAMP model, increases in glycolytic flux were observed in higher histological grade PCa [bib_ref] Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection..., Albers [/bib_ref] and was reduced after hormone therapy [bib_ref] Kinetic modeling of hyperpolarized 13 C 1 -pyruvate metabolism in normal rats..., Zierhut [/bib_ref].
A key nutrient for most cancers is glutamine, and one of the critical steps in its utilization is its conversion to glutamate through the glutaminase enzyme [bib_ref] Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds..., Deberardinis [/bib_ref] [bib_ref] Therapeutic targets in cancer cell metabolism and autophagy, Cheong [/bib_ref] [bib_ref] Glutamine addiction: a new therapeutic target in cancer, Wise [/bib_ref] [bib_ref] Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B..., Le [/bib_ref]. The GLS gene encodes two splice variants of glutaminase, kidney-type glutaminase (KGA) and glutaminase C (GAC), collectively called GLS1 [bib_ref] A novel glutaminase isoform in mammalian tissues, De La Rosa [/bib_ref]. The GLS2 gene encodes two glutaminase isoforms, LGA (liver-type) and GAB, collectively called GLS2 [bib_ref] A novel glutaminase isoform in mammalian tissues, De La Rosa [/bib_ref]. GLS2 isozymes have been (a) In this schematic, partial pathways of glycolysis, glutaminolysis, and the citric acid cycle are shown: red type is specific enzymes, red arrows depict the conversion of cofactors such as NAD + , blue type is transporters, and black type is specific metabolites. The dashed box highlights the production of lactate through lactate dehydrogenase (LDH). There are several steps between fructose-6-phosphate and the generation of two equivalences of pyruvate not shown. We were specifically focused on determining the differences in aerobic and anaerobic glycolytic metabolites in PCa cell lines. Abbreviations: HK (hexokinase), PGI (phosphoglucose isomerase), GLDH (glutamate dehydrogenase), GLS (glutaminase), MCT4 and MCT1 (monocarboxylic acid transporters), GLUT1 (glucose transporter), SLC1A5 (glutamine transporter), MPC (mitochondrial pyruvate carrier), MAE (malic enzyme). (b) To compare extracellular metabolite changes among cell lines, concentrations of metabolites within a 24-hour period were determined and changes over that period were graphed as shown. Positive bars are metabolites excreted into the media and negative bars are metabolites consumed by the cells over the 24-hour period (n = 5 samples per cell type). Glutamine uptake by PC3M cells was over three fold higher than that by PC3 cells (P < 0.0001). Lactate production by PC3 was 1.3 fold higher than that by PC3M (P < 0.0001). Statistical significance between metabolite levels was determined using twoway ANOVA with Tukey's multiple comparisons. (c) To compare intracellular metabolite levels in cultured cells, metabolite concentrations were determined and the fold change compared to the indolent cell line (RWPE1) was plotted. The levels of metabolites succinate and phosphocholine (PCho) were significantly lower in PC3M cell lysates than in PC3 lysates (P < 0.0004). Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons. (d) To compare levels of metabolites in ex vivo tumor tissues, PC3 and PC3M tumors were excised from mice and samples prepared for analysis (100 mg per sample, PC3 n = 4, PC3M n = 5). Levels of lactate and taurine were significantly higher in PC3 tumors than in PC3M tumors, while levels of aspartate, glutamate, glutamine and succinate were significantly higher in PC3M tumors than in PC3 tumors. Unpaired t-test using Holm-Sidak method was used to determine statistical significance (**P < 0.02, *P < 0.04).
shown to be downregulated in several cancers 38 , including acute myeloid leukemia 39 and breast [bib_ref] Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications..., Cassago [/bib_ref] [bib_ref] Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer, Gross [/bib_ref] and lung cancers [bib_ref] Altered glutamine metabolism and therapeutic opportunities for lung cancer, Mohamed [/bib_ref]. In contrast, GLS1 is typically upregulated in cancers [bib_ref] Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with..., Jacque [/bib_ref]. Glutamine can be used not only as a nitrogen source but also as a carbon source and for energy production [bib_ref] Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds..., Deberardinis [/bib_ref] [bib_ref] Therapeutic targets in cancer cell metabolism and autophagy, Cheong [/bib_ref] [bib_ref] Glutamine addiction: a new therapeutic target in cancer, Wise [/bib_ref]. A recent publication reported that GLS1 was expressed in 68 of 107 (64%) PCa specimens but in only 9 of 37 (24%) benign hyperplastic prostate specimens [bib_ref] Elevated expression of glutaminase confers glucose utilization via glutaminolysis in prostate cancer, Pan [/bib_ref].
Androgen receptor (AR) signaling is one of the most critical pathways for maintaining prostate growth and normal function; however, AR activation is also important in prostate cancer pathogenesis and progression [bib_ref] The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis, Massie [/bib_ref] [bib_ref] Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively..., Shafi [/bib_ref]. Androgen ablation is usually a first-line drug with and without resection. However, a subset of patients does not benefit from androgen ablation or in time their cancer evolves and begins to be non-responsive. Androgen insensitive PCa, termed castration resistant, can occur by multiple mechanisms including point mutations in AR gene, generation of splicing variants (e.g. AR-V7), amplification of AR gene, or complete ablation of the AR receptor [bib_ref] The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis, Massie [/bib_ref] [bib_ref] Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively..., Shafi [/bib_ref].
We are specifically interested in determining the metabolic differences in castration resistant and metastatic PCa. Our purpose was to further our understanding in the metabolic differences in the extracellular and intracellular metabolic profiles of four PCa cell lines with varying degrees of aggressiveness specifically focusing on glutamine and glucose utilization. Our in vitro metabolic results led us to determine the hyperpolarized metabolic flux of 1-13 C pyruvate in xenograft PC3 and PC3M animal models and the testing of CB-839. CB-839 (Calithera Biosciences) is a nanomolar binder to both major isoforms of glutaminase 1 (GLS1), has good oral bioavailability, and is currently in a Phase I study for solid tumors and leukemia (NCT02071862, NCT02071927) [bib_ref] Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with..., Jacque [/bib_ref] [bib_ref] Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer, Gross [/bib_ref].
# Results
## Production of lactate and uptake of glucose and glutamine differ in prostate cancer cells.
Changes in the concentrations of extracellular metabolites (consumed and excreted) over 24 hours of growth were measured in four human PCa cell lines: RWPE-1, RWPE-2, PC3, and PC3M [bib_ref] Human prostate cancer cell lines, Russell [/bib_ref]. [fig_ref] Figure 1: Metabolic differences between PCa cell lines [/fig_ref] summarizes all of the metabolic profiling data both in cell culture and tumor tissue. Changes observed in selected extracellular metabolites among the four cell lines in 24 hours are shown in [fig_ref] Figure 1: Metabolic differences between PCa cell lines [/fig_ref]. Positive values represent secreted metabolites, while negative values represent consumed metabolites. Glutamine uptake was over 3 times higher in PC3M cells than in PC3 (P < 0.0001), while lactate production was 1.3 times higher in PC3 cells than in PC3M (P < 0.0001). However, glucose uptake and glutamate utilization is similar in RWPE2, PC3, and PC3M cell lines. Significant differences in intracellular metabolite levels were found between PC3 and PC3M both in cell culture and excised tumor tissues. In cell culture, levels of phosphocholine (PCho) and succinate were significantly different (P < 0.0001) in PC3 and PC3M [fig_ref] Figure 1: Metabolic differences between PCa cell lines [/fig_ref]. In addition, intracellular glutamine levels were observed to be lower in the aggressive cell lines (PC3 and PC3M) compared to unaggressive lines but were not statistically significant by two-way ANOVA analysis. The metabolic profile of tumor tissue samples [fig_ref] Figure 1: Metabolic differences between PCa cell lines [/fig_ref] , which includes both the cells (intracellular) and interstitial spaces (extracellular), revealed the same differences found in PC3 and PC3M in vitro assays: lactate was higher in PC3 tumors than in PC3M tumors, and succinate, aspartate, glutamate and glutamine were higher in PC3M tumors.
## Carbon-13 tracing experiments reveal differences in glutamine utilization.
To further explore the differences in glutamine and glucose utilization in PC3 and PC3M cells lines, carbon-13 feeding/tracing experiments were performed. Similar lactate values were seen in both cell types and in both tracing experiments; however with the carbon-13 glutamine feeding experiments we selectively found the label in intracellular succinate and proline in the PC3M cell line [fig_ref] Figure 2: Figure 2 [/fig_ref]. This result, along with our proton spectroscopy results, emphasizes the differences in the utilization of glutamine between PC3 and PC3M.
Hyperpolarized pyruvate studies reveal increased glycolytic flux in PC3 tumors. We utilized hyperpolarized 1-13 C pyruvate to determine the differences in glycolysis of PC3 and PC3M subcutaneous tumors in nude mice. This conversion rate can be measured in real time and in vivo by measuring the integration of the lactate signal over the overall hyperpolarized signal. A series of slice-selective 13 C spectra were collected immediately after injection of tumor-bearing mice with hyperpolarized 1-13 C pyruvate to observe the arrival of the compound to the tumor and its conversion to lactate. Pyruvate to lactate conversion was greater in PC3 tumors than in PC3M tumors (0.44 ± 0.09 [n = 5] versus 0.29 ± 0.02 [n = 3], P < 0.03) [fig_ref] Figure 3: Increased glycolysis observed in PC3 tumors with hyperpolarized pyruvate [/fig_ref]. This finding corresponds with the greater extracellular lactate production over 24 hours by PC3 cells than by PC3M cells in culture.
## Drug inhibition assays reveal glutamine utilization variability. pc3 and pc3m cells were grown
with and without added glutamine [fig_ref] Figure 4: Therapeutic vulnerabilities of PC3M cell line observed in drug assays [/fig_ref] or in the presence of 0.1% DMSO (vehicle control) or 1 μM of GLS1 inhibitor CB-839 for 72 hours [fig_ref] Figure 4: Therapeutic vulnerabilities of PC3M cell line observed in drug assays [/fig_ref]. Cells were counted after the 72-hour period. Both treatments reduced proliferation of the PC3M cells. Interestingly, both cell lines proliferated without glutamine. Cell viability measurements were performed in both cell lines after incubation for 72 hours with vehicle control or 1 μM CB-839. Levels of ATP were lower in the PC3M cells incubated with CB-839 [fig_ref] Figure 4: Therapeutic vulnerabilities of PC3M cell line observed in drug assays [/fig_ref]. The ATP level could be rescued by adding 4 mM dimethyl 2-oxoglutarate to the media [fig_ref] Figure 4: Therapeutic vulnerabilities of PC3M cell line observed in drug assays [/fig_ref]. Dimethyl 2-oxoglutarate is a hydrophobic analog of α-ketoglutarate and has been shown to be metabolized in the citric acid cycle [bib_ref] Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with..., Jacque [/bib_ref] [bib_ref] Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer, Gross [/bib_ref]. In cells treated with mTOR inhibitor rapamycin (3 nM or 25 nM) for 12 hours, the ratios of ATP levels in treated and control cells showed reduced ATP in PC3M cells but not in PC3 cells [fig_ref] Figure 4: Therapeutic vulnerabilities of PC3M cell line observed in drug assays [/fig_ref]. These results confirm that glutamine dependence is higher in the metastatic subline PC3M than in PC3 cells and that glutamine is being utilized to fuel the citric acid cycle. These results also suggest that mTOR activation is more prevalent in the PC3M cells than in the PC3 cells.
## Protein levels indicate differences in signaling and glutamine metabolism pathways.
Reverse-phase protein array (RPPA) technology 46 was used to analyze the cellular protein activity in the lysates from PC3 and PC3M cells (n = 3). highlights a few of the proteins whose levels varied by >0.1 between the cell lines and specifically in the signaling pathway of AKT and AMPK. Levels of phosphorylated S473-AKT (Akt-pS473, P < 0.01, unpaired t-test using Holm-Sidak method for multiple comparisons) was higher in the PC3M cell lysates than in the PC3 cell lysates, while total AKT levels were equal. Levels of p-AMPK were higher in the PC3 lysates than in the PC3M lysates. Both AMPK and AKT are master regulators of cell growth and cellular metabolism. The deviation between the levels of these two proteins could be the underlying cause of the metabolic variability between the two lines.
The levels of GLS1 (GAC and KCA) and GLS2 were determined by Western blot in PC3 and PC3M cell lysates treated or not treated with 1 μM CB-839 . Expression of GLS2 was higher in PC3 lysates than in PC3M lysates in both treated and untreated samples. Levels of GLS1 were approximately equivalent in both cell lines. GLS2 overexpression is observed in both PC3 and PC3M cells with drug incubation indicating that GLS2 could be compensating for GLS1 inhibition. GLS2 overexpression after drug treatment is the highest in PC3 cells. Reduced GLS2 levels with and without drug present may lead to the greater susceptibility of PC3M to CB-839 treatment.
# Discussion
Our results illustrate the metabolic reprogramming that can occur between similar cell lines. Our metabolic results are intriguing because the PC3M line was derived by isolating and growing a lung metastasis of PC3 in a nude mouse [bib_ref] Metastatic behavior of human tumor cell lines grown in the nude mouse, Kozlowski [/bib_ref]. The PC3M line is more metastatic and aggressive than the initial parent cell line [bib_ref] Metastatic behavior of human tumor cell lines grown in the nude mouse, Kozlowski [/bib_ref]. Both lines are androgen receptor-null and therefore castration resistant. Glycolysis was greater in PC3 cells than in PC3M cells both in vivo and in vitro as evaluated by metabolic imaging and metabolic profiling. Furthermore, variations in 13 C-labeled glutamine uptake and metabolic products in cultured PC3M cells compared to cultured PC3 cells indicate that glutamine is being utilized to a greater extent to feed the citric acid cycle (succinate labeling) in PC3M cells. In addition, higher levels of succinate and aspartate (both metabolites formed from the citric acid cycle) were observed in PC3M tumor tissue. The level of GLS2 expression was lower in PC3M cells than in PC3. Together, these differences lead to the greater vulnerability of PC3M cells to GLS1 inhibitor CB-839. To our knowledge, this is the first study showing that CB-839 reduces cell proliferation and viability in an isogenic PCa line.
This increase in glutamine utilization in more aggressively metastatic cell lines has been observed also in ovarian cancer [bib_ref] Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian..., Yang [/bib_ref] , suggesting that glutamine utilization might be directly related to metastatic progression [bib_ref] Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian..., Yang [/bib_ref]. Jiang et al. reported that cells in culture require glutamine reductive decarboxylation to form spheroid structures from anchorage-dependent growth [bib_ref] Reductive carboxylation supports redox homeostasis during anchorage-independent growth, Jiang [/bib_ref]. CB-839 could have a larger effect on metastatic progression than on cell proliferation. Our results reveal that both PC3 and PC3M proliferate even in the complete absence of glutamine, illustrating the dynamic nature of metabolism and the capacity for multiple compounds to be utilized as carbon and nitrogen sources to provide the building blocks for replication.
Our RPPA data revealed differences in levels of phosphorylated AKT (pS473P) and mTOR in PC3M versus PC3 lysates. Incubation with 3 nM rapamycin (an mTOR inhibitor) or 1 μM CB-839 reduced ATP production only in PC3M cells. Phosphorylated AKT is known to activate mTOR, which can stimulate glutamine utilization [bib_ref] The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4, Csibi [/bib_ref] , and increases in glutamine concentration can directly modulate the mTOR pathway [bib_ref] Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway, Yuan [/bib_ref]. In future studies, we will determine whether regulation of this pathway can further explain the differences in glutamine utilization between these two cell lines.
In conclusion, we observed greater glycolytic flux in PC3 tumors than in PC3M tumors in mice and see increased glutamine utilization in the PC3M cell line. Once we understand the mechanisms underlying this Carbon-13 feeding studies reveal differences in glutamine utilization.C-labeled feeding studies were done to elucidate glucose and glutamine utilization by PC3 and PC3M cells. Four plates of PC3 and PC3M cells were incubated for 24 hours with 1,6-13 C glucose or carbon-13 fully labeled glutamine. Metabolites were determined by 13 C high-resolution spectroscopy. The results show increased carbon-13 label from glutamine in succinate and proline in PC3M cells. Unpaired t-test using Holm-Sidak method was used to determine statistical significance.
SCIENTIfIC RepoRts | 7: 16159 | DOI:10.1038/s41598-017-16327-z metabolic variation between PCa subtypes, we may be able to use hyperpolarized metabolic imaging to provide a rationale for individualized combinations of metabolic pathway inhibitors and chemotherapy and potentially improve the outcome of prostate cancer patients.
# Materials and methods
Cell lines and culture conditions. Four human PCa lines were used: RWPE-1 (non-tumorigenic, considered benign), RWPE-2 (non-metastatic), PC3 (aggressive, castration resistant), and the aggressive subline PC3M (castration resistant) [bib_ref] Human prostate cancer cell lines, Russell [/bib_ref]. RWPE1, RWPE2, and PC3 cells were purchased from ATCC. PC3M cells were purchased from the MD Anderson cell line core facility, which validates all of the cell lines it provides. Cell lines were tested for mycoplasma every couple of months by MD Anderson cell line core facility. For intracellular metabolite analyses, RWPE1 and RWPE2 cells were cultured in keratinocyte serum-free media supplemented with bovine pituitary extract and recombinant human epidermal growth factor. PC3 cells were cultured in F12-K media supplemented with 10% fetal bovine serum (FBS), and PC3M cells were cultured in RPMI1640 media supplemented with 0.1 mM modified essential media (MEM) and 10% FBS. All media were supplemented with 10 U/mL penicillin and 10 µg/mL streptomycin. For extracellular metabolite determination, PC3 and PC3M cells were seeded in optimized media and, after 24 hours, exchanged into RWPE1/2 media. All media were purchased from Gibco and Corning.C spectroscopy was utilized to capture the 13 C signal in the tumor tissue after injection of hyperpolarized pyruvate. (d) Hyperpolarized pyruvate data were processed to generate dynamic curves characterizing the arrival of hyperpolarized pyruvate and its chemical conversion into lactate. Normalized lactate (nLac), defined as the ratio of total cumulative lactate signal to the total carbon-13 signal, was calculated for each 13 C scan. Normalized lactate conversion was greater in PC3 tumor-bearing animals (0.44 ± 0.09) than in PC3M tumor-bearing animals (0.29 ± 0.02). The statistical significance of the difference between tumor groups was determined by unpaired two-tailed t-test (P < 0.03).
For CB-839 studies and glutamine studies, PC3 and PC3M cells were grown in RPMI1640 media (with or without glucose/glutamine) supplemented with 10% FBS, 10 U/mL penicillin, and 10 µg/mL streptomycin. The media was supplemented with frozen aliquots of glutamine and glucose prior to experiments.
Mouse models. All experimental methods involving mice were performed in accordance with the guidelines and regulations of the MD Anderson Institutional Animal Care and Use Committee (IACUC), and the animal protocols used were approved by that committee. Male nude mice >5weeks old were each injected with ~5 × 10 6 cells subcutaneously on the rear flank. When the resulting tumors had grown to approximately 1 to 1.3 cm in size, the animals underwent imaging as described in a later subsection. The animals were sacrificed with isoflurane overdose and cervical dislocation 6-48 hours after imaging, and tumor tissues were removed and flash-frozen for metabolic studies.
High-resolution magnetic resonance spectroscopy. Magnetic resonance spectroscopy (MRS) was used for all metabolic analyses. MRS allows chemical resonances of metabolites to be determined in one experiment. For all high-resolution MRS, one-dimensional 1 H proton spectroscopy was performed with water suppression on a 500 MHz Bruker Biospin Avance III high-definition nuclear magnetic resonance (NMR) instrument equipped with a Prodigy BBO cryoprobe. The cryoprobe increases the sensitivity of the measurement 3-to 4-fold. All supplies (deuterium oxide [D 2 O], 3-(trimethylsilyl)-1-propanesulfonic acid-d 6 sodium salt [DSS-d [bib_ref] Citrate transport and metabolism in mammalian cells: prostate epithelial cells and prostate..., Mycielska [/bib_ref] , and potassium phosphate buffer [K 2 HPO 4 , pH 7.4]) were purchased from Sigma-Aldrich and used without further purification.
To determine intracellular metabolite levels, cells (0.5-3.0 × 10 7 per sample) were trypsinized and pelleted at approximately 80% confluence, and metabolites were extracted using an ice-cooled 2:1 methanol to water solution (3 mL) and MP Biomedicals lysing matrix D beads (~500 µL per 10 7 cells). The homogenates were then subjected to centrifugation for 10 min at 4000 g, the supernatant removed and lyophilized overnight, and the remaining metabolites dissolved in D 2 O with 0.5 mM DSS-d 6 and 50 mM K 2 HPO 4 . The one-dimensional 1 H-NMR spectra used 256 scans and a spectral width (SW) of 10245 Hz and was referenced to DSS at 0.00 ppm. Water suppression was performed with presaturation. For the metabolites assigned and quantified by using 1 H-NMR spectroscopy, four or five replicates of each cell type were analyzed. Data were processed/analyzed with Chenomx (Chenomx, Inc.), MestreNova (Mestrelab Research), and/or Topspin (Bruker Biospin) software. Integrated values of intracellular metabolites were determined by taking the ratio of the resonance for each metabolite over the DSS-d 6 peak to the total integration of all resonances in the spectra. This allowed normalization of the probe performance within each sample. Metabolite resonances were identified through reference to either of two online metabolomics databases, Human Metabolome Database (http://www.hmdb.ca) [bib_ref] HMDB 3.0-The Human Metabolome Database in 2013, Wishart [/bib_ref] or Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/metabolomics) and, when necessary, confirmed by spiking the sample with a known amount of the metabolite in question. To easily discern the metabolic profile of the more aggressive PCa lines, the fold difference of each metabolite to RWPE1 (indolent PCa) was graphed [fig_ref] Figure 1: Metabolic differences between PCa cell lines [/fig_ref].
## Figure 5.
Signaling pathway variation between PC3 and PC3M cells. Reverse-phase protein array (RPPA) analysis was used to identify differences in expression of various proteins in PC3 and PC3M cells (n = 3 samples per cell type). The x-axis labels represent selected proteins targeted by the antibodies used in the analysis; V is validated and C is course. The validated antibodies have been shown in multiple assays to target only the protein named and not to have off-target binding; course antibodies have off-target binding. PC3M cell lysates expressed higher levels of phosphorylated AKT (Akt-pS473, P < 0.01, unpaired t-test using Holm-Sidak method for multiple comparisons), 4E-BP1, and mTOR than PC3 cell lysates. Expression of AMPK and p-AMPK was higher in PC3 cell lysates. . Glutaminase protein expression in PC3 and PC3M cell lines before and after CB-839 treatment. PC3 and PC3M cells were treated (+) or not treated (−) with GLS1 inhibitor CB-839 (1 μM) and their expression of GLS1 and GLS2 proteins were determined by Western blot. Antibodies specific for GAC and KCA were utilized on two replicate samples, while GLS2 expression was determined on three replicates. PC3 cell lysates had higher levels of expression of GLS2 than PC3M cell lysates regardless of CB-839 treatment. The average density of bands in the GLS2 blots were determined by using ImageJ software. The statistical significance was determined by unpaired two-tailed t-test. The difference in GLS2 expression was significant in the untreated cells (P < 0.03) but because of high variability the difference was not significant in the drug-treated cells. Levels of GLS1 (GAC and KGA) were similar in all cell lysates. The full gels can be seen in the supplemental data.
SCIENTIfIC RepoRts | 7: 16159 | DOI:10.1038/s41598-017-16327-z To determine the differential uptake and excretion of some of the dominant media metabolites, five plates (145 × 20 mm) of each cell line were cultured so that there were five replicates at each time point. Because of the large difference in cell size, samples were not normalized to cell count; instead, cell cultures were carefully monitored to allow initiation of the media time course experiment when cells had reached 80% confluence. When cell cultures became approximately 80% confluent, uptake and excretion of metabolites were monitored in the four cell lines over a period of 24 hours in keratinocyte serum-free media. Aliquots of media were taken at the beginning (t = 0) and end of the 24-hour period (t = 24) from five separate tissue culture plates for each cell line. [fig_ref] Figure 1: Metabolic differences between PCa cell lines [/fig_ref] plots the fold change in the integrated value of each metabolite over a 24 hour period.
To determine intracellular metabolite values from subcutaneous tumors, flash frozen tissues (100 mg per sample) were homogenized using a liquid nitrogen-cooled mortar and pestle; metabolites were extracted by a method similar to that used for cell pellets. Integrated values for each metabolite were determined by taking the ratio of the resonance for each metabolite over the DSS-d 6 peak to the total integration of all resonances in the spectra. To visualize these differences easier, each integration value was multiplied by a scaling factor to account for the number of protons each chemical resonance corresponded to (1000 (1H), 500 (2H), 250 (4H), [fig_ref] Figure 1: Metabolic differences between PCa cell lines [/fig_ref].
Carbon-13 tracing experiments. PC3 and PC3M cells were cultured for 24 hours in glucose/glutaminefree Dulbecco MEM to which either fully labeled 13 C-glutamine (final concentration 3 mM) or 1,6-13 C glucose (final concentration 10 mM) was added [fig_ref] Figure 2: Figure 2 [/fig_ref]. After the 24-hour period, the media was removed, and the cells were washed twice with phosphate-buffered saline solution (PBS), trypsinized, pelleted, and then homogenized by the same method as for non-tracing experiments. Labeled compounds (Sigma-Aldrich) were used without any further purification. Lyophilized metabolites were redissolved in 50 mM phosphate buffer (pH 7.4) with 5 mM DSS-d 6 . Proton decoupled one-dimensional 13 C spectroscopy was run (4096 averages, relaxation delay 6s, SW 29760 Hz, 30° flip angle) on each sample. Two-dimensional heteronuclear single-quantum correlation experiments were run on each sample to confirm the assignment of each metabolite (16 averages, relaxation delay 3s, 256 points in 2 nd dimension, SW 6500 Hz for 1 H, SW 20800 Hz for 13 C).
## Protein array and western blotting analyses.
For reverse-phase protein array (RPPA) analysis, flash-frozen cell pellets comprising 5 × 10 6 cells were transferred to the MD Anderson RPPA core facility for processing and analysis. The pellets were subjected to lysis using RPPA lysis buffer, and lysates were serially diluted manually with five 2-fold dilutions of lysis buffer and then printed on nitrocellulose-coated slides [bib_ref] Reverse phase protein array: validation of a novel proteomic technology and utility..., Tibes [/bib_ref]. The NormLinear algorithm, which corrects for protein loading and antibody variations, was used to determine the difference in protein expression between groups of lysates [bib_ref] Functional proteomics can define prognosis and predict pathologic complete response in patients..., Gonzalez-Angulo [/bib_ref] [bib_ref] Non-parametric quantification of protein lysate arrays, Hu [/bib_ref].
For detection of specific proteins via Western blotting, PC3 and PC3M cells were grown in complete RPMI1640 media supplemented with 10% FBS, 10 U/mL penicillin, and 10 µg/mL streptomycin in 6-well plates. When cells became 60% confluent, fresh media was added with or without GLS1 inhibitor CB-839 (1 μM). After 72 hours, the media was removed and the cells were detached using trypsin, counted, and pelleted. Whole-cell lysates were prepared by using RIPA buffer (Thermo Scientific), and total protein concentration was determined by the Pierce BCA protein assay (Thermo Scientific). Protein lysates were denatured by boiling in sodium dodecyl sulfate (SDS) sample buffer for 5 min at 95 °C and then loaded onto a 4-12% mini-precast polyacrylamide gel (Nupage). Membranes were blocked with Odyssey blocking buffer for 60 minutes at room temperature, and then were incubated with antibodies recognizing KCA (20170-AB, ProteinTech) [bib_ref] Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with..., Jacque [/bib_ref] , GAC (199581-AP, ProteinTech) [bib_ref] Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with..., Jacque [/bib_ref] , or GLS2 (NBP1-76544, Novus Biologicals) overnight at 4 °C and with β-actin (SC477778, Santa Cruz Biotechnology) for 1 hour at room temperature. Molecular weights were determined using SeeBlue Plus2 Pre-stained protein standard (LC5925, Invitrogen). Secondary antibodies conjugated to infrared (IR) fluorescent dyes were utilized to image bands using the LI-COR Odyssey IR fluorescent system. Densitometry analysis was performed using ImageJ software .
Imaging procedures and hyperpolarized pyruvate. Ox063 trityl radical (Oxford Instruments) was mixed with neat 1-13 C pyruvic acid (Sigma-Aldrich) to a concentration of 15 mM. Aliquots of this solution (20 μL) along with 0.4 μL of 50 mM Gd 3+ relaxation agent (Magnevist, Bayer Healthcare) was loaded into a commercial HyperSense dynamic nuclear polarization (DNP) polarizer (Oxford Instruments) and irradiated at a microwave frequency of 94.100 GHz for 30-40 minutes (until the polarization plateau was reached) and then dissolved in 4 mL buffer solution containing 40 mM Tris (7.6 pH preset), 80 mM NaOH, 0.1 g/L EDTA, and 50 mM NaCl.
For imaging, the tumor-bearing mice were anesthetized with 3% isoflurane mixed with oxygen and then maintained with 0.5-1% isoflurane. Animals were placed on a heated pad and their respiration and heart rate monitored during imaging procedures. The neutral (pH 7-8) 80 mM hyperpolarized 1-13 C pyruvate solution was injected into each mouse via tail vein catheter.
All imaging and spectroscopy were performed with a dual tuned 1 H/ 13 C volume coil (Doty Scientific) or a 1 H/ 13 C volume coil (Bruker BioSpin) in a 7T Bruker Biospec horizontal bore MR scanner equipped with a single channel for carbon excitation/reception. Proton anatomic images were taken using a multi-slice T2-weighted RARE sequence. A small 8 M 13 C-urea phantom doped with gadolinium-DPTA was next to the tumor for chemical shift referencing. A series of slice-selectiveC spectra (field-of-view 40 × 40 mm, slice-thickness 8-12 mm) were collected immediately after injection of hyperpolarized 1-13 C pyruvate. The single slice was placed over the tumor using the multi-slice proton imaging sequence for placement. A total of 90 transients were acquired with a time delay of 2 seconds between each transient (total time 3 minutes). Each transient utilized a 15-20° degree flip angle excitation pulse (gauss pulse) and 2048 data points. Metabolic flux ratios of pyruvate to lactate were determined with a unidirectional model [fig_ref] Figure 3: Increased glycolysis observed in PC3 tumors with hyperpolarized pyruvate [/fig_ref]. Data were processed both in MATLAB (MathWorks Inc) or MestReNova. The dynamic spectra were manually phased and line-broadening was applied (10 to 15 Hz). The area under the spectral peaks for pyruvate and lactate were integrated over the whole array. Normalized lactate (nLac) ratio was calculated as lactate over the sum of pyruvate and lactate signals 56 .
CB-839, glutamine, and rapamycin cell proliferation and viability assays. For cell viability assays, PC3 or PC3M cells (2,000) in RPMI media were added to wells of a 96-well plate. GLS1 inhibitor CB-839 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. We prepared 100 μl aliquots of the solution which were kept frozen at −20 °C. For each experiment, a separate frozen aliquot of the 10 mM CB-839 solution was utilized to generate our experimental 1 μM CB-839 conditions and any remaining material was disposed of. The plates were incubated in the CO 2 incubator for 72 hours. Vehicle control wells were treated with 0.1% DMSO. The cells were then subjected to the Promega CellTiter-Glo Luminescent Cell Viability Assay (G7571). A similar assay was utilized to determine the effect of mTOR inhibitor rapamycin, except cells were seeded at 5000 cells per well at the beginning of the 12-hour incubation. For the cell proliferation assay, PC3 or PC3M in RPMI media were added to each well of a 24-well plate (5000 cells per well). Cells were treated with CB-839 (1 μM) or 0.1% DMSO. Plates were incubated in a CO 2 incubator for 72 hours. Cells were then trypsinized and counted using a Bio-Rad TC20 Cell Counter. In another set of experiments using the same parameters, cells were grown in the presence or absence of glutamine and counted after 72 hours [fig_ref] Figure 4: Therapeutic vulnerabilities of PC3M cell line observed in drug assays [/fig_ref].
Statistical analyses. All statistical analyses were performed with GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. P values are given for all analyses and statistical significance determined to be any P value below 0.05. All experimental data are shown as mean ±SEM.
Data availability. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
[fig] Figure 1: Metabolic differences between PCa cell lines. In all experiments, metabolite values were determined by MRS and values were normalized as stated per experiment. Statistical significance indicated by an asterisk (*). [/fig]
[fig] Figure 2: Figure 2. Carbon-13 feeding studies reveal differences in glutamine utilization. 13 C-labeled feeding studies were done to elucidate glucose and glutamine utilization by PC3 and PC3M cells. Four plates of PC3 and PC3M cells were incubated for 24 hours with 1,6-13 C glucose or carbon-13 fully labeled glutamine. Metabolites were determined by 13 C high-resolution spectroscopy. The results show increased carbon-13 label from glutamine in succinate and proline in PC3M cells. Unpaired t-test using Holm-Sidak method was used to determine statistical significance. [/fig]
[fig] Figure 3: Increased glycolysis observed in PC3 tumors with hyperpolarized pyruvate. (a) The schematic illustrates the different pathways through which hyperpolarized 1-13 C pyruvate can be metabolized in the cell. Because of the quick loss of polarization, resonances for lactate and alanine are the main metabolites detected in vivo. (b) Spectra from representative PC3M and PC3 tumor-bearing mice taken 21 s after tail vein injection of hyperpolarized pyruvate. The faster conversion rate in PC3 is readily seen. (c) 1 H image of a representative PC3M tumor-bearing mouse. Slice-selective [/fig]
[fig] Figure 4: Therapeutic vulnerabilities of PC3M cell line observed in drug assays. PC3 and PC3M cells were treated with various metabolites or inhibitors and the effects on cell viability and proliferation were assessed. Unless otherwise noted, the statistical significance of differences between groups was determined by unpaired two-tailed t-test.(a) Comparison of PC3 and PC3M cell counts after 72 hours of growth in the presence or absence of 2 mM glutamine (Gln). (b) Comparison of PC3 and PC3M cell counts after 72 hours of treatment with GLS1 inhibitor CB-839 or vehicle. Five to six replicates were used for each condition. In both experiments (a and b), PC3M proliferation was inhibited to a greater extent than PC3 proliferation (P < 0.03 [+/− Gln], P < 0.00001 [+/− CB-839]). PC3 proliferation was not inhibited by CB-839 but was inhibited by complete exclusion of glutamine in the media. Both cell lines proliferated in the absence of glutamine and in the presence of CB-839. (c) ATP levels in cells treated for 72 hours with vehicle or with 1 μM CB-839 over untreated control. Five to six replicates were used for each condition. Levels of ATP were reduced only in the treated PC3M samples (P < 0.0001). (d). ATP levels were equivalent to those in cells treated with vehicle when cells treated with 1 μM CB-839 were also incubated with 4 mM dimethyl 2-oxoglutarate, showing that DMKG rescued the citric acid cycle. (e) Treatment with mTOR inhibitor rapamycin (3 nM or 25 nM) significantly reduced ATP levels in PC3M cells compared to vehicle control but did not reduce ATP levels in PC3 cells. The statistical significance of the differences was determined by two-way ANOVA (P < 0.00001, seven replicate samples per condition). [/fig]
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The Pivotal Role of Invasive Functional Assessment in Patients With Myocardial Infarction With Non-Obstructive Coronary Arteries (MINOCA)
Myocardial infarction with non-obstructive coronary arteries (MINOCA) encompasses several pathophysiological mechanisms not yet fully understood. Among the latter, vasomotion abnormalities and coronary microvascular dysfunction (CMD) play a major role for both epidemiological and prognostic reasons. Despite current guidelines do not recommend routine physiological assessment of both epicardial and microvascular coronary compartments within the context of an acute myocardial infarction, several recent evidence support the critical role of a comprehensive invasive functional assessment in order to identify the underlying pathophysiological mechanism and consequently to select an appropriate therapeutic strategy. Unfortunately, optimal medical therapy for these patients is not currently established due to the lack of dedicated trials evaluating clinical outcomes of commonly used medications for secondary prevention in MINOCA patients. For this reason, additional research is warranted to provide personalized treatments for patients affected by this puzzling clinical entity.Keywords: MINOCA, coronary artery disease, acute coronary syndrome, coronary physiology, microvascular dysfunctionMINOCA: DEFINITION, PATHOGENESIS, AND UNMET CLINICAL NEEDSMyocardial infarction with non-obstructive coronary arteries (MINOCA) was first formally labeled as a clinical syndrome in an international expert opinion paper only in 2016 (1), though autoptic evidence potentially referring to this condition have been reported since the 1930s (2, 3). The epidemiological burden of MINOCA has been underestimated for decades due to the lack of a standardized definition, however the most recent epidemiological data suggest an overall prevalence of 6% out of all acute myocardial infarctions (AMI) events (4).The pathophysiological mechanisms underlying MINOCA are multiple and not fully understood, however vasomotion abnormalities and coronary microvascular dysfunction (CMD) play a prominent role for both epidemiological and prognostic reasons. Therefore, similarly to the algorithm formulated in the EAPCI consensus document on ischemia and nonobstructive coronary arteries (INOCA) (5), a comprehensive invasive functional assessment of both epicardial and microvascular coronary circulation is critical to establish the exact pathophysiological mechanisms of MINOCA and, therefore, to select an appropriate etiology-based therapeutic strategy.
Myocardial infarction with non-obstructive coronary arteries (MINOCA) encompasses several pathophysiological mechanisms not yet fully understood. Among the latter, vasomotion abnormalities and coronary microvascular dysfunction (CMD) play a major role for both epidemiological and prognostic reasons. Despite current guidelines do not recommend routine physiological assessment of both epicardial and microvascular coronary compartments within the context of an acute myocardial infarction, several recent evidence support the critical role of a comprehensive invasive functional assessment in order to identify the underlying pathophysiological mechanism and consequently to select an appropriate therapeutic strategy. Unfortunately, optimal medical therapy for these patients is not currently established due to the lack of dedicated trials evaluating clinical outcomes of commonly used medications for secondary prevention in MINOCA patients. For this reason, additional research is warranted to provide personalized treatments for patients affected by this puzzling clinical entity.
Keywords: MINOCA, coronary artery disease, acute coronary syndrome, coronary physiology, microvascular dysfunction
## Minoca: definition, pathogenesis, and unmet clinical needs
Myocardial infarction with non-obstructive coronary arteries (MINOCA) was first formally labeled as a clinical syndrome in an international expert opinion paper only in 2016 (1), though autoptic evidence potentially referring to this condition have been reported since the 1930s. The epidemiological burden of MINOCA has been underestimated for decades due to the lack of a standardized definition, however the most recent epidemiological data suggest an overall prevalence of 6% out of all acute myocardial infarctions (AMI) events.
The pathophysiological mechanisms underlying MINOCA are multiple and not fully understood, however vasomotion abnormalities and coronary microvascular dysfunction (CMD) play a prominent role for both epidemiological and prognostic reasons. Therefore, similarly to the algorithm formulated in the EAPCI consensus document on ischemia and nonobstructive coronary arteries (INOCA) (5), a comprehensive invasive functional assessment of both epicardial and microvascular coronary circulation is critical to establish the exact pathophysiological mechanisms of MINOCA and, therefore, to select an appropriate etiology-based therapeutic strategy.
The purpose of this review is to briefly summarize the main pathophysiological mechanisms of MINOCA and to elucidate the critical role of a comprehensive invasive functional assessment. We also report on the main currently available etiologytailored therapies.
## Definition
The growing body of evidence reporting cases of acute myocardial infarction (AMI) with no significant angiographic findings, particularly with the recent development of highsensitivity troponin assays, as well as the conflicting positions regarding the heterogeneous pathophysiological mechanisms of MINOCA and the absence of standardized guidelines of management, led the European Society of Cardiology (ESC) in 2017 (1) and the American Heart Association (AHA) in 2019to publish specific statements to address these issues. According to the latest statement by the AHA, three main criteria need to be met to fulfill the diagnosis of MINOCA:
(1) AMI criteria as defined by the "Fourth universal definition of myocardial infarction" (7); (2) non-obstructive coronary arteries on angiography (no coronary artery stenosis ≥ 50%); (3) no specific alternative diagnosis for the clinical presentation. With these more stringent criteria, several disorders that were initially considered as potential causes of MINOCA (e.g., heart failure, Takotsubo syndrome, myocarditis, renal failure, sepsis, arrhythmias, hypotension/shock, pulmonary embolism, stroke, adult respiratory distress syndrome [ARDS]) are no longer considered as such. Consequently, only ischemic causes of AMI are now considered in the pathogenesis of MINOCA, thus contributing to simplify the diagnostic work-up.
## Pathogenesis
Destabilization of vulnerable coronary plaques, spontaneous coronary artery dissection (SCAD), coronary microvascular dysfunction (CMD), coronary thromboembolism, and vasospasm are all potential causes of MINOCA and could be targeted by specific diagnostic tools and therapeutic strategies. The possible etiologies of MINOCA according to the pathological substrate (atherosclerotic/non-atherosclerotic), anatomical localization (epicardial/microvascular) and the type of MI is summarized in. In particular, the pathophysiological mechanisms of coronary plaque disease, CMD and vasomotion abnormalities will be more deeply discussed in the following paragraphs.
## Coronary plaque disease
Among MINOCA patients, plaque rupture and plaque erosion play a key role as acute triggers of myocardial infarction mainly producing intraluminal thrombosis with potential overlapping of coronary spasm, distal embolization, or a combination of these processes. Of note, plaque composition seems to be highly different in these two pathological entities. An abundance of proteoglycan and glycosaminoglycan in the extracellular matrix with dominant endothelial cells apoptosis is usually associated with plaque erosion. In contrast, ruptureprone plaques present a thin fibrous cap with interstitial collagen discontinuation, excess of lipid core and inflammatory cells. While plaque erosion more frequently leads to AMI via non-occlusive thrombosis causing distal embolization and possibly superimposed spasm, transient occlusive intraluminal thrombosis with prompt and spontaneous thrombolysis is more frequent in plaque rupture.
The introduction of coronary intravascular imaging in ordinary interventional practice allowed a more precise evaluation of the incidence of plaque vulnerability in MINOCA patients. Intravascular ultrasound (IVUS)-based studies have shown an incidence of plaque rupture of ∼40% in these patients. However, no data regarding the incidence of plaque erosion are currently available, because of the lack of analysis performed within the MINOCA population using optical coherence tomography (OCT), which is the only high-resolution coronary intravascular imaging technique capable of detecting plaque erosion. Furthermore, intracoronary imaging allowed the identification of a rare cause of coronary plaque disease, the calcified nodule, which has also been reported as an uncommon cause of MINOCA. Recently, the autoptic morphometric analysis of consecutive calcified nodules allowed to identify the fragmentation of the necrotic core calcifications caused by mechanical stress with subsequent overlying luminal thrombosis as a plausible potential mechanism of acute coronary thrombosis and sudden cardiac death.
## Coronary microvascular dysfunction
The potential epidemiological impact of CMD in women with ischemic heart disease in the absence of flow-limiting epicardial stenoses was proposed two decades ago after the WISE study, which showed that abnormal coronary flow reserve (CFR) was observed in approximately half of women with chest pain and non-obstructive coronary arteries. Similar estimates have been subsequently confirmed by other studies suggesting that 43-54% of MINOCA patients experience microvascular spasmand that CMD may be detected in up to 50% of patients with chest discomfort and non-obstructive coronary arteries on invasive coronary angiography.
Both endothelium-dependent and non-endotheliumdependent mechanisms participate in the pathogenesis of CMD. Endothelial dysfunction implies a dynamic dysregulation of coronary microvasculature vasoreactivity, which inhibits the physiological flow-mediated vasodilation occurring in conditions of increased myocardial oxygen consumption. The latter may even result in paradoxical vasoconstriction, thus determining microvascular spasm. The diagnostic approach to microvascular tone with the most reliable efficacy-safety profile is the acetylcholine test. According to the Coronary Vasomotion Disorders International Study Group (COVADIS) indications, this vasoreactivity test meets the criteria for microvascular vasospasm when it reproduces the symptoms usually experienced by the patients and triggers ischemic ECG changes in the absence of significative epicardial spasm. On the other hand, non-endothelium dependent mechanisms relate to structural remodeling of coronary microvasculature due to an increased wall-to-lumen ratio (intimal thickening and perivascular fibrosis) and a loss of myocardial capillary density (capillary rarefaction), which result in increased microvascular resistances and permanent decline of coronary flow through the microvasculature. In the acute setting of MINOCA, CMD develops as a consequence of two separate mechanisms: a chronic microvascular dysfunction causing prolonged ischemia with a superimposed acute trigger potentially driving to myocardial necrosis or an isolated, acute, hyper-adrenergic tone with prompt and sudden rise of microvascular resistance that may lead to cardiomyocyte death.
## Coronary vasospasm
Epicardial vasospasm shares with the microvascular counterpart the identical mechanism related to smooth muscle cells hyperreactivity to both endogenous (e.g., acetylcholine) and exogenous (e.g., cocaine, methamphetamines, fluorouracil) agents. According to the COVADIS definition, coronary epicardial vasospasm may be diagnosed if the administration of high-dose intracoronary acetylcholine boluses (20-100 µg) reproduces the symptoms usually experienced by the patients and triggers ischemic ECG changes with significative epicardial spasm (>90% diameter reduction). Epidemiologically, epicardial vasospasm is one the most frequent mechanisms of MINOCA. Its prevalence ranges between 3 and 95%, with significant variations based on ethnicity, showing a particular predilection for Asian rather than white patients. However, more recent investigations found epicardial vasospasm in 46% of MINOCA patients undergoing acetylcholine provocative tests. In addition, a recent contribution by Montone et al.assessed the relationship between myocardial bridging and coronary spasm in patients with MINOCA undergoing provocative Ach testing. The authors found that myocardial bridging is present in almost 21% of MINOCA patients with vasomotion abnormalities and represents within this population an independent predictor of a worse outcome.
## Unmet clinical needs
While the clinical management of AMI with obstructive coronary artery disease (CAD) is guided by frequently updated evidencebased guidelines released by the international scientific society, no specific guidelines or treatment recommendations are currently available for MINOCA due to the paucity of dedicated clinical trials focused on this population. In addition, optimal medical therapy for these patients is not currently established due to the lack of dedicated trials evaluating clinical outcomes of commonly used medications for secondary prevention in MINOCA patients. However, an etiology-based tailored approach should be followed as a general recommendation.
## Invasive functional assessment: why so critical?
In order to establish a tailored therapeutic strategy, an invasive evaluation aiming to identify the pathophysiological mechanism underlying MINOCA plays a crucial role. Once ruling out atherosclerotic causes of MINOCA (i.e., acute plaque destabilization due to rupture or erosion) via coronary angiography and intracoronary imaging techniques, one or more of the following tests could be used in the invasive diagnostic work-up of MINOCA: (1) functional assessment of angiographically intermediate coronary stenosis with fractional flow reserve (FFR) or non-hyperaemic pressure ratios (NHPR) to rule out obstructive CAD; (2) thermodilution-or Dopplerbased assessment of coronary microvascular function; (3) provocative test with acetylcholine/ergonovine to rule out coronary vasoreactivity.
Hemodynamic profiles of CMD and vasospasm emerging via invasive pressure-flow assessment are summarized in.
## Epicardial cad: hyperaemic and non-hyperaemic pressure ratios
The absence of obstructive CAD at coronary angiography is a major criterium for MINOCA diagnosis, therefore the presence of angiographically intermediate coronary stenosis deserves a functional evaluation via hyperaemic or nonhyperaemic indexes. Among hyperaemic tests, FFR (88% sensitivity, 100% specificity, 100% positive predictive value, 88% negative predictive value, and 93% accuracy, the ratio between mean distal pressure and mean aortic pressure during maximal hyperaemia ( Pdhyper Pahyper ), is the most widely used and accepted index for a physiological evaluation of epicardial coronary disease. International guidelines recommend a specific cut-off (FFR ≤ 0.80) for the detection of functionally relevant CAD.
On the other hand, resting physiological indexes may be adopted in order to assess the functional significance of coronary lesions with no pharmacological administration of hyperaemic agents: at this regard, instantaneous wave-free ratio [iFR−73% sensitivity, 87.8% specificity, 77% positive predictive value, 85.3% negative predictive value], the ratio between mean distal pressure and mean aortic pressure during the diastolic wavefree period ( Pdwave−free Pawave−free ) (35), resulted sufficiently accurate when compared to FFR with the pre-specified cut-off of 0.89and, more importantly, non-inferior to FFR in terms of clinical outcomes in two large scale, randomized, clinical trials.
Several other NHPR have been proposed for adenosine-free functional assessment of CAD: the resting full-cycle ratio (RFRthe lowest Pd/Pa within the entire cardiac cycle, diagnostic accuracy 97.4%, sensitivity 98.2%, specificity 96.9%, positive predictive value 94.5%, negative predictive value 99.0%) and diastolic pressure ratio (dPR-accuracy 87.5%, sensitivity 62.3%, specificity 95.6%, positive predictive value 82.0% and negative predictive value 88.7%) which is an averaged Pd/Pa ratio during a part or the entire diastolic period without selection of a wavefree period. These NHPR (pre-specified cut-off of 0.89) showed a significant correlation with iFR (39) and then emerged as reliable alternative tools to guide treatment strategy in patients with coronary artery disease.
## Microvascular cad: from coronary flow to microvascular resistance
A comprehensive coronary physiology assessment including the appraisal of both the epicardial and the microvascular compartments provides a detailed analysis of ischemic heart disease, particularly among patients with non-critical epicardial CAD who may benefit from a more accurate investigation of CMD in order to establish the pathophysiological mechanism underlying the ischemic symptoms. Although CFR cannot specifically assess the contribution of the microvasculature to ischemic heart disease, it is generally accepted that, in the absence of significant epicardial disease, an impairment of CFR reflects the presence of CMD. From a technical point of view, CFR may be measured invasively using Doppler flow velocity or thermodilution. The use of a Doppler flow-pressure wire allows the assessment of CFR as the ratio of hyperaemic [after 140 mg/kg/min of intravenous adenosine] to resting coronary flow velocity (CFV):
CFVhyper CFV rest. On the other hand, the use of a specific temperature-pressure wire enables CFR measurement through thermodilution as resting mean transit time (Tmn) divided by hyperaemic Tmn (CFR = Tmn baseline Tmn hyperemic ), showing a strong correlation with true coronary flow. Cut-off values of <2.0 for thermodilution-based measurementand <2.5 for Doppler-based measurementshowed the strongest prognostic impact (sensitivity 86-92%, specificity 89-100%, diagnostic accuracy 89-96%, positive predictive value 84-100%, negative predictive value 77-95%). Microvascular function can be specifically estimated with the thermodilution-based index of microvascular resistance (IMRsensitivity 64%, specificity 75%. According to Ohm's law, vascular resistance (R) is equal to driving pressure ( P) divided by flow rate (Q): R = P/Q. P is the pressure difference across the myocardium (Pd -Pv) and Q represents the coronary flow, which is known to be inversely related to Tmn ( 1 Tmn ). Therefore, coronary microvascular resistance (R) can be calculated as follows: Pd-Pv/ 1 Tmn = (Pd-Pv) × Tmn. Assuming that venous pressure is close to zero (Pv = 0), the final equation will be: Pd × Tmn. Therefore, the index of coronary microvascular resistance (IMR) is calculated with thermodilution as the product of distal coronary pressures (Pd) and Tmn during maximal hyperemia (IMR = Pd × Tmn . Of note, a strong correlation between IMR and true microcirculatory resistance (TMR) was found. In particular, IMR values ≥25 suggest high microvascular resistance and, therefore, CMD.
Both CFR and IMR are thermodilution-based physiological indexes in which coronary flow and microvascular resistances are indirectly estimated via the Tmn of a manually injected saline bolus, thus implying that its measurement depends to a certain degree on the injection technique. Moreover, both CFR and IMR require the achievement of adenosine-induced stable hyperemia. The measurement of absolute coronary flow (Q) and microvascular resistance (R) has been proposed to overcome these limitations by adopting a thermodilution technique that requires a continuous infusion of saline through a dedicated monorail catheter. Such equipment allows the infusion of saline at room temperature infused at a pre-specified flow rate (Qi, 20 mL/min for the left anterior descending [LAD] and left circumflex artery [LCx] and 15 mL/min for the right coronary artery [RCA]), resulting in a hyperaemic state similar to that produced by adenosine. The temperature of the infused saline (Ti) and of the saline/blood mixture (T), and the distal coronary pressure (Pd) are measured with a pressure/temperature sensortipped guide wire. Q is calculated as 1.08 x (Ti/T) x Qi, and expressed in ml/min. R is calculated as Pd/Q, and expressed as mm Hg/L/min, or Wood units. These measurements present several advantages over the traditional CFR and IMR, as they are safe, reproducible, and not operator-dependent. Moreover, they do not require pharmacological-induced hyperemia since continuous saline injection produces a prolonged and steady physiological hyperemic state within seconds.
The physiological finding of an impaired CFR in patients with ischemic heart disease and non-obstructive CAD currently meets a general agreement. Multiple contributions support the hypothesis that, even in the absence of critical epicardial CAD, microvascular dysfunction implies impairment of blood flow across coronary vessels, which may also reflect an abnormal resting coronary flow velocity, with a subsequent adverse myocardial performance, thus potentially leading to unfavorable prognosis. Interestingly, in a cohort of patients with ischemia and non-obstructive coronary arteries (INOCA) who underwent invasive physiological assessment, also including FFR, acetylcholine testing, and adenosine administration, absolute coronary flow measured with continuous thermodilution, resulted as the best predictor of self-reported angina.
However, the body of evidence concerning coronary flow and flow reserve measurement among the MINOCA population is currently limited. Similarly, the role and the clinical implications of continuous thermodilution-derived indexes within MINOCA patients are not yet established. A recent contribution by Mochula et al.showed that MINOCA patients develop a mild reduction of myocardial blood flow and perfusion as assessed by SPECT myocardial perfusion scintigraphy (MPS). Indeed, it provides a further proof that despite the absence of obstructive CAD this subset of patients has more pronounced risk of cardiac events needing of more aggressive observation and treatment. Yet, the prognostic impact of CFR impairment as well as the reference values to adequately establish it are still matter of debate.
A recent work by Konst et al.reported that absolute microvascular resistance, as assessed by continuous thermodilution, was significantly increased in INOCA, thus suggesting that a functional impairment of the coronary microvascular district plays a central role in INOCA pathophysiological mechanisms.
According to the microvascular resistance status, several combinations of epicardial and microvascular disease are available. The prognostic impact of discordant coronary physiology indexes has been largely debated. Focusing on ischemic heart disease with non-obstructive CAD, patients with preserved FFR (>0.80) but reduced CFR (<2) have been shown to experience a higher incidence of unfavorable outcomes compared with those with preserved FFR and CFR, thus highlighting the critical prognostic role of CMD in ischemiadriven adverse events.
On the other hand, contributions investigating the potential prognostic impact of CMD among MINOCA population are limited to exploratory results deriving from observational studies. In particular, a recent study by Abdu et al. is worth mentioning. Coronary microvascular function was assessed in a small cohort of 109 MINOCA patients with the coronary angiography-derived index of microvascular resistance (caIMR), a novel pressure-wire-free index of CMD evaluation. Interestingly, a caIMR value >43U has been reported in most of the patients and resulted a strong independent predictor of major adverse outcomes as well as a useful tool for risk stratification among MINOCA patients (60), although adequately powered randomized clinical trials specifically comparing the prognostic impact of both caIMR and traditional IMR within MINOCA population are warranted to address this issue. Similarly, there are limited evidence evaluating whether the cut-off values for both CFR and IMR in the acute setting of MINOCA are equivalent to those commonly established in patients with stable ischemic heart disease.
In addition, several non-invasive methods of coronary microvascular function assessment have been tested: particularly, a recent study by De Vita et al. enrolling a small cohort of MINOCA patients found an abnormal coronary flow velocity response to both ergonovine and adenosine as assessed by trans-thoracic Doppler echocardiography, thus indirectly suggesting a significant microvascular impairment within this population.
## Provocative tests
Although current guidelines do not recommend provocative tests in patients with AMI, several pieces of evidence support their adoption in the presence of a reasonable clinical suspect of coronary vasomotion abnormalities for epidemiological and prognostic reasons. In this regard, the epidemiological burden of coronary vasospasm among MINOCA patients has not yet been clarified. However, according to the most recent estimates, clinically relevant vasomotion abnormalities have been found in a large cohort of MINOCA patients undergoing acetylcholine provocative tests (∼46%).
In addition, although the impact of a positive provocative test is limited in patients presenting with unstable angina and nonobstructive coronary arteries, the prognostic implications of a positive test in patients with AMI appear to be relevant since patients with MINOCA and underlying impaired coronary vasomotor tone show a more frequent occurrence of death for all causes, cardiac death and readmission for ACS as well as worse angina status. The latter evidence supports the hypothesis that a positive provocative test among the MINOCA population identifies a sub-group of patients at higher risk.
Multiple safety concerns have risen since invasive provocative tests showed a potential risk of malign ventricular arrhythmias as well as bradyarrhythmias development. However, several studies offer encouraging evidence showing that the overall incidence of arrhythmic complications is comparable with that occurring during a spontaneous anginal attack, thus suggesting that provocative tests do not provide significant additional arrhythmic risk.
## Etiology-tailored therapeutic strategies
The recent trend toward tailored therapeutic management guided by a comprehensive invasive evaluation of the pathophysiological mechanisms of ischemia in patients with non-obstructive coronary arteries has been set by the British Heart Foundation Coronary Microvascular Angina (CorMicA) trial. In this pivotal study, the authors demonstrated that a strategy of adjunctive invasive assessments of coronary function followed by a stratified and etiology-based therapy led to a reduction in angina severity and improved quality of life. In particular, consecutive patients with angina in the absence of angiographically relevant obstructive CAD were randomized 1:1 to the interventional group (invasive functional evaluation with tailored stratified therapeutic strategy) or control group (standard care). At 1 year of follow-up, the intervention significantly improved angina and quality of life, even without relevant differences in MACE.
Although antiplatelet therapy represents one of the cornerstones of secondary prevention of AMI with obstructive coronary arteries, considerable evidence has suggested a neutralor even detrimental (69) effect of antiplatelet therapy among the overall heterogeneous cohort of MINOCA patients, further highlighting that the management strategies should be strictly based on the findings arising from a careful invasive evaluation. Selectively focusing on patients with coronary plaque disruption (i.e., plaque rupture or erosion), there is unequivocal evidence of a benefit from lifetime single antiplatelet therapy with aspirin. Moreover, 1 year of dual antiplatelet therapy with the addition of a P 2 Y 12 receptor inhibitor should be considered in these patients in light of the significant role of thrombosis and distal embolization in the pathogenesis of MINOCA with plaque disruption.
The optimal management of SCAD is still a matter of debate since no randomized clinical trials have compared medical therapy to revascularization strategies. However, according to the available data, except for type-1 SCAD obstructing coronary flow or hemodynamically unstable patients presenting with STEMI, a conservative approach aiming at limiting the risk of dissection propagation following percutaneous coronary interventions (PCI) is associated with acceptable outcomes and should therefore be preferred. Hypertension is a wellknown independent predictor of recurrent SCAD, and strict blood pressure control with ACE/ARBs and/or beta-blockers associated with single antiplatelet therapy is the mainstay of conservative SCAD management. In contrast, dual antiplatelet therapy should be reserved for patients undergoing PCI and stent implantation.
In patients with evidence of either epicardial or microvascular spasm following provocative tests, calcium channel blockers (dihydropyridine, non-dihydropyridine, or both) should be considered first-line therapy due to their ability to induce smooth muscle cell relaxation and decrease myocardial oxygen consumption. Conversely, in patients with CMD due to arteriolar remodeling and capillary rarefaction presenting with reduced CFR, increased IMR/HMR, and negative acetylcholine test, ACE-I and beta-blockers seem a more reasonable option.
Lastly, when coronary thromboembolism is suspected, the standard treatment is strictly related to the cause of embolism. According to dedicated guidelines, long-term or even lifetime anticoagulation therapy may be suggested in patients affected by acquired/inherited thrombophilia. In patients with paradoxical embolism due to ASD, transcatheter device closure or surgical repair is recommended (78), whereas secondary prevention of PFO-related embolism include the administration of long-term antiplatelet therapy or trans-catheter device closure (79, 80).
# Conclusion
MINOCA is a complex clinical syndrome with a broad spectrum of pathophysiological mechanisms, among which vasomotion abnormalities and coronary microvascular dysfunction (CMD) play a significant role. Invasive functional assessment has a limited body of evidence in the acute setting, and therefore current guidelines do not recommend routine physiological investigation of epicardial and microvascular coronary compartments within the context of AMI. Furthermore, several invasive tests may indirectly alter microvascular function and status: for example, the pharmacological stimuli with acetylcholine conventionally used to evaluate epicardial vasomotion abnormalities may also have constrictor effects on smooth muscle cells at the microvascular level, thus contributing to an impaired microvascular response. Therefore, it is not always possible to attribute CMD to a specific mechanism since it may frequently recognize multiple underlying causes potentially overlapping. However, despite all these limitation, several studies suggest that a comprehensive invasive functional assessment may help identify the underlying pathophysiological mechanism of myocardial ischemia and consequently select an appropriate therapeutic strategy. Although current guidelines do not recommend routine physiological assessment of epicardial and microvascular coronary compartments within the context of AMI, several pieces of evidence support the pivotal role of a comprehensive invasive functional assessment to identify the underlying pathophysiological mechanism and consequently to select an appropriate therapeutic strategy. Unfortunately, the optimal medical therapy for these patients is not currently established; however, a tailored etiology-based approach should be followed as a general recommendation. Lastly, there is an urgent need for randomized trials to evaluate the short and long-term effects of secondary prevention measures and etiology-targeted therapies to improve the management and prognosis of this heterogeneous population.
# Author contributions
FM and MV wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. |
Quorum-Sensing Regulation of Antimicrobial Resistance in Bacteria
Quorum sensing is a cell-to-cell communication system that exists widely in the microbiome and is related to cell density. The high-density colony population can generate a sufficient number of small molecule signals, activate a variety of downstream cellular processes including virulence and drug resistance mechanisms, tolerate antibiotics, and harm the host. This article gives a general introduction to the current research status of microbial quorum-sensing systems, focuses on the role of quorum-sensing systems in regulating microbial resistance mechanisms, such as drug efflux pump and microbial biofilm formation regulation, and discusses a new strategy for the treatment of drug-resistant bacteria proposed by using quorum quenching to prevent microbial resistance.
# Introduction
Quorum sensing (QS) is also called density sensing, which controls a variety of physiological behaviors in bacteria. Whether in Gram-negative or Gram-positive bacteria, quorum-sensing mechanisms exist, but the signal molecules they use to transmit information are different. Bacteria control the behavior of the entire bacterial population by synthesizing and secreting signal molecules (also known as self-inducing molecules). When the concentration of signal molecules reaches a certain threshold with the bacterial population density, the expression of certain specific genes can be started to regulate the bacterial population adaptation. The QS system regulates various cellular processes, which mainly involve the regulation of bacterial luminescence, virulence factors, disinfectants tolerance, spore formation, toxin production, motility, biofilm formation, and drug resistance.
Antibiotics are now widely used around the world, and antibiotic resistance is spreading faster than ever before [bib_ref] Antimicrobial resistance: A priority for global health action, Chioro [/bib_ref] [bib_ref] National action for global gains in antimicrobial resistance, Abdula [/bib_ref]. Since the introduction of antibiotics, the use of millions of tons of antibiotics has caused selection pressure, and almost all pathogenic bacteria have developed resistance to commonly used antibiotics [bib_ref] Antibiotic resistance-the need for global solutions, Laxminarayan [/bib_ref] [bib_ref] Interplay of antibiotic resistance and food-associated stress tolerance in foodborne pathogens, Liao [/bib_ref]. Most antibiotics currently used are designed to directly kill pathogenic bacteria, such as destroying cell membranes and interfering with key protein synthesis [bib_ref] Multidrug Resistance in Bacteria, Nikaido [/bib_ref]. This "life or death" selection pressure promotes the evolution of microbial resistance, and the large-scale use of antibiotics has brought serious microbial resistance issues. In recent years, "superbugs" that can resist various commonly used antibiotics have appeared around the world [bib_ref] Discovery, research, and development of new antibiotics: The WHO priority list of..., Tacconelli [/bib_ref] [bib_ref] Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in..., Cassini [/bib_ref] [bib_ref] A review of the influence of treatment strategies on antibiotic resistant bacteria..., Sharma [/bib_ref]. The increasing resistance of bacteria to antibacterial compounds and the spread of resistant pathogens have become serious threats to human health [bib_ref] Stress tolerance of Staphylococcus aureus with different antibiotic resistance profiles, Ma [/bib_ref] [bib_ref] Novel antibacterial modalities against methicillin resistant Staphylococcus aureus derived from plants, Li [/bib_ref]. At present, most antibacterial compounds target the necessary bacterial physiological processes, thereby exerting strong selection pressure on bacteria and promoting the emergence and spread of drug-resistant strains [bib_ref] Pharmacological Targeting of the Host-Pathogen Interaction: Alternatives to Classical Antibiotics to Combat..., Munguia [/bib_ref]. A report commissioned by the British government recently estimated that by 2050, antimicrobial resistance could cause 10 million deaths each year and cause cumulative losses of US$ 100 trillion to world GDP [bib_ref] Tackling Drug-Resistant Infections Globally: Final Report and Recommendations, O'niel [/bib_ref] [bib_ref] Progress in and promise of bacterial quorum sensing research, Whiteley [/bib_ref]. Recently, some studies at home and abroad indicate that the QS system may be related to bacterial resistance [bib_ref] Developments in strategies for Quorum Sensing virulence factor inhibition to combat bacterial..., Haque [/bib_ref] [bib_ref] Quorum Sensing: A Prospective Therapeutic Target for Bacterial Diseases, Jiang [/bib_ref] [bib_ref] Current Perspectives on Viable but Non-culturable State in Foodborne Pathogens, Zhao [/bib_ref]. Therefore, inhibiting bacterial QS has become a new promising antibacterial strategy, which can not only prevent the development of bacterial resistance, but also eliminate the expression of virulence factor genes related to population density.
## Microbial resistance mechanisms
The large-scale use of antibiotics in clinical treatment has led to the formation of corresponding multi-resistance mechanisms by microorganisms against the target of antibiotics [bib_ref] Production of diacylglycerols through glycerolysis with SBA-15 supported Thermomyces lanuginosus lipase as..., Zhao [/bib_ref]. The main mechanisms are passivation of antibiotics through chemical modification, efflux pump systemic elimination of antibiotics, and modification of drug-targeting genes [fig_ref] Figure 1: Common mechanisms of microbial resistance [/fig_ref]. At the same time, many pathogenic bacteria can form a dense biofilm [bib_ref] A resource of anti-biofilm agents and their potential implications in targeting antibiotic..., Rajput [/bib_ref] , which makes the bacteria highly resistant.
(1) Chemical modification inactivates the mechanism of antibiotic activity, that is, the secretion of a modified enzyme to change the chemical structure of antibiotic drugs, which leads to antibiotic inactivation and loss of activity. Its enzymatic mechanism includes antibiotic degradation and derivatization of antibiotic chemical groups. One of the best strategies to deal with the existence of antibiotics is to produce enzymes. By adding specific chemicals to the compounds to inactivate the drugs or destroy the molecules themselves, the antibiotics cannot interact with the target substances.
(2) Microorganisms can also use antibiotic pumps to expel antibiotics. Antibiotic drugs usually must enter the cells through the cell membrane of the microorganism in order to effectively attack specific targets. Antibiotic drug efflux resistance is an important mechanism of microbial resistance, which is accomplished by drug efflux pumps. Microorganisms assemble an efflux pump protein on the cell membrane to expel antibiotic drugs in the cell. The excretion rate is usually faster than the drug penetration rate, thereby controlling the drug level in the cell to a non-sensitive level. So far, there have been a variety of microbial efflux pump systems that have been discovered, such as lipophilic and hydrophilic efflux systems, which are targeted at drugs of different chemical properties [bib_ref] Biochemistry of Bacterial Multidrug Efflux Pumps, Kumar [/bib_ref] [bib_ref] Active efflux as the multidrug resistance mechanism, Wasaznik [/bib_ref]. (3) Another important resistance mechanism is the modification of drug-targeting genes. This mechanism mainly changes the drug-targeting genes through modification, which makes the drug lose its target. Another common strategy for bacteria to develop antibiotic resistance is to avoid the effects of antibiotics by interfering with the target site. As a result, bacteria have evolved different strategies, including protecting the target (avoiding the antibiotic from reaching its binding site) and modifying the target site, thereby reducing affinity for antibiotic molecules. β-lactam antibiotics play a bactericidal role by inhibiting the mucopeptide synthase, penicillin binding proteins (PBPs) of the bacterial cell wall trans peptide process, which prevents bacteria from forming a complete cell wall and dies. (4) Drug resistance is due to cellular adaptation. Over the years, bacteria have developed complex mechanisms to cope with environmental stress and stress in order to survive in the harshest environments, including the human body. To gain an advantage, bacteria need to compete for nutrients and avoid attacks from molecules produced by other competing organisms. In a particular host, bacteria are constantly attacked by the host's immune system, and in order to establish themselves in a particular biological environment, it is essential that they adapt and cope with these stressful situations. Thus, bacteria have devised complex mechanisms to avoid disrupting key cellular processes, such as cell wall synthesis and membrane homeostasis. The development of resistance to daptomycin (DAP) and vancomycin (at low levels in Staphylococcus aureus) is the most clinically relevant example of a resistance phenotype that is the result of an overall cellular adaptive response to bacterial attack.
The existence of multiple drug resistance mechanisms has made it difficult to overcome and solve the problem of microbial resistance. In the struggle between microorganisms and antibiotics, more and more microorganisms have evolved multiple resistance mechanisms and have become "super bacteria".
For example, the super resistant bacterium Staphylococcus aureus can specifically degrade penicillin by producing β-lactamase, and on the other hand, it can deprive methicillin of its ability to bind cell wall mucin synthase by producing PBP2a protein [bib_ref] Mechanisms of Methicillin Resistance in Staphylococcus aureus, Peacock [/bib_ref]. In another super bacterium Pseudomonas aeruginosa, in addition to being able to produce different drug efflux pumps to resist multiple types of antibiotics [bib_ref] Inhibiting Bacterial Drug Efflux Pumps via Phyto-Therapeutics to Combat Threatening Antimicrobial Resistance, Shriram [/bib_ref] [bib_ref] Efflux-mediated multiresistance in Gram-negative bacteria, Poole [/bib_ref] , resistance genes can also be obtained through horizontal gene transfer [bib_ref] Horizontal gene transfer and the origin of species: Lessons from bacteria, De La Cruz [/bib_ref] [bib_ref] Ecology and Evolution of Chromosomal Gene Transfer between Environmental Microorganisms and Pathogens, Martinez [/bib_ref] , and its bacteria changes in body shape and dense biofilms can resist almost all antibiotics on the market [bib_ref] A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance, Mah [/bib_ref] [bib_ref] Can Biofilm Be Reversed Through Quorum Sensing in Pseudomonas aeruginosa?, Yan [/bib_ref]. In recent years, researchers have found that the formation of bacterial biofilm resistance to bacteria is particularly important. The drug resistance produced by bacterial biofilm is a systematic and complex drug resistance mechanism. The principle of drug resistance has at least the following three points [bib_ref] Mechanisms of antibiotic resistance in bacterial biofilms, Stewart [/bib_ref] [bib_ref] The role of biofilms as environmental reservoirs of antibiotic resistance, Balcazar [/bib_ref] : (1) The biofilm itself is an effective drug barrier, which can significantly reduce it antibiotic permeability. Bacteria are connected to each other through proteins and DNA, especially extracellular polysaccharides, forming an insurmountable barrier, which can greatly reduce the permeability of antibiotic drugs and improve the survival rate of bacteria in the biofilm. (2) The special microenvironment in the biofilm makes the bacteria in the membrane produce heterogeneity and regulates the antibiotic resistance of the bacteria. The study found that the concentrations of nutrients and bacterial secretions in different areas of the biofilm were not the same, which led to the inconsistent growth status of the bacterial bodies in different areas of the biofilm, that is, the heterogeneity of the bacteria, which led to different levels of drug resistance bacterial cells. (3) The extreme environment outside the biofilm promotes drug resistance within the membrane. Some drastic environmental changes outside the biofilm, such as changes in temperature, pH, and the concentration of certain chemicals, may affect the functions of the bacteria in the biofilm to regulate its physiological and biochemical functions, reduce its growth efficiency, and form a state of resistance to antibiotics. The microbial resistance mechanisms are summarized in [fig_ref] Table 1: Microbial resistance mechanisms [/fig_ref]. Therefore, it is advantageous to study bacterial resistance through inhibiting the formation of bacterial biofilms, inhibiting the bacterial quorum, reducing the barrier effect of biofilms, and inhibiting the phenotypic changes of bacteria in biofilms to weaken the resistance of bacterial biofilms to antibiotics.
## Resistance mechanism action mechanism
Chemical modification Change the chemical structure of antibiotic drugs Efflux pump system Discharge intracellular antibiotic drugs Modification of drug-targeting genes Change drug-targeting genes Global cellular adaptation Adapt and cope with stress Biofilm itself
Reduce the permeability of antibiotics Special internal environment in biofilm Intracellular thallus produce heterogeneity Extreme environment outside the biofilm Intracellular thallus produce resistance
## Microbial quorum sensing system and its regulation mechanism
In recent years, the discovery of microbial quorum-sensing systems has provided new hope for studying the regulatory mechanism of drug resistance mechanisms and overcoming drug resistance. The results show that the quorum-sensing system regulates various cellular processes of microorganisms, such as pathogenic gene expression, toxin production and extracellular polysaccharide synthesis, and plays an important regulatory role in the process of drug efflux pumps and the formation of microbial biofilms [bib_ref] Self-organization of active particles by quorum sensing rules, Bauerle [/bib_ref] [bib_ref] Quorum sensing: Little talks for an effective bacterial coordination, Turan [/bib_ref] [bib_ref] Induction of Viable but Nonculturable Escherichia coli O157:H7 by Low Temperature and..., Wei [/bib_ref]. According to different self-inducible molecules, bacterial QS systems are divided into three types. One is the QS system with acyl-homoserine lactone (AHL) as the self-inducible molecule, which exists in Gram-negative bacteria. The oligopeptides are QS systems that are self-inducing molecules and exist in Gram-positive bacteria. The other types are QS systems that use furan borate diesters as self-inducing molecules and exist in Gram-negative and Gram-positive bacteria.
Signal molecules represented by small molecule oligopeptides, that is, autoinducing oligopeptide (AIP), are mainly used as quorum-sensing signal molecules in Gram-positive bacteria. AIP precursor molecules are generally formed by modification of the leader peptide, and it can be transported across the body with the help of the ATP-binding cassette (ABC) transporter for extracellular secretion. For example, note the Agr system in Staphylococcus aureus [fig_ref] Figure 2: Agr quorum-sensing system in Staphylococcus aureus [/fig_ref]. With the increase of cell density, bacteria begin to synthesize a large number of virulence factors, thereby increasing their pathogenicity. This process is a response made by oligopeptide signal molecules to regulate gene expression and stimulate cells. When the signal molecule oligopeptide secreted to the outside reaches a certain concentration, it will bind to the receptor protein on the cell membrane and pass the phosphorylation/dephosphorylation cascade to pass the oligopeptide to the intracellular binding promoter to start the transcription mechanism and post-translational modifications that activate or inhibit the expression of the gene of interest [bib_ref] Quorum-sensing regulators in Gram-positive bacteria: "cherchez le peptide, Monnet [/bib_ref].
The quorum-sensing signal molecules represented by acyl-homoserine lactones (AHLs), represented by autoinducer-l (AI-1), are mainly used in Gram-negative bacteria [bib_ref] Exploiting Quorum Sensing Interfering Strategies in Gram-Negative Bacteria for the Enhancement of..., Zhang [/bib_ref]. AHL, as a synthetic product in the LuxR-LuxI system, which is widely present in Gram-negative bacteria, can diffuse freely into and out of bacterial cells, and it can also accumulate in the surrounding environment [fig_ref] Figure 3: Acyl-homoserine lactone [/fig_ref]. LuxR, which encodes the LuxR-binding protein, is a transcriptional activator that induces a response, and it can also be called a transcription regulator. Its regulation requires the activation of an acyl-AHL molecule; luxI, which encodes the LuxI protein, is an AHL synthetase. Function requires S-adenosylmethionine (SAM) and acyl-acyl carrier protein (acyl-ACP) as substrates. After AHL binds to LuxR, dimerization or multimerization occurs, and the multimerization product binds to the upstream regulatory region of the target gene to activate or inhibit the expression of the target gene [bib_ref] Quorum sensing signal-response systems in Gram-negative bacteria, Papenfort [/bib_ref] [bib_ref] Advances in the research of LuxR family protein in quorum-sensing system of..., Chen [/bib_ref] [bib_ref] The talking language in some major Gram-negative bacteria, Banerjee [/bib_ref]. The binding specificity of AHL and LuxR is very strong because the specificity of AHL is controlled by different acyl side chain groups. Therefore, Gram-negative bacteria use this method to achieve the transfer of information between cells within the species, which will avoid interference caused by external bacteria [bib_ref] The Quorum Sensing System of Yersinia enterocolitica 8081 Regulates Swimming Motility, Host..., Ng [/bib_ref]. The signal molecule represented by furan borate diester [bib_ref] Production, detection and application perspectives of quorum sensing autoinducer-2 in bacteria, Zhao [/bib_ref] , which is represented by AI-2, can be used as a quorum-sensing signal molecule in both Gram-negative and Gram-positive bacteria. What is different is that AI-2 mediates the interspecies quorum-sensing system [bib_ref] AI-2-mediated signalling in bacteria, Pereira [/bib_ref] [bib_ref] Bacterial interspecies quorum sensing in the mammalian gut microbiota, Xavier [/bib_ref] , that is, the physiological phenomenon that bacteria receive signals from AI-2 released by foreign bacteria and regulate corresponding gene expression [fig_ref] Figure 4: AI-2 quorum-sensing system [/fig_ref]. In the activated methyl cycle (AMC), AI-2 is produced from S-adenosylmethionine (SAM) in a three-step enzymatic reaction. Among them, SAM is a methyl donor, and the intermediate product S-adenosylhomocysteine (SAH) is hydrolyzed by 5'-methylthioadenosine/S-adenosylhomocysteine ribozyme (Pfs) to S-ribosylhomocysteine (SRH) and adenine [bib_ref] Making 'sense' of metabolism: Autoinducer-2, LuxS and pathogenic bacteria, Vendeville [/bib_ref]. LuxS (S-ribosylhomocysteinase), the encoded product of luxS, is an AI-2 synthetase [bib_ref] Short communication: The role of autoinducer 2 (AI-2) on antibiotic resistance regulation..., Xue [/bib_ref]. LuxS catalyzes the cleavage of SRH into DPD and homocysteine. DPD (4,5-dihydroxy-2,3-pentanedione) is a precursor substance for the synthesis of AI-2 [bib_ref] Ac2-DPD, the bis-(O)-acetylated derivative of 4,5-dihydroxy-2,3-pentanedione (DPD) is a convenient stable precursor..., Frezza [/bib_ref] [bib_ref] A Versatile Strategy for the Synthesis of 4,5-Dihydroxy-2,3-Pentanedione (DPD) and Related Compounds..., Stotani [/bib_ref]. The molecular structure of DPD is very unstable, and cyclization, rearrangement, and other reactions may change at any time. This shows that DPD can derive a variety of AI-2 molecules with different structures, compositions, and similarities, which can be recognized by different species of bacteria. It can also be said that Al-2 can be understood as a mixture of several similar small molecules with different conformations. There are two types of AI-2 structures that have been discovered. The molecular structure of AI-2 in Vibrio harveyi is a furanosyl borate diester discovered by Chen et al. [bib_ref] Structural identification of a bacterial quorum-sensing signal containing boron, Chen [/bib_ref]. Miller et al. [bib_ref] Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal..., Miller [/bib_ref] found that the molecular structure of AI-2 of Salmonella typhimurium is furanosyl borate diester that lacks boron.
## Quorum sensing and biological competition
Ecological competition refers to the process by which an organism reduces the survival or reproduction of other organisms for its own survival needs. This competition can be divided into exploitative competition and interference competition [bib_ref] Competition sensing: The social side of bacterial stress responses, Cornforth [/bib_ref] [bib_ref] Multifaceted interfaces of bacterial competition, Stubbendieck [/bib_ref]. Exploitation competition occurs indirectly, and it manifests itself as one organism consumes another organism's resources. Exploitation competition also occurs in microorganisms, especially when they aggregate and form dense communities (such as biofilms), mainly as nutritional restrictions strong competition for exploitation occurs between cells of the same genotype and different genotypes. Interference competition refers to competition that occurs when individuals directly harm each other. In microorganisms, it refers to the secretion of metabolites that harm other cells, including the secretion of antibiotic compounds and asphyxiating polymers [bib_ref] Bacterial communities: Interactions to scale, Stubbendieck [/bib_ref]. Co-culture experiments have shown that these secreted factors often determine which genotypes can prevail in mixed communities [bib_ref] A fitness trade-off between local competition and dispersal in Vibrio cholerae biofilms, Nadell [/bib_ref]. Exploitation and interference competition are common in bacterial communities, which strongly affect the results of bacterial diversity [bib_ref] Multifactorial competition and resistance in a two-species bacterial system, Khare [/bib_ref]. At present, bacteria have evolved methods that can directly detect and respond to ecological competition. In the bacterial stress response, bacteria interact with each other and regulate a series of favorable behaviors (such as reproduction), which is called bacterial quorum sensing [bib_ref] Quorum sensing and bacterial social interactions in biofilms: Bacterial cooperation and competition, Li [/bib_ref]. QS is a social trait that controls bacterial population and diversity by regulating the production of extracellular public goods, including beneficial public goods and harmful public goods [bib_ref] Quorum-sensing control of antibiotic resistance stabilizes cooperation in Chromobacterium violaceum, Evans [/bib_ref].
The stress response to ecological competition is usually associated with the release of toxins because in bacterial community ecological competition usually has foreign genotypes and evolved genotypes [bib_ref] Bacterial stress responses as determinants of antimicrobial resistance, Poole [/bib_ref]. Bacterial populations describe QS's role in regulating microbial diversity through the role of toxins. Toxins produced by bacteria cause bacterial damage, and bacteria usually release toxins that kill other bacteria to break down single cells of other genotypes. Toxins are particularly useful in identifying evolutionary functions that have evolved to affect the activity of other bacteria and ultimately kill them, such as bacteriocin, a narrow-spectrum antibiotic that targets other bacteria, and pyocyanin, which has multiple potential effects on metabolism and nutrition [bib_ref] Competition sensing: The social side of bacterial stress responses, Cornforth [/bib_ref]. The microbial diversity regulated by toxins is as follows: On the one hand, bacteria sense ecological competition through information related to the number of microorganisms in the community mediated by QS. The density of self-cells is a key factor in regulating the effects of toxins. QS regulation ensures that there are enough bacteria in the community with the same genotype to secrete toxins or promote growth products [bib_ref] Toxin production spontaneously becomes regulated by local cell density in evolving bacterial..., Doekes [/bib_ref]. On the other hand, QS information can also predict the intensity of ecological competition. The diversity potential of microbial communities means that the population density information not only acts on itself, but also may be the genotype of specific signals generated by other cells, and the signal molecules are highly specific. It is possible to distinguish population density information because it can identify specific strains or species as evolutionary competitors, such as Proteus mirabilis. It can detect other genotype strains in the species and identify future competitors, or there are strains of quorum-receptor molecules that they do not produce. The luxR and its homologues can respond to AHLs signals of other genotypes in the community, identify competitors, and regulate biofilm formation, luminescence, release of virulence factors, swimming capacity, and expression of protease activity genes, etc., and reduce interference by interfering with competition The number of genotype strains can finally regulate the order of different genotypes in the microbial community to achieve the purpose of regulating the biodiversity in the community.
## Regulation of microbial resistance by quorum sensing
## Regulation of bacterial efflux pump by qs
Bacterial active efflux pumps can effectively discharge antibiotics into the bacteria, and they play an important role in multidrug resistance. Bacterial active efflux pumps are generally composed of three parts: from the outside to the inside are the outer membrane channel protein, the fusion protein, and the cytoplasmic membrane efflux protein. The fusion protein connects the outer membrane channel protein and the cytoplasmic membrane efflux with the participation of energy. Protein, foreign substances including antibiotics, and their metabolites are selectively or non-selectively eliminated from bacteria. At present, QS system's regulating effect on bacterial efflux pump has been confirmed [bib_ref] RND efflux pump systems in Acinetobacter, with special emphasis on their role..., Subhadra [/bib_ref] [bib_ref] Regulatory Mechanisms of the LuxS/AI-2 System and Bacterial Resistance, Wang [/bib_ref]. The regulation effect of QS system on the expression of multidrug-resistant pumps is that both the expression of multidrug-resistant pumps can be regulated, and the QS system itself is also affected by the expression level of multidrug-resistant pumps. On the one hand, the QS system can regulate the expression of efflux pump genes. For example, Bacteroides fragilis ATCC25285 was cultured in vitro in the presence or absence of self-inducible molecules C 6 -HSL and C 8 -HSL. Its growth-resistant drug-sensitive efflux pump gene bmeB expression and the biofilm structure was tested, and it was found that LuxR, a self-inducing molecule receptor of the QS system, can respond to exogenous AHL, upregulate the expression of bmeB efflux pump, and form resistance to antibiotics [bib_ref] Presence of quorum-sensing systems associated with multidrug resistance and biofilm formation in..., Pumbwe [/bib_ref]. Similarly, some researchers have found that autoinducer can up-regulate the multidrug resistance pump MexAB-OprM, causing bacteria to develop multidrug resistance [bib_ref] Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and..., Maseda [/bib_ref]. On the other hand, the QS system itself is also affected by the expression level of the efflux pump. However, some researchers have found that the overexpression of the MexCD-OprJ multidrug efflux pump shuts down the QS response of P. aeruginosa [bib_ref] Role of the Multidrug Resistance Efflux Pump MexCD-OprJ in the Pseudomonas aeruginosa..., Alcalde-Rico [/bib_ref]. While some efflux pumps (such as RND) expel the drug out of the cell to form drug resistance, QS system self-inducible molecules can also be expelled from the cell, increasing the concentration of extracellular self-inducible molecules, which is manifested by the exacerbation of bacterial infections [bib_ref] The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria, Li [/bib_ref]. This suggests that the high expression of the efflux pump may promote the further activation of the QS system, promote the QS system's regulation of toxin infection factor synthesis and efflux pump expression, and enhance the infectivity and invasiveness of bacteria. At the same time, researchers have found that the QS system regulates biofilm-forming genes and also regulates bacterial resistance-related genes. The LuxR receptor of the QS system in Bacteroides fragilis has been shown to regulate biofilm formation and bmeB expression in efflux pumps [bib_ref] Presence of quorum-sensing systems associated with multidrug resistance and biofilm formation in..., Pumbwe [/bib_ref]. Similarly, in the presence of imipenem, the resistance gene ampC can be highly expressed in biofilms [bib_ref] Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression..., Bagge [/bib_ref] [bib_ref] Imipenem Treatment Induces Expression of Important Genes and Phenotypes in a Resistant..., Dhabaan [/bib_ref]. This situation of high biofilm-forming bacteria, but not free or low-expression, suggests that the QS system may also regulate the expression of these resistance-related genes while regulating biofilm formation. This has attracted people's attention and may be another way for the QS system to directly regulate the related genes to form drug resistance [fig_ref] Table 2: Regulation of microbial resistance by quorum sensing [/fig_ref]. Permeation limitation Lack of nutrients Less sensitive to antibioticslasl/lasR and rhlI/rhlI signaling system Activate related transcription regulators Form biofilms [bib_ref] Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: A tale of regulatory..., Williams [/bib_ref] Interspecies signal analogues Regulate or inhibit enzyme activity Altered biofilm formation [bib_ref] Modulation of antibiotic sensitivity and biofilm formation in Pseudomonas aeruginosa by interspecies..., An [/bib_ref] WalK/WalR two-component system Directly regulates biofilm formation [bib_ref] New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a..., Dubrac [/bib_ref] Stimulating factors Promote QS system adjustment Regulate biofilm formation [bib_ref] Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System, Tan [/bib_ref]
## Regulation of bacterial biofilm formation by qs
Biofilm is a special structure formed by bacteria adsorbed on the surface of inert or active materials in order to adapt to the living environment. It is composed of itself and extracellular matrix such as polysaccharides and proteins. It has a certain spatial structure and shows new biological traits and stronger environmental adaptability. Recent studies have shown that the majority of human bacterial infections are related to biofilm, and the formation of biofilm is one of the important reasons why clinical bacterial infections are difficult to cure and relapse [bib_ref] Spatial structure facilitates the accumulation and persistence of antibiotic-resistant mutants in biofilms, France [/bib_ref]. The formation of bacterial biofilms is closely related to the development of drug resistance. It is generally believed that bacterial biofilms can lead to bacterial drug resistance through penetration restriction mechanisms, nutrition restriction mechanisms, and drug resistance phenotypic mechanisms [bib_ref] Biofilms: Architecture, Resistance, Quorum Sensing and Control Mechanisms, Saxena [/bib_ref]. The molecular barrier and charge barrier (mostly negatively charged) formed by the polysaccharides in the biofilm can prevent or delay the penetration of certain antibiotics, which is the main mechanism of limiting permeability [bib_ref] Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their..., Roy [/bib_ref]. The nutrient limitation mechanism is closely related to the permeation limitation mechanism. Due to the existence of the biofilm's permeation limitation, nutrients cannot easily pass through the biofilm, which makes the nutrient in the biofilm lack nutrients and slows the growth of the inner bacteria. The slow-growing state is also called starvation, and the starvation of bacteria is less sensitive to antibiotics [bib_ref] Antimicrobial Resistance of Bacteria in, Chebotar [/bib_ref] [bib_ref] Densely adherent growth mode, rather than extracellular polymer substance matrix build-up ability,..., Qu [/bib_ref]. In addition, the study also found that the formation of drug resistance is related to the differential expression of some biofilm-related phenotype-related genes, which suggests that the formation of biofilm resistance may also have a drug-resistant phenotype mechanism [bib_ref] 12:i:-Portuguese Isolates: A Phenotypic, Genotypic, and Socio-geographic Analysis, Seixas [/bib_ref] [bib_ref] Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus..., Pozzi [/bib_ref].
In the process of biofilm formation, the QS system plays an important regulatory role. QS system regulates the biofilm formation of Gram-negative bacteria and Gram-positive bacteria. The formation of Gram-negative bacteria's biofilm is regulated by the QS system using AHL as a signal molecule, which is composed of signal molecules and corresponding signal molecule receptors. For example, in P. aeruginosa, the QS system has two signaling systems: lasl/lasR and rhlI/rhlI. The lasl/rhlI and lasR and rhlR genes encode different signal molecule synthetases and signal molecule receptors, respectively [bib_ref] Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: A tale of regulatory..., Williams [/bib_ref] [bib_ref] Modulation of antibiotic sensitivity and biofilm formation in Pseudomonas aeruginosa by interspecies..., An [/bib_ref]. Signal molecules increase in secretion as the density of bacteria increases. When a signal molecule reaches a certain threshold, the signal molecule binds to the corresponding signal molecule receptor and activates the receptor. The activated receptor then activates the relevant transcriptional regulators to synthesize extracellular polysaccharides, toxic factors, alginates, etc., thereby causing bacteria to form biofilms. The QS system regulates the biofilm of Gram-positive bacteria by using oligopeptides as signal molecules, which can be recognized by the two-component sensing protein after modification, and it regulates the expression of the target gene through phosphorylation and dephosphorylation of the protein. This further regulates the formation of biofilms. Different types of bacteria have different oligopeptide signaling molecules and QS system regulation pathways. For example, the two-component system in Streptococcus is the histidine protein kinase and response regulatory protein [bib_ref] A two-component system that controls the expression of pneumococcal surface antigen A..., Mccluskey [/bib_ref] , while the QS system in Staphylococcus aureus is highly conserved. WalK/WalR is also called the YycG/YycF two-component system, which directly regulates the formation of biofilms [bib_ref] New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a..., Dubrac [/bib_ref]. In addition to two-component systems, there are also known regulatory factors that influence biofilm formation. For example, RNA nucleic acid polymerase III can promote the formation of S. aureus biofilms, while RNA nucleic acid polymerase III inhibits protein peptides and can significantly inhibit the production of its biofilms [bib_ref] Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System, Tan [/bib_ref] [bib_ref] agr RNAIII divergently regulates glucose-induced biofilm formation in clinical isolates of Staphylococcus..., Coelho [/bib_ref]. In Streptococcus, competent stimulating factors can promote the regulation of the biofilm by the QS system [bib_ref] Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System, Tan [/bib_ref]. In Staphylococcus epidermidis, QS system-related comprehensive regulator sarA is closely related to biofilm formation and is a positive regulator of Staphylococcus epidermidis biofilm formation [bib_ref] SarZ Promotes the Expression of Virulence Factors and Represses Biofilm Formation by..., Tamber [/bib_ref] [fig_ref] Table 2: Regulation of microbial resistance by quorum sensing [/fig_ref].
## Regulation of bacterial secretion system by qs
Pathogenic bacteria secrete proteins through the cell membrane. This basic process enables them to attack other microorganisms, evade the host's immune system, cause tissue damage, and invade host cells. Secreted proteins can act as toxic factors, produce toxic substances to host cells, and may also promote adhesion to these cells. Proteins are transported on phospholipid membranes by several secretory systems [bib_ref] Bacterial secretion systems: An overview, Green [/bib_ref]. The secretory system plays an important role in the spread of bacteria. So far, the structure, composition, and activity of eight secretory systems (T1, T2, T3, T4, T5, T6, T7, T9) have been determined. These differences are due to the differences between Gram-positive and Gram-negative bacteria [bib_ref] Secretion systems in Gram-negative bacteria: Structural and mechanistic insights, Costa [/bib_ref] [bib_ref] Secretion Systems Used by Bacteria to Subvert Host Functions, Rapisarda [/bib_ref].
The type I secretion system (T1SS) is widely distributed in Gram-negative bacteria. It has three structural elements: ABC transporter, membrane fusion protein, and outer membrane factor [bib_ref] The type 1 secretion pathway-The hemolysin system and beyond, Thomas [/bib_ref]. So far, there are two systems that can regulate the expression and secretion of T1SS substrates: the Has system of Serratia marcescens and Pseudomonas aeruginosa, and the hemolysin system of Vibrio cholerae, Neisseria meningitidis, and E. coli [bib_ref] The type 1 secretion pathway-The hemolysin system and beyond, Thomas [/bib_ref]. In the transcriptional study of P. aeruginosa, T1SS is positively regulated by QS because the expression of its effector alkaline protease AprA depends on QS [bib_ref] Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors, Hentzer [/bib_ref]. The type II secretion system (T2SS) is responsible for secreting folded proteins from the periplasm in Gram-negative bacteria [bib_ref] Bacterial secretion systems: An overview, Green [/bib_ref]. The main function of T2SS is to obtain nutrition. It is responsible for secreting a large amount of exoproteins. The Xcp system in P. aeruginosa secretes QS-regulated virulence factors elastase and exotoxin, which itself is also positively regulated by QS [bib_ref] Evidence for direct control of virulence and defense gene circuits by the..., Maura [/bib_ref]. The QS system directly controls biofilm production related to the Vibrio cholerae type II secretion system [bib_ref] Living in the matrix: Assembly and control of Vibrio cholerae biofilms, Teschler [/bib_ref]. The type IV secretion system is widely present in Gram-negative and Gram-positive bacteria. T4SS is the most worldwide secretion system. It can transfer not only proteins, but also DNA [bib_ref] Type IV secretion in Gram-negative and Gram-positive bacteria, Grohmann [/bib_ref]. The QS system is directly related to T4SS in Brucella abortus. For the virB operon encoding T4SS regulated by VjbR, LuxR-type QS is responsible for the virulence characteristics of Brucella abortus [bib_ref] Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic..., Li [/bib_ref].
## New strategy for preventive treatment of microbial resistance
The QS system plays an important role in the formation of bacterial drug resistance mechanisms by regulating the formation of biofilms and the direct regulation of drug efflux pumps. The increase in bacterial resistance has aggravated the difficulty of disease prevention, and the side effects caused by excessive use of drugs may also endanger human health. The discovery of control strategies for quorum quenching diseases in recent years has provided new possibilities for overcoming and solving the problem of microbial resistance [bib_ref] Natural products as biofilm formation antagonists and regulators of quorum sensing functions:..., Ciric [/bib_ref]. By interfering with the QS system of specific microorganisms, hindering the exchange of information between microorganisms, and reducing the expression level of hazard factors, this phenomenon is called quorum quenching (QQ) [bib_ref] Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase, Dong [/bib_ref] [bib_ref] Quorum sensing for population-level control of bacteria and potential therapeutic applications, Wu [/bib_ref]. The properties of quorum quenching molecular roles (chemical compounds, enzymes), modes of action (competition, inhibition, QS signal interdict, etc.), and targets are diverse and are all major steps in the QS pathway, including from synthesis to diffusion to accumulation, and sensing of a portion of the QS signal may be affected. Normally, the enzymes that inactivate the QS signal are called quorum-quenching enzymes, while the chemicals that disrupt the QS pathway are called QS inhibitors. The active substances with quorum quenching are collectively referred to as quorum-sensing inhibitors (QSIs). Unlike currently commonly used antibiotics, quorum-quenching agents reduce microbial infections by inhibiting microbial quorum induction, and they generally do not affect microbial growth. There are three main ways of quorum quenching: (1) inhibition of signal molecule production, (2) degradation of signal molecule, and (3) inhibition of signal molecule conduction or binding to receptors [bib_ref] Regulatory Mechanisms of the LuxS/AI-2 System and Bacterial Resistance, Wang [/bib_ref] [bib_ref] Bacterial quorum sensing inhibitors: Attractive alternatives for control of infectious pathogens showing..., Bhardwaj [/bib_ref] [fig_ref] Figure 5: Schematic diagram of the quorum-sensing inhibitor mechanism [/fig_ref]. The QS system suppression strategies are summarized in [fig_ref] Table 3: Review of QS system inhibition strategies [/fig_ref]. Compared with the traditional prevention and control methods, which mainly aim at inhibiting and killing microorganisms, QQ will not pressure the growth of microorganisms, nor will it induce the development of microbial resistance. Therefore, the research and development of QQ-based QSI has gradually attracted the attention of researchers and has become a new strategy for controlling harmful microorganisms.
## Inhibition of signal molecule production
In the QS system, the synthesis of signal molecules plays a vital role in the communication between cells [bib_ref] Presence of quorum-sensing systems associated with multidrug resistance and biofilm formation in..., Pumbwe [/bib_ref]. Interfering with the synthesis of signal molecules is direct way to inhibit quorum sensing. In short, if no signal molecules are produced, quorum sensing will not occur. However, there are few studies on signal molecule synthesis inhibitors, and the data are very limited. Recombinant PS and LuxS can successfully convert SAH to homocysteine and DPD. The LuxS compound is chemically modified to generate QS signal AI-2, and its activity can be determined by monitoring the luminescence of Vibrio harvey BB170. The researchers screened peptides obtained after three rounds of selection to inhibit LuxS enzyme activity. The corresponding synthetic peptide TNRHNPHHLHHV showed a specific inhibitory effect on LuxS [bib_ref] Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus..., Han [/bib_ref]. Schramm et al. synthesized MT-DADMe-ImmA, a picomolar inhibitor, through 5 -methylthioadenosine phosphorylase (MTAP) transition state structure [bib_ref] Transition state analogues in quorum sensing and SAM recycling, Schramm [/bib_ref]. It blocks QS in Vibrio cholerae without affecting the growth rate of the bacteria.
The compounds based on (2-nitrophenyl) methanol are considered promising PqsD inhibitors. PqsD is a key enzyme for signal molecule biosynthesis in Pseudomonas aeruginosa in intercellular communication. Studies have found that (2-nitrophenyl) methanol derivatives have improved cell efficacy and provide new prospects for the application of PqsD inhibitors as anti-infective drugs [bib_ref] From in vitro to in cellulo: Structure-activity relationship of (2-nitrophenyl)methanol derivatives as..., Storz [/bib_ref]. A study revealed a well-proven but underdeveloped novel inhibitor of the target enoyl-ACP reductase that promotes the acyl chain length of N-acyl homoserine lactones, of which an ester is the main signaling molecule in Gram-negative bacteria.
## Degradation of signal molecule
Degradation of signal molecules is a more studied quenching method. This method mainly uses quorum-quenching enzymes produced by microorganisms or other organisms to degrade quorum-sensing signal molecules, so that the concentration of signal molecules is lower than the threshold, and pathogenic bacteria cannot express pathogenic genes and produce pathogenic factors, thus losing the ability to infect the host.
Dong et al. [bib_ref] Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase, Dong [/bib_ref] showed that the aiiA gene encoding the AHL degrading enzyme in Bacillus sp. 240B1 was expressed in potato and tobacco. The expression of aiiA gene in transgenic tobacco and potato significantly increased resistance to Pseudomonas legume. After inoculation with pathogenic bacteria, potato tubers and tobacco leaves did not present with disease spots nor was the appearance of disease spots significantly delayed, which proved that enzymatic degradation of AHL signal molecules is indeed an effective disease prevention and control method. The quorum-sensing system using AHLs as a signal for many pathogenic bacteria is an important regulator of virulence and an attractive target for anti-infective drugs. This method of prevention and control does not act on the pathogenic bacteria itself, but it acts on the signal molecules produced by the pathogenic bacteria so that the pathogenic genes are not expressed. Thus, the "life or death" selection pressure is not exerted on the pathogenic bacteria, and the possibility of bacteria becoming resistant and causing disease is greatly reduced, which is favorable for use as a highly effective and long-lasting drug [bib_ref] Quorum quenching: Role in nature and applied developments, Grandclement [/bib_ref] [bib_ref] Viable but non-culturable state and toxin gene expression of enterohemorrhagic Escherichia coli..., Liu [/bib_ref] [bib_ref] Advances in Rapid Detection Methods for Foodborne Pathogens, Zhao [/bib_ref]. Researchers identified an AidB from a soil bacterium, Bosea sp. strain F3-2. It is a new AHL lactonase that hydrolyzes the ester bonds of the homoserine lactone (HSL) ring. The expression of AidB reduced the AHL signal and the production of QS-dependent virulence factors by Pseudomonas aeruginosa and Pectobacterium carotovorum. It is worth noting that AidB is a thermostable enzyme, which retains its catalytic activity after being treated at 80 - C for 30 min, and it shows reliable storage stability at 4 - C and room temperature. These characteristics may make it more suitable for practical applications. Zhang et al. [bib_ref] Heterologous Expression of the Marine-Derived Quorum Quenching Enzyme MomL Can Expand the..., Zhang [/bib_ref] connected the target fragment MomL with pNCMO2 to obtain a recombinant strain named BbMomL. The BbMomL can not only degrade the exogenous signal molecule C6-HSL, but it also degrades AHL signal molecules produced by the Gram-negative pathogen botulinum. Compared to wild-type Bifidobacterium breve, BbMomL not only inhibits fungal and Gram-positive bacterial pathogens, but it also significantly inhibits Gram-negative bacterial pathogens. The results show that BbMomL has a wide antibacterial spectrum. In addition, the BbMomL significantly reduced the secretion of pathogenic factors and the pathogenicity of Pseudomonas aeruginosa. Research by showed that crude extracts from Lactobacillus crustorum ZHG 2-1 can degrade AHL [bib_ref] Lactobacillus crustorum ZHG 2-1 as novel quorum-quenching bacteria reducing virulence factors and..., Cui [/bib_ref].
Signal molecule inactivation or denaturation can be achieved through a variety of mechanisms, which is the most basic approach to using QS to prevent bacterial resistance and to study new antibacterial strategies. Some microorganisms can metabolize AI-2, thereby inhibiting the function of QS. The addition of ATP and LsrK (AI-2 kinase) in bacterial culture can achieve the degradation of signal molecules, and AI-2 is phosphorylated outside the cell; bacterial crosstalk controlled by AI-2 is significantly reduced [bib_ref] Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells, Quan [/bib_ref]. AI-2 molecules are more hydrophilic after phosphorylation and are thought to be unable to pass through the cell membrane and serve as QS signals [bib_ref] Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation, Zhu [/bib_ref]. AI-2 molecules that are phosphorylated in vitro prevented the QS response of E. coli, Haber's bacillus, and Salmonella typhimurium [bib_ref] Cross species quorum quenching using a native AI-2 processing enzyme, Roy [/bib_ref]. This strategy may be useful in mixed infections because LsrK can phosphorylate DPD as a precursor molecule of AI-2 [bib_ref] Phosphorylation and processing of the quorum-sensing molecule autoinducer-2 in enteric bacteria, Xavier [/bib_ref]. In addition, it may be effective regardless of the structure and transport/sensor mechanism of AI-2 for different bacterial QS systems [bib_ref] DPD-inspired discovery of novel LsrK kinase inhibitors: An opportunity to fight antimicrobial..., Stotani [/bib_ref]. It has been reported that exogenous imidazole is a furan ring analog of AI-2, which reduces the resistance of E. coli to β-lactam antibiotics by inhibiting the function of AI-2 [bib_ref] Imidazole decreases the ampicillin resistance of an Escherichia coli strain isolated from..., Yu [/bib_ref].
## Inhibition of signal molecule conduction or binding to receptors
In addition, quorum-inducing signal inhibitors also play an important role in reducing the pathogenicity of bacteria. Studies have found that many organisms can secrete quorum-sensing signal analogs, competitively combine with bacterial quorum-sensing signal receptors, interfere with the regulation of the quorum-sensing system, and significantly reduce the pathogenicity of bacteria. Wei et al. [bib_ref] Chinese medicinal herb extract inhibits PQS-mediated quorum sensing system in Pseudomonas aeruginosa, Wei [/bib_ref] discovered a medicinal herb extract (MHE). The Pseudomonas quinolone signaling (PQS) system was completely suppressed, the rhlR/rhlI QS system was moderately suppressed, and the lasR/lasI QS system was only slightly affected, suggesting that MHE may selectively target the PQS system to inhibit bacterial toxicity. In addition, electrophoretic mobility shift assays showed that MHE inhibited the binding of MvfR to the corresponding pqsA promoter region, suggesting that MHE acts as a competitor to quench the QS function in P. aeruginosa. Truchado et al. [bib_ref] Inhibition of Quorum Sensing (QS) in Yersinia enterocolitica by an Orange Extract..., Truchado [/bib_ref] found that flavonoids rich in Citrus sinensis have the function of inhibiting quorum-sensing signals, which can significantly reduce the concentration of quorum-sensing signals secreted by Yersinia enterocolitica and the formation of quorum-sensed biofilms without affecting bacterial growth.
The results showed that D-galactose inhibited AI-2 activity, which could inhibit periodontal pathogens from forming biofilms [bib_ref] D-Galactose as an autoinducer 2 inhibitor to control the biofilm formation of..., Ryu [/bib_ref]. The D-galactose-binding protein is highly similar to ribose-binding protein (RbsB), which is the AI-2 receptor for Actinomycetes. A small peptide 5906 was identified, which inhibited the LuxS activity of Edwardsiella tarda by specifically binding LuxS in a manner that might prevent the formation of functionally identical LuxS dimers. The QS response mediated by AI-2 usually occurs in a bacterial community composed of different types of microorganisms. Multiple studies have shown that the QS phenotype in a variety of microbial communities mediates activity between normal flora and pathogens [bib_ref] Quorum sensing signal-response systems in Gram-negative bacteria, Papenfort [/bib_ref] [bib_ref] Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing, Neiditch [/bib_ref]. It is precisely because LuxS/AI-2 regulates pathogen virulence in a variety of microbial communications that disrupting and preventing signaling in these networks provides an excellent target for QS quenchers [bib_ref] Regulatory Mechanisms of the LuxS/AI-2 System and Bacterial Resistance, Wang [/bib_ref] [bib_ref] LuxS-dependent AI-2 regulates versatile functions in Enterococcus faecalis V583, Shao [/bib_ref] [bib_ref] Autoinducer-2-mediated quorum sensing partially regulates the toxic shock response of anaerobic digestion, Xiao [/bib_ref].
Although quorum-sensing signal antagonistic activity has been found in many bacterial, fungal, plant, and animal metabolites, only a few active molecules have been isolated and identified. Therefore, many researchers have used artificial synthesis to synthesize quorum-sensing signal analogs to antagonize the quorum-sensing signal of pathogenic bacteria and have achieved certain results [bib_ref] Mechanisms and Synthetic Modulators of AHL-Dependent Gene Regulation, Stevens [/bib_ref]. Five different haloquinone analogs were tested carrying different positions of methoxy and hydroxyl [bib_ref] Synthesis of novel inhibitors blocking Wnt signaling downstream of beta-Catenin, Halbedl [/bib_ref]. Tests of Wnt activity in cell culture and Xenopus embryos have shown that two of these compounds can be effective inhibitors of abnormally activated Wnt/β-catenin signaling. Researchers have resolved a 2H-pyran-3(6H)-one derivative to develop a new asymmetric catalytic method for synthesizing collections of compounds inspired by iridescent compounds [bib_ref] Discovery of Inhibitors of the Wnt and Hedgehog Signaling Pathways through the..., Takayama [/bib_ref]. The desired product is efficiently formed with high diastereoselectivity and enantioselectivity. Evaluation of the obtained compound set led to the discovery of novel Wnt and Hedgehog signaling pathway inhibitors. Smith et al. [bib_ref] Library screening for synthetic agonists and antagonists of a Pseudomonas aeruginosa autoinducer, Smith [/bib_ref] chemically synthesized a series of structural analogues of LasR, a receptor protein of the Pseudomonas aeruginosa quorum sensing system, and subsequent experiments showed that this series of analogues can produce antagonistic effects and inhibit the expression of virulence factors of pathogenic bacteria. In one study, researchers synthesized a group of six compounds based on a scaffold (alkylquinoxaline-2(1H)-one), which is a new anti-QS feature of the Aeromonas caviae Sch3 [bib_ref] Synthesis, and Evaluation of Alkyl-Quinoxalin-2(1H)-One Derivatives as Anti-Quorum Sensing Molecules, Inhibiting Biofilm..., Blocher [/bib_ref]. This preliminary study will help develop anti-QS compounds to overcome the clinical challenges of drug-resistant strains. In addition, some researchers have synthesized known QS inhibitors designed on the N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL) QS molecular scaffold [bib_ref] Conjugation of LasR Quorum-Sensing Inhibitors with Ciprofloxacin Decreases the Antibiotic Tolerance of..., Bortolotti [/bib_ref]. The LasR antagonist interacts with the N-terminal ligand binding domain of LasR, thereby blocking the binding site of the QS molecule. They were coupled with ciprofloxacin to inhibit the formation of P. aeruginosa biofilms and increase the antibiotic sensitivity of clinical strains.
As mentioned earlier, in addition to affecting the expression of pathogenic genes, the quorum-sensing system also plays an important role in regulating biofilm formation and overexpression of drug efflux pumps. Therefore, in addition to being used directly for the prevention and control of microbial diseases, population quenchers can also be used simultaneously with antibiotics to reduce the resistance of microorganisms and increase the bactericidal efficacy of antibiotics.
## Future outlook
Quorum sensing, as an emerging research area in microbial research, has gradually received attention since the 1990s. At present, many related studies are still incomplete, and the field of drug resistance is relatively late in the field of quorum sensing. A lot of fruitful work has been done, but the related regulatory mechanism of quorum sensing in microbial resistance is still unclear. It can be seen that future research on quorum sensing in the field of microbial resistance is full of opportunities and challenges. In order to better adapt to this development, future research on quorum sensing in the field of microbial resistance should focus on the following areas: (1) In view of the incompleteness of the current QS-related regulatory mechanisms, related research should be further improved by means of molecular biology. [bib_ref] National action for global gains in antimicrobial resistance, Abdula [/bib_ref] In view of the complexity of the microbial drug resistance system, we should focus on strengthening relevant research on bacterial quorum sensing. (3) In view of the inefficiency of current QSI screening, it is important to focus on establishing new and efficient QSI screening technologies. (4) In view of the broad prospects of QS systems in synthetic biology, research and discovery of multiple QS regulation components should focus on rigorous regulation of target genes to meet the diversity of engineering bacteria in actual production.
The diversity of microbial resistance mechanisms has undoubtedly brought great challenges to research and solve the problem of resistance. Therefore, it is of great practical significance to study the formation and regulation of the main resistance mechanisms of microorganisms for the prevention and control of microbial diseases. At present, research on the regulation mechanism of microbial resistance is scarce. Although studies have shown that, in Pseudomonas aeruginosa and several other pathogens, the quorum-sensing system is involved in the regulation of biofilm formation and drug efflux pump gene expression, the formation and regulation of drug resistance mechanisms of more clinically important pathogens still need further study. Therefore, advances and breakthroughs in research on quorum sensing and other regulatory systems will likely inject new vitality into the study of microbial resistance.
The study of microbial quorum sensing has its complexities. At the same time, research on the relationship between quorum sensing and drug resistance also faces many challenges. The quorum-sensing mechanism in many microorganisms is not singular. If the same phenotype of bacteria is regulated by multiple signaling networks, will the inhibition of one signaling mechanism be replaced by other communication mechanisms? Will inhibition of one communication mechanism cause other previously disabled regulatory mechanisms to be disabled? Recovery is a problem that may be faced in future scientific research. Finding quorum-sensing regulatory systems that are critical to microbial pathogenicity and drug resistance will be the focus of future research.
# Conclusions
Although there are still many shortcomings in research on QS, with continuous maturation of molecular biology, synthetic biology, and omics, future research on QS will definitely enter a new stage, and this will also make new contributions to the development of the microbial resistance industry.
Author Contributions: X.Z., conceiving the structure of the manuscript, writing and replying the comments; Z.Y., critical comments, writing and editing the manuscript; T.D., project design, reviewing and revising the manuscript. All authors have read and agreed to the published version of the manuscript.
[fig] Figure 1: Common mechanisms of microbial resistance. [/fig]
[fig] Figure 2: Agr quorum-sensing system in Staphylococcus aureus. [/fig]
[fig] Figure 3: Acyl-homoserine lactone (AHL) quorum-sensing system. [/fig]
[fig] Figure 4: AI-2 quorum-sensing system. [/fig]
[fig] Figure 5: Schematic diagram of the quorum-sensing inhibitor mechanism. [/fig]
[table] Table 1: Microbial resistance mechanisms. [/table]
[table] Table 2: Regulation of microbial resistance by quorum sensing. [/table]
[table] Table 3: Review of QS system inhibition strategies. [/table]
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The iHealth-T2D study: a cluster randomised trial for the prevention of type 2 diabetes amongst South Asians with central obesity and prediabetes—a statistical analysis plan
## Measure
Expected SD 0% Dropout 10% Dropout 20% Dropout
Waist ( |
Frequent mechanical stress suppresses proliferation of mesenchymal stem cells from human bone marrow without loss of multipotency
Mounting evidence indicated that human mesenchymal stem cells (hMSCs) are responsive not onlyto biochemical but also to physical cues, such as substrate topography and stiffness. To simulate the dynamic structures of extracellular environments of the marrow in vivo, we designed a novel surrogate substrate for marrow derived hMSCs based on physically cross-linked hydrogels whose elasticity can be adopted dynamically by chemical stimuli. Under frequent mechanical stress, hMSCs grown on our hydrogel substrates maintain the expression of STRO-1 over 20 d, irrespective of the substrate elasticity. On exposure to the corresponding induction media, these cultured hMSCs can undergo adipogenesis and osteogenesis without requiring cell transfer onto other substrates. Moreover, we demonstrated that our surrogate substrate suppresses the proliferation of hMSCs by up to 90% without any loss of multiple lineage potential by changing the substrate elasticity every 2nd days. Such "dynamic in vitro niche" can be used not only for a better understanding of the role of dynamic mechanical stresses on the fate of hMSCs but also for the synchronized differentiation of adult stem cells to a specific lineage.Human mesenchymal stem cells (hMSCs) can undergo both self-renewal and differentiation into multiple lineages, which makes them highly attractive for applications in regenerative medicine and tissue engineering 1 . Recent experimental evidence indicates that mechanical properties of the microenvironment, as well as biochemical stimuli, determine the long-term fate of stem and progenitor cells 2,3 . Cells can actively sense and respond to the mechanical properties (elasticity) of the surrounding extracellular environments by the clustering of integrin receptors. This leads to the formation of focal adhesions that facilitate the downstream cascades of intracellular signaling pathways. Such adhesion-induced signaling pathways, called as outside-in signaling, trigger the generation of forces by the contracting actin-myosin (actomyosin) complexes 4 . The resistance of substrates against the applied traction force controls signaling molecules, such as talin-vinculin complexes, which mediate the connection between integrin clusters and actomyosin complexes 5 . On the other hand, the stimulation of actomyosin contraction can also lead to the conformational change in the cytoplasmic domains of integrin molecules, which increases the binding affinity of the extracelluar domain (inside-out signaling) 6 .
Scientific RepoRts | 6:24264 | DOI: [bib_ref] Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft..., Engler [/bib_ref].1038/srep24264
To date, various extracellular matrix (ECM) models based on chemically cross-linked hydrogels have been developed in order to understand how such a positive feedback in mechano-sensing regulates the fate of stem and progenitor cells. Fine tuning of the cross-linker concentration and the reaction time [bib_ref] Microelastic gradient gelatinous gels to induce cellular mechanotaxis, Kidoaki [/bib_ref] [bib_ref] Interplay of matrix stiffness and protein tethering in stem cell differentiatio, Wen [/bib_ref] enables one to control the elastic modulus of a given gel substrate. Such "ex situ" regulation of the mechanical microenvironment of cells provides important insight into the vital role of elasticity compliance in optimizing the cell morphology [bib_ref] Substrate compliance versus ligand density in cell on gel responses, Engler [/bib_ref] [bib_ref] Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft..., Engler [/bib_ref] [bib_ref] Matrices with compliance comparable to that of brain tissue select neuronal over..., Georges [/bib_ref] , as well as regulating the migratory behavior [bib_ref] Cell movement is guided by the rigidity of the substrate, Lo [/bib_ref] [bib_ref] Repositioning of cells by mechanotaxis on surfaces with micropatterned Young's modulus, Gray [/bib_ref] [bib_ref] Elasticity boundary conditions required for cell mechanotaxis on microelastically-patterned gels, Kidoaki [/bib_ref] , and the differentiation fate of human mesenchymal stem cells (hMSCs) [bib_ref] Matrix elasticity directs stem cell lineage specification, Engler [/bib_ref] [bib_ref] Growth factors, matrices, and forces combine and control stem cells, Discher [/bib_ref]. Engler et al. [bib_ref] Matrix elasticity directs stem cell lineage specification, Engler [/bib_ref] functionalized the surface of chemically cross-linked polyacrylamide gels with type I collagen, and demonstrated that the multiple lineage differentiation of hMSCs can be controlled via the substrate elasticity. Winer et al. [bib_ref] Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but..., Winer [/bib_ref] reported that hMSCs seeded on very soft polyacrylamide gels (E ~ 250 Pa) with fibronectin coatings sustained their multi-lineage differentiation capacity and suppressed cell proliferation. Zemel et al. [bib_ref] Optimal matrix rigidity for stress-fibre polarization in stem cells, Zemel [/bib_ref] calculated the nematic order parameters of actomyosin complexes by staining both actin and non-muscle myosin II (NMMII) for hMSCs on polyacrylamide gels, and found that the maximum order parameter correlated directly with the optimal substrate elasticity for muscular tissues. However, these ex situ approaches have a fundamental problem to model dynamic mechanical microenvironments of stem cells [bib_ref] Signals from the Sympathetic Nervous System Regulate Hematopoietic Stem Cell Egress from..., Katayama [/bib_ref]. As suggested by in vivo studies, cells are highly sensitive to dynamic changes in their mechanical environment both during their development and when subjected to disease. One biologically relevant example is the significant influence of ECM density on the transition of cancer cells from a protease-dependent movement to an amoeboid movement [bib_ref] Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular..., Wolf [/bib_ref]. To provide stem cells with dynamically tunable mechanical environments, stimulus-responsive polymers have been the focus of increasing attention for biomedical applications [bib_ref] Block copolymer micelles for drug delivery: design, characterization and biological significance, Kataoka [/bib_ref] [bib_ref] Lessons from nature: stimuli-responsive polymers and their biomedical applications, Jeong [/bib_ref] [bib_ref] Stimuli responsive polymers for biomedical applications, Alarcon [/bib_ref] [bib_ref] Shape-changing polymer assemblies, Grubbs [/bib_ref] [bib_ref] Functional block copolymer assemblies responsive to tumor and intracellular microenvironments for site-specific..., Ge [/bib_ref] [bib_ref] Reversible Inside− Out Micellization of pH-responsive and Water-Soluble Vesicles Based on Polypeptide..., Rodríguez-Hernández [/bib_ref]. For example, Okano and co-workers designed substrates based on thermo-responsive poly(N-isopropylacrylamide)-based hydrogels for the formation of two-dimensional cell sheets, which can be readily detached from culture dishes below the low critical solution temperature for transplantation [bib_ref] Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces, Okano [/bib_ref] [bib_ref] Corneal rerconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium, Nishida [/bib_ref]. More recently, Yang et al. [bib_ref] Mechanical memory and dosing influence stem cell fate, Yang [/bib_ref] reported the control of hMSC on poly(ethylene glycol) hydrogel, whose elastic modulus can be lowered from 10 kPa-2 kPa by UV illumination.
In our previous study, we utilized a copolymer hydrogel that exhibits a substantial change in mechanical properties in response to subtle changes in solution pH [bib_ref] Live cell tracking of symmetry break in actin cytoskeleton triggered by abrupt..., Inoue [/bib_ref] [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref]. This copolymer gel comprises a highly biocompatible, pH-responsive, physically cross-linked ABA-type triblock copolymer [A = poly(2-(diisopropylamino)ethyl-methacrylate), (PDPA) and B = poly(2-(methacryloyloxy)ethyl-phosphorylcholine), (PMPC)] [fig_ref] Figure 1: Schematic illustration of stimulus responsive substrates [/fig_ref]. The central PMPC block (for which the mean degree of polymerization n, is 250) confers excellent biocompatibility [bib_ref] Synthesis and characterization of biocompatible thermo-responsive gelators based on ABA triblock copolymers, Li [/bib_ref] [bib_ref] Phosphorylcholine-based polymers and their use in the prevention of biofouling, Lewis [/bib_ref] [bib_ref] Phosphorylcholine-containing polymers for biomedical applications, Iwasaki [/bib_ref] [bib_ref] Biocompatible wound dressings based on chemically degradable triblock copolymer hydrogels, Madsen [/bib_ref] , while the mean degree of ionization of the two outer PDPA blocks (for which n = 50 for each block) changes significantly at around neutral pH [bib_ref] Synthesis of biocompatible, stimuli-responsive, physical gels based on ABA triblock copolymers, Ma [/bib_ref]. We reported that the Young's modulus, E, can be modulated reversibly from 1.4 kPa-40 kPa by adjusting the pH between 7.0 and 8.0 [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref] [bib_ref] Fine Adjustment of Interfacial Potential between pH-Responsive Hydrogels and Cell-Sized Particles, Monzel [/bib_ref] , this is an ideal range since it encompasses the gel stiffness of various tissues. The kinetics of elasticity modulation is determined by that of pH titration, which is in order of minutes [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref]. Mouse myoblast cells (C2C12) repeatedly undergo transition between contractile and hemi-spherical morphologies, demonstrating that such pH-responsive hydrogels enable appropriate dynamic mechanical cues, which are applied to cells [bib_ref] Live cell tracking of symmetry break in actin cytoskeleton triggered by abrupt..., Inoue [/bib_ref] [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref]. In this study, we utilized PDPA-PMPC-PDPA copolymer gels as a novel tool to manipulate the stiffness of environmental substrates, hence providing dynamic mechanical cues to the hMSCs as surrogate niche models, with subsequent modification of the stem cell fate. By avoiding covalent coupling of adhesion motifs (such as type I collagen) [bib_ref] Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal..., Salasznyk [/bib_ref] [bib_ref] Growth of mesenchymal stem cells on electrospun type I collagen nanofibers, Tsai [/bib_ref] , we minimize the adhesion-induced interference with the fate of hMSCs and address the following important questions: Can PDPA-PMPC-PDPA gels sustain the multiple lineage potential of hMSCs? How do hMSCs sense the dynamic stiffening/softening of gel substrates? Is it possible to influence the fate of hMSCs by applying periodic mechanical stresses?
# Results
Morphological Response of hMSCs to Surrogate Elasticity (ex situ). [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] ,b represent the phase contrast images obtained for hMSCs placed on "stiff (pH 8.0, E = 40 kPa)" and "soft (pH 7.2, E = 2 kPa)" PDPA-PMPC-PDPA copolymer gels, respectively. hMSCs became anisotropically spread on stiff gels after 10 d. In fact, when the actin cytoskeleton was labeled with phalloidin-Alexa Fluor 488 after fixation, a pronounced stress fiber formation was observed [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref]. In contrast, hMSCs spreading on the soft copolymer gel was much more isotropic, and no distinct order of stress fibers could be identified [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref].
The difference in cell morphology can be further assessed by calculating the nematic order parameter of actin cytoskeletons, θ = S cos 2 with the aid of an elongated Laplace function of a Gaussian filter to obtain maximum response [bib_ref] Optimal matrix rigidity for stress-fibre polarization in stem cells, Zemel [/bib_ref] [bib_ref] Live cell tracking of symmetry break in actin cytoskeleton triggered by abrupt..., Inoue [/bib_ref] [bib_ref] Morphology and adhesion strength of myoblast cells on photocurable gelatin under native..., Yoshikawa [/bib_ref]. As presented in the insets shown in [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] ,d, hMSCs on the stiff copolymer gel exhibit a much higher order parameter, = . S 0 33 stiff , than on the soft copolymer gel, = . S 0 06 soft . Previous reports demonstrated that hMSCs adapt their shape and cytoskeletal order in response to the elasticity of the underlying substrate, and hence commit to the specific lineage via such mechanical cues [bib_ref] Matrix elasticity directs stem cell lineage specification, Engler [/bib_ref] [bib_ref] Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but..., Winer [/bib_ref]. Thus, the observation of a clear difference in cell morphology [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] led to the next question. Do hMSCs cultured on stiff and soft substrates differentiate into the terminal lineage? In other words: does the difference in morphology coincide with the lineage commitment?
Maintenance of hMSC STRO-1 Expression is Independent from Substrate Elasticity. [fig_ref] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness [/fig_ref] represents the flow of 4 × series of experiments to unravel the influence of substrate elasticity on the fate of hMSCs. hMSCs categorized as Type A were cultured always on substrates whose elasticity was always kept "stiff " (E = 40 kPa), while Type B hMSCs were cultured always on soft substrates (E = 2 kPa). Type C hMSCs were first cultured on soft substrates (E = 2 kPa) for 10 d, and the elasticity was switched to E = 40 kPa (stiff). In case of Type D hMSCs, the substrate elasticity was switched from "stiff " to "soft" at t = 10 d. Prior to the experiments, we confirmed that pH modulation did not interfere with the viability (SI [fig_ref] Figure 1: Schematic illustration of stimulus responsive substrates [/fig_ref] and morphology (SI [fig_ref] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness [/fig_ref] of hMSCs. [fig_ref] Figure 1: Schematic illustration of stimulus responsive substrates [/fig_ref] represents the immunofluorescence images of Type A hMSCs (3b1') and Type B hMSCs (3b1") at t = 20 d, labeled with antibody to a mesenchymal stem cell marker STRO-1 [bib_ref] The effect of mesenchymal stem cell shape on the maintenance of multipotency, Zhang [/bib_ref] [bib_ref] Identification of stromal cell precursors in human bone marrow by a novel..., Simmons [/bib_ref]. A substantial majority (~90%) of the hMSCs out of approx. 40 cells cultured on both stiff and soft substrates exhibited positive STRO-1 signals. This is in contrast to the same hMSCs cultured on plastic dishes, where we found the fraction of cells exhibiting positive STRO-1 signals decreased to 15-20% already within 10 d (SI [fig_ref] Figure 6: Dynamic morphological response of hMSCs to mechanical stresses over time [/fig_ref] , which agrees well with the previous account [bib_ref] Identification of stromal cell precursors in human bone marrow by a novel..., Simmons [/bib_ref]. To further verify the multiple lineage potential of hMSCs cultured on PDPA-PMPC-PDPA copolymer gels, the media were exchanged to induction media for osteogenesis and adipogenesis at t = 20 d. After hMSCs were further cultured for 28 d, hMSCs were subjected to the labeling with Oil Red O (ORO) for adipogenesis [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] and Alizarin Red S (ARS) for osteogenesis [fig_ref] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness [/fig_ref] , respectively. Both Type A hMSCs and Type B hMSCs showed positive signals for ORO and ARS, suggesting hMSCs retain multiple lineage capability irrespective of the substrate stiffness. The Type C and Type D hMSCs showed the same tendency for the anti STRO-1 labeling, adipogenesis and osteogenesis (SI [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref]. It should be noted that the size of lipid droplets in hMSCs cultured on hydrogel substrates [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] seemed smaller, and the number of droplets in cells seemed much less compared to those on confluent hMSCs cultured on plastic dishes (SI [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref]. Different from conventional culture conditions of hMSCs, we modulated pH using HEPES-buffered medium and seeded hMSCs at a density (10 4 cell/cm 2 ). The former is essential because pH modulation between 7.2 and 8.0 is not possible by using commonly used hydrogen carbonate buffer. The latter condition was selected because the main focus of the study is to elucidate the mechanical response of individual hMSCs. In order to understand if such a difference potentially be attributed to the influence of culture media (hydrogen carbonate buffer vs. HEPES buffer), we first compared hMSCs cultured on plastic dishes in two different media. Lipid droplets in hMSCs cultured in hydrogen carbonate-buffered medium (SI [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref] were much more pronounced compared to those in hMSCs cultured in HEPES-buffered medium (SI [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref] , suggesting that the use of HEPES caused a poor lipid droplet formation. Moreover, we found that the decrease in cell density resulted in a clear decrease in the lipid droplet formation in both media (SI [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref] , which agrees well with the previous report by McBeath et al. [bib_ref] Cell Shape, Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment, Mcbeath [/bib_ref]. Thus, it has been concluded that an apparently poorer lipid droplet formation on gels [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] was caused by the use of HEPES buffer as well as the low cell density. In case of osteogenetic induction, the formation of calcinated extracellular matrix could be identified by positive ARS signals for hMSC cultured on both stiff and soft hydrogel substrates [fig_ref] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness [/fig_ref]. Although ARS signals in SI [fig_ref] Figure 5: Morphological phenotypes of hMSCs [/fig_ref] also seemed weaker compared to the signal intensity from hMSCs cultured on plastic dishes kept in the same induction medium (SI [fig_ref] Figure 5: Morphological phenotypes of hMSCs [/fig_ref] , a negative control experiment of the same hMSC in growth medium (SI [fig_ref] Figure 5: Morphological phenotypes of hMSCs [/fig_ref] confirmed the specificity of the labeling. Based on the above mentioned results, we concluded that hMSCs cultured on PDPA-PMPC-PDPA copolymer gels sustained the capability to undergo both adipogenesis and osteogenesis after t = 20 d, independent from the substrate elasticity.
The next question we wanted to address if the efficiency of chemical induction is influenced by the mechanical environment. As presented in [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref] , 26 ± 11% of Type A hMSC (approx. 50 cells for each condition) exhibited positive ORO signals, while the corresponding value for Type B hMSC was 38 ± 4%. When hMSCs experienced a change in substrate elasticity at t = 10 d, the fractions of cells showing positive ORO signals were found to be 24 ± 8% for Type C hMSCs and 30 ± 14% for Type D hMSCs, respectively. Despite of relatively large errors in [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref] , the statistical analysis (Wilcoxon test) suggest that the adipogenic induction efficiencies of Type B and Type D hMSCs were distinctly higher compared to those of Type A and Type C hMSCs. This suggests that Type C and Type D can readopt to their new mechanical environments and cancel the mechanical memory of initial substrates. It should be noted that the fraction of hMSCs undergoing osteogenesis could not be quantified in case of osteogenic induction, because ARS identifies calcium compounds of the extracellular matrix but not osteogenic cells. Our finding clearly differs from the previous account [bib_ref] Matrix elasticity directs stem cell lineage specification, Engler [/bib_ref] , which reported that hMSCs commit to terminal differentiation after just 14 d. Currently, this apparent discrepancy can be attributed to the lack of collagen type I on the surface. In fact, several studies suggested that type I collagen and vitronectin on the surface could bias the MSC lineage towards osteogenesis [bib_ref] Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal..., Salasznyk [/bib_ref] [bib_ref] Growth of mesenchymal stem cells on electrospun type I collagen nanofibers, Tsai [/bib_ref] [bib_ref] Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM..., Chastain [/bib_ref]. In contrast, the surface of copolymer gel substrates was coated by fibronetin physisorbed from the cell culture medium [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref] [bib_ref] Live cell tracking of symmetry break in actin cytoskeleton triggered by abrupt..., Inoue [/bib_ref].
## Reversible morphological dynamics of hmscs to mechanical stresses (in situ).
The fact that the hMSCs could undergo multiple lineage commitments even after 20 d [fig_ref] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness [/fig_ref] suggests that the morphology is not the decisive parameter that direct the fate of hMSCs. As presented in [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref] , hMSCs were able to adopt their shape in response to the change in substrate elasticity and cancel the mechanical memory of initial environments after 10 d. To identify the characteristic morphological patterns, the morphology of Types A-D hMSCs was categorized by plotting the circularity γ π = A L (4 )/( ) projected p erimeter 2 versus aspect ratio AR, which is the length/width ratio defined by an ellipsoidal fit at t = 20 d [fig_ref] Figure 5: Morphological phenotypes of hMSCs [/fig_ref]. The γ-AR map of Type A hMSCs attains a maximum γ value of ~0.6 at AR = 1-5. This main group (89%) is accompanied with a very broad tail towards larger aspect ratios (AR ~ 20), suggesting that the cells were anisotropically stretched. On the other hand, AR values observed for Type B hMSCs are concentrated within a narrower range (AR = 1-3), showing widely scattered γ values. This pattern covering all Type B hMSCs coincides with the isotropic spreading of hMSCs on soft gels, with extended spiky protrusions (filopodia). Once the substrate stiffness is switched from "soft" to "stiff " at t = 10 d (Type C), the morphological pattern and the order parameter at t = 20 d appear to be very similar to that of Type A cells: the value of actin order parameter for the Type A does not differ significantly from Type C <S A > = 0.33 ± 0.17 and <S C > = 0.36 ± 0.12 (SI [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref]. In contrast, the morphology of Type D hMSCs (experiencing the change in substrate stiffness from "stiff " to soft" at t = 10 d) becomes almost identical to that of Type B, which can also be characterized by a very low order parameter, <S B > = 0.02 ± 0.18 and <S D > = 0.18 ± 0.18 (n > 9 cells). The values of the actin order parameter for the Type B and Type D are not significantly different (SI [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref]. In summary, these results indicate that hMSCs retain their ability to adopt not only the nematic order of actin stress fibers [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] as well as their morphological patterns in both directions ("soft to stiff " and "stiff to soft") after 10 d.
In a subsequent step, dynamic changes in morphological patterns of hMSC induced by the change in substrate stiffness were monitored over time. [fig_ref] Figure 6: Dynamic morphological response of hMSCs to mechanical stresses over time [/fig_ref] represents the histograms of aspect ratio AR measured at t = 10 d (red), 16 d (green), and 20 d (blue) for the four aforementioned four types of hMSCs. Type A hMSCs exhibit three characteristic peaks at AR ~ 3, 7, and 12 over time (Pattern A), while Type B hMSCs are characterized by a single peak near AR ~ 1 (Pattern B). When hMSCs experience a change in the substrate stiffness from "soft-to-stiff " (Type C), hMSCs undergo a transition from Pattern B (soft) to Pattern A (stiff) over time. On the other hand, AR of Type D hMSCs changed from Pattern A to Pattern B. If one generalizes the morphological transition of hMSCs upon an abrupt change in the substrate elasticity as a non-equilibrium relaxation process, the characteristic time constants in both directions were estimated from the change in intensity of the principal peaks with the smallest AR to be τ = 3-4 d. The hMSC response to such an "elasticity jump" appears to be much slower than that of myoblasts C2C12 (τ = 10-30 min) [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref] [bib_ref] Live cell tracking of symmetry break in actin cytoskeleton triggered by abrupt..., Inoue [/bib_ref]. The projected area of hMSCs (n = 30 cells, t = 10 d), A hMSC (soft) = (1.8 ± 0.9) × 10 4 μm 2 and A hMSC (stiff) = (1.1 ± 0.7) × 10 4 μm 2 , was about two orders of magnitude larger than that of C2C12 [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref]. This is the first demonstration of the reversible switching of STRO-1 positive hMSC morphology, which offers a unique advantage to understand the mechanism regulating the dynamics of morphological response and remodeling actin cytoskeletons. This is of particular interest, not only for maintaining hMSC multiple lineage potential, but also for optimization of the hMSC-based feeder layers for the expansion of human hematopoietic [bib_ref] Molecular and Secretory Profiles of Human Mesenchymal Stromal Cells and Their Abilities..., Wagner [/bib_ref]. From this context, a recent study indicated that the cytoskeletal protein nestin may play a critical role in the maintenance of stemness of mouse HSC in the bone marrow niche 47 .
## Frequent mechanical stresses suppress proliferation of hmscs. consideration of the above results
naturally leads to the following two questions. Is it possible to influence the fate of hMSCs by applying periodic mechanical stresses? Is there a characteristic frequency that is particularly effective for influencing cell fate? To address these fundamental questions, we examined the incorporation of bromodeoxyuridine (BrdU) into the DNA of hMSCs subjected to mechanical jumps of different frequencies. [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] -d depict fluorescence images of hMSCs that were fixed and stained with 4′ ,6-diamidino-2-phenylindole (DAPI, blue) and anti-BrdU (magenta) after overnight incubation with BrdU at t = 20 d. Comparing the proliferative behavior of hMSCs experiencing no change on a stiff gel substrate (Type A, [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] , one change at t = 10 d (Type C, [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] , nine changes at t = 2, 4, 6, 8, 10, 12, 14, 16 and 18 d [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] , and hMSCs cultured on a plastic dish (control, [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] , it is clear that the fraction of proliferating cells at t = 20 d exhibits a dramatic reduction as the frequency of mechanical switching f −1 is increased. As summarized in [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] , the fraction of proliferating cells χ plotted as a function of the duration of one mechanical step (and thus f −1 ) at t = 10 d (blue) and 20 d (red) shows a non-linear behavior. The resulting χ vs. f −1 relationship can be fitted with an empirical Hill equation [bib_ref] The possible effects of the aggregation of the molecules of haemoglobin on..., Hill [/bib_ref] : where χ max and χ min stand for the maximum and minimum values of χ, − f half 1 = 6.5 d for the characteristic duration of the transition between proliferative and no-proliferative cells, and n ~ 7 for the cooperativity at t = 10 d. We found that a frequent mechanical stress applied to hMSCs can suppress proliferation by approximately 90 ± 5% after maintaining hMSCs on copolymer gel substrates for 10 d. Moreover, the suppression of proliferation was still prominent even after 20 d, where we found 85 ± 9% of cells remained non-proliferative.
[formula] χ χ χ χ = + − − − − − f f f ( ) 1 ( / ) ,(1) [/formula]
# Discussion
There have been an increasing number of papers reporting the vital roles of mechanical properties of stem cell niche in directing the fate of stem cells. For example, Katayama et al. [bib_ref] Signals from the Sympathetic Nervous System Regulate Hematopoietic Stem Cell Egress from..., Katayama [/bib_ref] reported that the sympathetic nervous system induced the stiffening and flattening of osteoblasts in bone marrow, which regulates the egress of hematopoietic stem cells. Cameron et al. [bib_ref] The influence of substrate creep on mesenchymal stem cell behaviour and phenotype, Cameron [/bib_ref] demonstrated that the substrates possessing a high loss modulus (viscosity) can keep the contractility of actin cytoskeleton low. More recently, Zhang et al. [bib_ref] The effect of mesenchymal stem cell shape on the maintenance of multipotency, Zhang [/bib_ref] reported that the multipotency of hMSC is sustained if the contractility of actomyosin is kept low. However, despite major achievements in the last decade, most of experimental studies have been performed on gel substrates whose elasticity was fixed as a function of the degree of chemical cross-linking. This raised the issue if such in vitro niche models are biologically relevant, as cellular micro-environments are known to be highly dynamic. Recently, Guvendiren et al. [bib_ref] Stiffening hydrogels to probe short-and long-term cellular responses to dynamic mechanics, Guvendiren [/bib_ref] prepared hydrogels with tunable elasticity by functionalizing hyaluronic acid with methacrylic anhydride (HA-MA). Such gels became dynamically stiffened (from E = 3-30 kPa) after injection of dithiothreitol (DTT) by cross-linking the pendent MA groups via radical polymerization under UV irradiation. After functionalization of surfaces with RGD motifs, they found that adipogenesis is favored for later stiffening (t > 8 d) while osteogenesis is preferred for earlier stiffening (t < 8 d).
Within this context, PDPA-PMPC-PDPA copolymer gels offer a unique advantage over the above mentioned materials owing to its capability of switching the elasticity in a bidirectional reversible manner. As the hMSC marker, we utilized the surface marker STRO-1, as Simmons et al. [bib_ref] Identification of stromal cell precursors in human bone marrow by a novel..., Simmons [/bib_ref] reported that colony forming unit factor (CFU-F) is present exclusively in STRO-1 positive populations. Other authors have confirmed that STRO-1 could be used to sort hMSCs that possess multiple lineage potentials [bib_ref] The STRO-1+ Marrow Cell Population Is Multipotential, Dennis [/bib_ref]. On the other hand, it has been reported that the fraction of STRO-1 positive hMSCs significantly decreased down to 25% when the cells were cultured on plastic dishes for 10 d 43 . Therefore, although STRO-1 expression level does not necessarily coincide with the level of multipotency, it is remarkable that about 90% of the hMSCs exhibited positive signals for STRO-1 at t = = 20 d both on stiff and soft substrates [fig_ref] Figure 1: Schematic illustration of stimulus responsive substrates [/fig_ref]. In fact, the percentages of STRO-1+ cells found on gel substrates were far beyond of the corresponding level on plastic dishes (15-20%, SI [fig_ref] Figure 6: Dynamic morphological response of hMSCs to mechanical stresses over time [/fig_ref]. In the next step, we evaluated the functional parameters of multipotency of the hMSCs on copolymer gel substrates by biochemically inducing adipogenesis and osteogenesis at t = 20 d [fig_ref] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness [/fig_ref]. After hMSCs were cultured in induction media for 28 d, we found that hMSCs could undergo both adipogenesis [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref]. The apparently weaker ORO signals (for adipogenesis) could be attributed to the use of HEPES buffer necessary for pH modulation as well as to the low surface density of cells (10 4 cells/cm 2 ) necessary for the identification of single cell response. The maintenance of multiple lineage potentials over 20 d is in striking contrast to previous studies, which showed that hMSCs were committed to specific lineages after incubation for 14 d on cross-linked polyacrylamide gel substrates with fixed elasticity [bib_ref] Matrix elasticity directs stem cell lineage specification, Engler [/bib_ref] [bib_ref] Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but..., Winer [/bib_ref]. Previously, Winer et al. [bib_ref] Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but..., Winer [/bib_ref] suggested that hMSCs remain "quiescent" on very soft polyacrylamide gels (E = 250 Pa) functionalized by covalent immobilization of collagen type I and fibronectin. They demonstrated adipogenesis on soft gels, but osteogenesis could only be induced after hMSCs were transferred onto a glass substrate which has about 7 orders of magnitude larger Young's modulus. The maintenance of multiple lineage potentials of hMSCs on PDPA-PMPC-PDPA gels over 20 d could be attributed to a decrease in the interference with the MSC lineage by avoiding the covalent immobilization of collagen type I or vitronectin, which tend to interfere with the lineage towards osteogenesis [bib_ref] Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal..., Salasznyk [/bib_ref] [bib_ref] Growth of mesenchymal stem cells on electrospun type I collagen nanofibers, Tsai [/bib_ref] [bib_ref] Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM..., Chastain [/bib_ref].
As presented in [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref] , the fraction of Type C hMSCs showing positive adipogenetic ORO signals was very similar to that of Type A hMSCs, and the levels in Type B and Type D hMSCs were also comparable. The fact that the efficiency of adipogenesis does not depend on the original substrate elasticity at t = 0 d suggests that hMSCs would lose the mechanical memory of their contact substrates once they are cultured on either stiff or soft substrates for 10 d. The plot of circularity γ vs. aspect ratio AR [fig_ref] Figure 5: Morphological phenotypes of hMSCs [/fig_ref] enables one to discriminate the morphological phenotypes of Type A and Type B hMSCs. As presented in [fig_ref] Figure 5: Morphological phenotypes of hMSCs [/fig_ref] , the about 70% of Type C hMSCs can be categorized in the pattern of Type A hMSCs, while more than 90% of Type D hMScs are in the pattern of Type B, respectively. Moreover, it has been demonstrated that the nematic order parameter of actin stress fibers exhibited the same tendency (SI [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] as the morphological phenotypes. From the results presented in [fig_ref] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells [/fig_ref] , we therefore concluded that 10 d would be sufficient for hMSCs to adopt their morphology and cytoskeletal order, which finally determines their lineage commitment. Maintenance of multiple lineage potentials on PDPA-PMPC-PDPA copolymer gels found both on stiff and soft substrates clearly indicates that the substrate elasticity alone may not be sufficient to determine whether hMSCs retain multiple lineage potential or undergo terminal differentiation.
To further investigate the characteristic response time for hMSCs to adopt their morphology to an abrupt change in substrate elasticity, we have plotted the change in aspect ratio AR as a function of time [fig_ref] Figure 6: Dynamic morphological response of hMSCs to mechanical stresses over time [/fig_ref]. From the change in intensity of the principal peaks with the smallest AR, the characteristic time constants of τ = 3-4 d could be obtained for both Type C (soft to stiff) and Type D (stiff to soft). Recently, Yang et al. [bib_ref] Mechanical memory and dosing influence stem cell fate, Yang [/bib_ref] utilized poly(ethylene glycol)-based gels with a photodegradable cross-linker, and examined the effect of substrate softening on the expression of transcription factors YAP/TAZ in hMSCs. When hMSCs were seeded on stiff gel substrates (E ~ 10 kPa), these transcription factors were localized in the cell nucleus already within 1 d. Upon softening of substrates by UV irradiation (E ~ 2 kPa), the location of YAP/TAZ was shifted to the cytosol of hMSCs. Interestingly, such changes in transcription disappeared if hMSCs were cultured on stiff substrates for more than 7 d. Although they could change the substrate elasticity only in one direction (stiff to soft), the observed plasticity in the expression of transcription factors seemed to agree with our experimental finding: the level of adipogenetic lineage does not depend on the initial substrate elasticity at t = 0 d, but on the elasticity of last 10 d [fig_ref] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28... [/fig_ref].
Finally, we investigated whether the proliferation of hMSCs could be influenced by applying periodic mechanical stresses. As presented in [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] , an increase in the frequency f of mechanical stress, i.e. a decrease in the duration of a mechanical step f −1 , led to a monotonic decrease in the fraction of proliferating stem cells, labeled with anti-BrdU [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref]. In fact, periodic mechanical stresses at high frequency (f −1 = 2 d) significantly suppressed the proliferation: only 15 ± 9% of hMSCs proliferated even after 20 d. This is distinctly different from the results from Types A and B hMSCs, where the fraction of BrdU positive cells was 25 ± 3.7%. The fitting of proliferation levels with an empirical Hill's equation [fig_ref] Figure 1: Schematic illustration of stimulus responsive substrates [/fig_ref] yields the characteristic duration of a mechanical step causing the suppression of hMSC proliferation, f −1 = 6.5 d. Although the readouts are different, this is comparable to the critical time windows for the plasticity of adipogenesis vs. osteogenesis reported by the UV-induced stiffening (8 d) [bib_ref] Stiffening hydrogels to probe short-and long-term cellular responses to dynamic mechanics, Guvendiren [/bib_ref] and the location of YAP/TAZ expression by the UV-induced softening (7 d) [bib_ref] Mechanical memory and dosing influence stem cell fate, Yang [/bib_ref]. Though there have been several studies that utilize the expansion and contraction of silicon rubber substrates for osteogenic lineage [bib_ref] Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via..., Simmons [/bib_ref] [bib_ref] Osteogenic Differentiation of Human Mesenchymal Stem Cells in Collagen Matrices: Effect of..., Sumanasinghe [/bib_ref] , this is the first quantitative demonstration of the existence of a characteristic frequency of elasticity changes beyond which hMSCs suppress their proliferation.
The use of biocompatible, stimulus-responsive synthetic copolymer gels as in vitro surrogate substrates has enabled us to apply periodic mechanical stresses at high frequencies, which eventually push hMSCs into a non-proliferating state without losing their multiple lineage potentials. Note that the adhesion of hMSC to PDPA-PMPC-PDPA gel substrates was solely mediated through non-covalently anchored serum fibronectin and the switching of substrate elasticity was triggered by pH modulation that does not interfere with the cell viability. Since the copolymer substrates have no reactive unhydride side chains or covalently coupled adhesion motifs (e.g. collagen type I or RGD motifs) and do not require UV irradiation that might damage stem cells, the in vitro niche model based on PDPA-PMPC-PDPA copolymers can provide hMSCs with not only a dynamic but also a biologically safe micro-environment for expansion: Our findings have the potential to solve some fundamental problems in clinical applications of cultured stem and progenitor cells. Expansion of stem cells such as MSCs upon long-term culture in vitro is confounded by ill-defined factors that invariably induce a profound impairment on the functional integrity of the cell products, as evidenced by replicative senescence, and loss of differentiation potentials after incubation on plastic dishes [bib_ref] The effect of mesenchymal stem cell shape on the maintenance of multipotency, Zhang [/bib_ref] [bib_ref] Mesenchymal Stem Cells for Bone Repair and Metabolic Bone Diseases, Undale [/bib_ref] [bib_ref] Aging of mesenchymal stem cell in vitro, Bonab [/bib_ref] [bib_ref] Replicative Senescence of Mesenchymal Stem Cells: A Continuous and Organized Process, Wagner [/bib_ref]. The ablitiy to suppress the proliferation of hMSCs without losing their multiple lineage potential might enable us to synchronize differentiation of stem cells to a specific target lineage by providing a specific biochemical induction cue at a defined time point.
# Methods
Preparation of Surrogate Substrates. The PDPA 50 -PMPC 250 -PDPA 50 triblock copolymer (M n = 60,500; M w /M n = 1.43) was synthesized by atom transfer radical polymerization (ATRP) as reported previously [bib_ref] Biocompatible wound dressings based on chemically degradable triblock copolymer hydrogels, Madsen [/bib_ref] [bib_ref] Synthesis of biocompatible, stimuli-responsive, physical gels based on ABA triblock copolymers, Ma [/bib_ref]. Glass cover slides were cleaned according to a modified RCA method [bib_ref] Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels, Yoshikawa [/bib_ref] and glued into a cell culture dish (Ø 35 mm). After spin-coating and annealing of substrates with a methanolic solution of PDPA 50 -PMPC 250 -PDPA 50 , residual methanol was removed by immersing in the culture medium (DMEM low glucose, HEPES buffered, 10% FCS). The elastic modulus of PDPA 50 -PMPC 250 -PDPA 50 copolymer gels measured as a function of pH was presented in our previous report hMSCs on Stimulus-Responsive Hydrogels. Human mesenchymal stem cells (hMSCs) were isolated and cultured as described before [bib_ref] The heterogeneity of human mesenchymal stem cell preparations-Evidence from simultaneous analysis of..., Wagner [/bib_ref]. Briefly, bone marrow from healthy donors for allogeneic transplantation was taken after written consent using guidelines approved by the Ehtic Committee on the Use of Human Subjects at the University of Heidelberg. The mononuclear cell fraction was isolated by density gradient centrifugation and seeded in plastic culture flasks at a density of 100,000 (MNC)/cm 2 in MSCGM TM (Mesenchymal Stem Cell Growth Medium, Lonza, Basel, Switzerland). After 10-14 days evolving colonies were separated and MSC further expanded. Cells of early passages were seeded on copolymer gels. Since the proliferation rate of hMSCs was very low on hydrogel substrates [fig_ref] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation [/fig_ref] , a seeding density of 10 4 cell/cm 2 was used to monitor the morphological response of single cells. To modulate pH, we used a culture medium of DMEM low glucose medium (Sigma-Aldrich, Schnelldorf, Germany) buffered with HEPES, supplemented with 10% FCS (PAA-GE-Healthcare, Munich, Germany), 1% L-glutamine and penicillin/streptomycin (100 U/ml) (Sigma-Aldrich, Schnelldorf, Germany), and adjusted pH using either 1 M NaOH aqueous solution or 1 M HCl, respectively. Cells first adhered on stiff gels (E = 40 kPa), and the substrate elasticity was adjusted according to the experimental flow presented in [fig_ref] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness [/fig_ref]. Throughout the study, this time point was defined as t = 0 d. hMSCs were cultured at 37 °C in a humidified atmosphere and the culture medium was exchanged every second day.
Immunocytochemistry. STRO-1 was labeled with FITC-conjugated anti-STRO-1 (BioLegend) for 1 h at room temperature. Actin filaments were stained with Alexa Fluor 488 Phalloidin (Invitrogen), and cell nuclei were stained with DAPI (Sigma). For osteogenic induction, hMSCs were cultured in osteogenic induction medium, supplemented with dexamethason (100 nM), L-ascorbic acid-2-PO 4 (200 μM), and β -glycerophosphate. The adipogenic induction medium was supplemented with dexamethason (1.0 mM), 1-methyl-3-isobutylxanthine (0.50 mM), and insulin (10 mg/L). The differentiated hMSCs were stained with alizarin red S for osteogenesis and oil red O for adipogenesis [bib_ref] Replicative Senescence of Mesenchymal Stem Cells: A Continuous and Organized Process, Wagner [/bib_ref] [bib_ref] Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue,..., Wagner [/bib_ref]. For quantification of adipogenic cells the stained cells with oil red and total cell numbers were counted [bib_ref] Comprehensive characterization of four different populations of human mesenchymal stem cells as..., Li [/bib_ref]. To determine the fraction of proliferating hMSC, the cells were incubated with BrdU reagent overnight. After cell fixation with 4% (w/v) paraformaldehyde, cells were permeabilized with 0.5% Triton X100 and incubated with Cy5-conjugated anti-BrdU antibody for 1 h, while cell nuclei were stained with DAPI. Images of DAPI and Cy5-BrdU channels were overlaid and the fraction of BrdU-positive cells was calculated manually.
## Calculation of order parameters of cytoskeletons.
The pixel orientational map where each orientational angle is coded with different colors was obtained by image analysis with a series of elongated Laplace functions of Gaussian filters [bib_ref] Optimal matrix rigidity for stress-fibre polarization in stem cells, Zemel [/bib_ref] [bib_ref] Live cell tracking of symmetry break in actin cytoskeleton triggered by abrupt..., Inoue [/bib_ref] [bib_ref] Morphology and adhesion strength of myoblast cells on photocurable gelatin under native..., Yoshikawa [/bib_ref]. Order parameters were determined from the histogram of the pixel numbers multiplied by the corresponding pixel fluorescence intensities at each orientation, which reflects the amounts of actin fibers at each orientation. Shape Analysis. Morphological response of MSCs to mechanical stimuli was monitored using an inverted microscope (TE-2000U, Nikon, Düsseldorf, Germany). Cell images were recorded with a CCD camera (A602f, Basler, Ahrensburg, Germany) using 4 × air objective with phase L (N.A. 0.13, Nikon). Shape analysis of MSCs, aspect ratio (ratio between major and minor axis) and circularity (4π A projected )/(L perimeter ) 2 ) determination was done using ImageJ software.
Statistics. Each data point corresponds to means ± standard deviations from more than five independent experiments. The number of cells taken for the data analysis is stated in the figure captions. The statistical significance was evaluated by the Wilcoxon test, which is indicated in the figures.
[fig] Figure 1: Schematic illustration of stimulus responsive substrates. Chemical structure of the pH sensitive triblock copolymer (PDPA-PMPC-PDPA) and the changes within the micellar structure induced by the pH change. pH titration between 7.2 and 8.0 enables one to reversibly switch the elastic modulus by a factor of 20. [/fig]
[fig] Figure 2: Morphological response of bone marrow-derived, human mesenchymal stem cells (hMSCs) on PDPA 50 -PMPC 250 -PDPA50 copolymer gels. Phase contrast images of hMSCs at t = 10 d on (a) stiff (E = 40 kPa) and (b) soft (E = 2 kPa) substrates. Fluorescence images of phalloidin-labeled actin (green) at t = 10 d on (c) stiff (E = 40 kPa) and (d) soft (E = 2 kPa) substrates. The order parameters <S> of actin cytoskeletons can be extracted from the pixel orientation maps calculated from the original images are presented as insets. [/fig]
[fig] Figure 3: hMSCs sustains multipotency after 20 d independent from substrate stiffness. (a) Flow of experiments: Type A; substrate elasticity was always kept "stiff " (E = 40 kPa) for 20 d, Type B; substrate elasticity was always kept "soft" (E = 2 kPa) for 20 d, Type C; substrate elasticity was switched from "soft" to "stiff " at t = 10 d, Type D; substrate elasticity was switched from "stiff " to "soft" at t = 10 d.(b) Fluorescence images of hMSCs always cultured on stiff substrates (Type A) and soft substrates (Type B). Labels: (b1) anti-STRO-1, (b2) Oil Red O (ORO), and (b3) Alizarin Red S (ARS). Scientific RepoRts | 6:24264 | DOI: 10.1038/srep24264 [/fig]
[fig] Figure 4: Fraction of ORO positive hMSCs cultured in adipogenic induction medium for 28 d. Prior to induction hMSCs were cultured for 20 days on hydrogel substrates as illustrated in Fig. 3a. Significance levels p < 0.05 by Wilcoxon-test. [/fig]
[fig] Figure 5: Morphological phenotypes of hMSCs. (a-d) Plot of circularity γ vs. aspect ratio AR for Type A-Type D hMSCs at t = 20 d. Representative fluorescence images (actin: green, nucleus: blue) are presented in insets. Each data set represents from n > 30 cells. Morphological populations were grouped by two ellipses characteristic for Type A hMSCs (89%) and Type B hMSCs (100% [/fig]
[fig] Figure 6: Dynamic morphological response of hMSCs to mechanical stresses over time. (a-d) Change in aspect ratio (AR) of hMSCs vs. time for four types of hMSCs (Type A-Type D). Each histogram was extracted from n > 30 cells. Phase contrast images of representative cells for the four types (Type A-D) at t = 20 d are presented as insets. The cell contour was highlighted in yellow. Scientific RepoRts | 6:24264 | DOI: 10.1038/srep24264 [/fig]
[fig] Figure 7: Impact of the frequency of mechanical stress f −1 on hMSC proliferation. (a-d) Dual staining with DAPI (blue) and anti-BrdU (magenta) of hMSCs (t = 20 d) experiencing: (a) no change (Type A), (b) f −1 = 10 d (Type C), (c) f −1 = 2 d. (d) hMSCs cultured on plastic dishes (control). (e) Fractions of anti-BrdU positive (proliferating) cells χ plotted as function of duration of a mechanical step f −1 at t = 10 d (blue) and 20 d (red) exhibiting a non-linear relationship between χ and f −1 . Each data point represents mean values ± SD for n > 30 cells. [/fig]
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Analysis of Single Nucleotide Polymorphism in Adolescent Idiopathic Scoliosis in Korea: For Personalized Treatment
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Purpose: The incidence of adolescent idiopathic scoliosis (AIS) has rapidly increased, and with it, physician consultations and expenditures (about one and a half times) in the last 5 years. Recent etiological studies reveal that AIS is a complex genetic disorder that results from the interaction of multiple gene loci and the environment. For personalized treatment of AIS, a tool that can accurately measure the progression of Cobb's angle would be of great use. Gene analysis utilizing single nucleotide polymorphism (SNP) has been developed as a diagnostic tool for use in Caucasians but not Koreans. Therefore, we attempted to reveal AIS-related genes and their relevance in Koreans, exploring the potential use of gene analysis as a diagnostic tool for personalized treatment of AIS therein. Materials and Methods: A total of 68 Korean AIS and 35 age-and sex-matched, healthy adolescents were enrolled in this study and were examined for 10 candidate scoliosis gene SNPs. Results: This study revealed that the SNPs of rs2449539 in lysosomal-associated transmembrane protein 4 beta (LAPTM4B) and rs5742612 in upstream and insulin-like growth factor 1 (IGF1) were associated with both susceptibility to and curve severity in AIS. The results suggested that both LAPTM4B and IGF1 genes were important in AIS predisposition and progression. Conclusion: Thus, on the basis of this study, if more SNPs or candidate genes are studied in a larger population in Korea, personalized treatment of Korean AIS patients might become a possibility.
# Introduction
Adolescent idiopathic scoliosis (AIS) is characterized by a structural lateral curvature of the spine with a rotary component deviation in otherwise healthy individuals. When defined as a Cobb's angle of at least 10°, AIS is the most common pediatric spinal deformity, with reports of incidences ranging from 0.5% to 10% worldwide. [bib_ref] Adolescent idiopathic scoliosis, Weinstein [/bib_ref] A report published in Korea revealed a scoliosis prevalence of 3.26% among 1134890 school children, higher than those of other countries. [bib_ref] Idiopathic scoliosis in Korean schoolchildren: a prospective screening study of over 1..., Suh [/bib_ref] According to a notification released by the Korea Health Insurance Review and Assessment Service, or exercise only often improve Cobb's angle. In some cases, progression of deformity occurs rapidly, and physicians and their patients may miss the proper timing of surgery. To solve this problem, a more accurate diagnostic tool is needed to determine whether or not deterioration will occur.
A prognostic test with a sufficient negative predictive value (NPV) can alter the monitoring schedule of a patient. Moreover, such test can reveal patients whose Cobb's angle will not undergo serious progression. Accordingly, low risk patients with a definite NPV might be spared the long-term negative effects of repeated exposure to radiation, unnecessary treatments, emotional stress, cost of direct care (physician's fee, hospital charge) and indirect cost of care, such as time away from a patient's studies and hospital visit expenses. [bib_ref] Cancer risks following diagnostic and therapeutic radiation exposure in children, Kleinerman [/bib_ref] In those who might undergo serious progression, however, it is important that AIS does not get worse. An effective prognostic test would allow physicians to develop the number of scoliosis patients and expenditures related to its treatment increased by 12.2% and 40.3% over the past five years, respectively.AIS is usually noticed while bathing or during physical activity by a child's parents. Sometimes, early detection of AIS occurs during periodic school screening upon discovery of a waist or rib hump. [bib_ref] Screening school children for scoliosis, Renshaw [/bib_ref] The Adams forward bending test is helpful in detecting a thoracic rib hump or lateralized lifting of the buttock, but exact diagnosis is difficult. A hump and/or the Adams forward bending test are sensitive but not specific. Upon suspicion of AIS, radiographs can be used to confirm the diagnosis.
Clinical features and radiographic findings suggestive of minimal or mild idiopathic scoliosis do not adequately predict the future progression of the deformity. Even mild idiopathic scoliosis has been found to progress, so that regular medical assessment over a period of years has become necessary to detect and assess progression of the deformity. [bib_ref] Adolescent idiopathic scoliosis, Weinstein [/bib_ref] [bib_ref] Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes, Sharma [/bib_ref] The current standards of care suggest that children and teens with AIS should be monitored until they cease growing, usually with serial radiographs, for curve progression. If serial radiographs confirm progression of Cobb's angle (20<Cobb's angle<45), a brace should be administered as the next step in the primary treatment thereof. Approximately 8% to 10% of all patients with adolescent idiopathic scoliosis are prescribed a brace at some point during their treatment. If the spinal curve continues to progress (Cobb >45), spinal fusion is often recommended.
Brace treatment comprising wearing of a brace for a minimum of 15 hours per day is considered effective. However, practically, it is difficult to ensure that adolescents wear a brace 15 hours a day. [bib_ref] Brace wear control of curve progression in adolescent idiopathic scoliosis, Katz [/bib_ref] In order to overcome non compliance, electronic monitoring of these individuals is now followed in certain places. [bib_ref] Electronic monitoring of scoliosis brace wear compliance, Rahman [/bib_ref] Due to difficulties faced during brace treatment, a prognostic method is direly needed to assess whether the deformity will progress or not. Such methods could help clinicians plan the treatment schedule of AIS on an individual and more effective manner.
Factors known to predict curve progression include maturity, curve size, and position of the curve apex. Progression equations have also been developed to quantify the risk of progression. Peterson and Nachemson 9 used a method combining Risser sign, level of apex, presence of trunk imbalance, and chronological age to the aforementioned list of factors to predict curve progression. Although this method might be of some help to clinicians and patients, some patients do not follow this equation, and a short period brace Cobb's of 12.5 degrees. She was kept under observation but the Cobb's angle deteriorated to 27 degrees. Therefore, she was treated with brace and exercise. After 2 years and 4 months of non-surgical treatment, Cobb's angle improved to 11.4 degrees. (B) Female patient (8 years 5 month) when first diagnosed, had 27.5 degrees Cobb's and was treated with brace and exercise but the Cobb's angle deteriorated to 140 degrees. Finally, her scoliosis and pulmonary functions deteriorated enough to cause serious danger to her life.
## A b
nese than Caucasians. Therefore, from a database of Japanese SNPs, we selected SNPs for study with a minor allele frequency of more than 5%. Other controversial SNPs in Asian populations were also included for confirmation. Common SNPs in the genes of matrilin 1 (MATN1) and insulin-like growth factor 1 (IGF1) are reported to be associated with AIS in Chinese, but studies to uncover candidate genes or marker SNPs for AIS in Koreans have yet to be performed. Furthermore, only one paper has reported on a relationship between AIS and osteoporosis in Korea, [bib_ref] Polymorphism in vitamin D receptor is associated with bone mineral density in..., Suh [/bib_ref] and the lack of data has limited the ability of physicians in Korea to personalize treatment of AIS, unlike Caucasian populations. The objective of this study was to reveal AIS-related genes and their relevance in Koreans, exploring the potential use of gene analysis as a diagnostic tool for personalized treatment of AIS therein.
# Materials and methods
Prior to conducting our study, we received Institutional Review Board and Clinical Research Ethics Committee approval of our study protocols. A total of 68 Korean adolescents (8-18 years of age), newly diagnosed with AIS at the authors' institution, in addition to 35 age-and sex-matched, healthy adolescents were enrolled in our study after applying the exclusion criteria detailed below. Some of the healthy controls comprised students from the school where the authors conducted the study. The following candidates were excluded: those with a history of congenital anomalies, neuromuscular diseases, endocrine disorders, skeletal dysplasia or connective tissue disorders, or mental retardation or mental illness, as well as psychiatric patients on medication af-new clinical protocols for managing high risk AIS patients.
Genetic associations in AIS have been described for decades.Recent etiologic studies revealed that AIS is a complex genetic disorder that results from the interaction of multiple gene loci and the environment. [bib_ref] Top theories for the etiopathogenesis of adolescent idiopathic scoliosis, Wang [/bib_ref] [bib_ref] Adolescent idiopathic scoliosis and genetic testing, Ogilvie [/bib_ref] Linkage studies of these families revealed multiple potential genetic loci, chromosomes 6, 9, 16 and 17, that may predispose individuals to the condition. 14 Several genes such as bone morphogenetic protein 4, interleukin-6, leptin, matrix metalloproteinase-3, melatonin 1B receptor (MTNR1B), etc., have been shown to be associated with a combinatorial effect.Recently, Ward insisted that by using 53 gene marker SNPs known to be associated with AIS, a NPV greater than 99% could be obtained.Ward, et al.also reported that these 53 SNPs were closely associated with AIS severity. The odds ratios for 5 of these SNPs (rs2449539, rs1437480, rs448013, rs10493083, rs16945692) with severe scoliosis ranged from 0.35 to 1.28, as shown in [fig_ref] Table 1: Selected SNP Markers in This Study CHL1, close homolog of L1 [/fig_ref]. Based on their study, a diagnostic kit was developed that could predict the progression of Cobb's angle and was sold commercially. Subsequently, several studies were carried out in Oriental countries concerning AIS candidate genes and SNP markers thereof. [bib_ref] The association analysis of TBX6 polymorphism with susceptibility to congenital scoliosis in..., Fei [/bib_ref] Sharma, et al. [bib_ref] Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes, Sharma [/bib_ref] genotyped the SNPs of an additional chromosome, 3p26.3, and tested replication in two follow-up case-control cohorts, obtaining stronger results when all three cohorts were combined (rs10510181 odds ratio of 1.49). In addition, other significant associations in their genome-wide association study (GWAS) were discovered for SNPs (rs2222973) in the Down syndrome cell adhesion molecule (DSCAM) gene, which encodes for an axon guidance protein in the same structural class.
Ethnically, Koreans are much more similar to the Japa- teocalcin and serum carboxy-terminal collagen crosslinks (CTX) levels in heparinized plasma were measured using a solid-phase, two-site chemiluminescent enzyme-labeled immunometric assay (Immulite Osteocalcin, Diagnostic Product Corporation, Los Angeles, CA, USA). Additionally, serum alkaline phosphatase (ALP) by radio immune essay (RIA) (Tandem-R Ostase, Beckman Coulter, Fullerton, CA, USA) and serum 25(OH)D3 levels by RIA using insulin-depleted saline (Immunodiagnostic Systems Limited, Boldon, UK) were measured. [bib_ref] Polymorphism in vitamin D receptor is associated with bone mineral density in..., Suh [/bib_ref] The intra-assay and inter-assay variability for 25(OH)D3 were below 10%. 17
## Snp marker selection
Reviewing many papers, we selected for study the most significant SNPs documented in the most recent genome-wide association studies, as well as other important studies. Two SNP markers (rs10510181, rs2222973) selected from Sharma, et al.'s paper, 5 SNP markers (rs2449539, rs1437480, rs448013, rs10493083, rs16945692) from Ward, et al.'s paper, and 3 SNP markers (rs1149048, rs4753426, rs5742612) from the aforementioned Chinese paper were selected.
# Genotyping method
Genotypes were screened via single base primer extension assay using an ABI PRISM SNaPShot Multiplex kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's recommendations. Briefly, genomic DNA flanking the SNPs of interest was amplified via polymerase chain reaction (PCR) with forward and reverse primer pairs [fig_ref] Table 2: Primer Sets and Tm for the SNaPshot Assay [/fig_ref] and standard PCR reagents in a 10 microliter reaction volume, containing 10 ng of genomic DNA, 0.5 pM of each oligonucleotide primer, 1 microliter of 10X PCR buffer, 250 M dNTP (2.5 mM each) and 0.25 units of i-StarTaq DNA Polymerase (5 unit/µL) (iNtRON Biotechnology, Seongnam, Korea). The PCR conditions were as follows: 10 min at 95°C for 1 cycle, and 35 cycles at 95°C for 30s, 60°C for 1 min, 72°C for 1 min, followed by 1 cycle of 72°C for 10 min. After amplification, the PCR products were treated with 1 unit each of shrimp alkaline phosphatase (SAP) (USB Corporation, Cleveland, OH, USA) and exonuclease I (USB Corporation, Cleveland, OH, USA) at 37°C for 75 minutes and 72°C for 15 minutes to purify the amplified products. One micro liter of the purified amplification products were added to a SNaPshot Multiplex Ready reaction mixture containing 0.15 pmols of genotyping primer for primer extension reaction. The primer extension reaction was carried out for 25 cycles at 96°C for 10 seconds, fecting bone metabolism. From the 68 patients in the scoliosis group, 33 patients with a Cobb's angle between 10-40 degrees allowing for non-surgical treatment were subdivided into a lower risk group, while 35 patients whose Cobb's angle was more than 40 degrees were divided into the high risk group as they required surgery.
## Evaluation of scoliosis angle
Normal standing postero-anterior radiographs were taken for each AIS patient upon their first visit. Standard techniques for measuring Cobb's angle were used, and if more than one curve was discovered, the most severe curve was selected for measurement. A Cobb's angle of less than 10 degrees was considered normal.
## Anthropometric measurements
Anthropometric measurements were done about body height and weight. Calculation of height for patients was corrected by use of Bjure's formula [Log y=0.011x-0.177, where y is the loss of trunk height (cm) due to a deformed spine and x is the greatest Cobb's angle of the primary curve]. [bib_ref] Polymorphism in vitamin D receptor is associated with bone mineral density in..., Suh [/bib_ref] [bib_ref] Abnormal peri-pubertal anthropometric measurements and growth pattern in adolescent idiopathic scoliosis: a..., Cheung [/bib_ref] Calculation of body mass indices (BMIs) was done by dividing weight (kg) by uncorrected height squared (m 2 ). 17
## Dual-energy x-ray absorptiometry
Lumbar spinal bone mineral density (LSBMD) and femoral neck BMD (FNBMD) of the non-dominant proximal femur were measured by dual-energy X-ray absorptiometry (XR-36; Norland Corp., Fort Atkinson, WI, USA). LSB-MDs were measured at L1-L4 in the anterior-posterior view. [bib_ref] Polymorphism in vitamin D receptor is associated with bone mineral density in..., Suh [/bib_ref] There was no difficulty in measuring BMD in the control group and lower risk group, but severely rotated vertebrae made the measurements difficult in the high risk group. To minimize this problem, each spine of the high risk group was pre-scanned once, after which a reference line was drawn to join the highest points of the iliac crests, which usually passed between the third and fourth lumbar spinous processes. On that reference line, a rectangle was erected to include L2-L4, and this was defined as the scan area. [bib_ref] Polymorphism in vitamin D receptor is associated with bone mineral density in..., Suh [/bib_ref] As the standardized T score for adolescents has not yet been exactly defined in Korea, total bone mass was used for bone mineral density.
## Biochemical markers of bone turnover
Blood samples were collected between 8:00 and 10:00 a.m. after an overnight fast. Plasma and serum samples were analyzed in a routine laboratory using standard procedures. Os-priate. p-values <0.05 were considered significant for clinical parameters such as BMI.
The Hardy-Weinberg equilibrium was tested for each SNP in the patient and control groups using the chi-square test. The Hardy-Weinberg equilibrium principle states that genetic variation in a population will remain constant from one generation to the next in the absence of disturbing factors. Frequency distributions of genotypes in patients and controls were compared using the chi-square test for each SNP studied. One-way ANOVA was used to compare Cobb's angles among different genotypes. Inter-group comparisons were made using t-test, a p-value of <0.01 was considered significant in genotype or allele comparisons.
# Results
In this study, the following three groups were compared: 35 subjects in the control group, 33 patients in the lower risk 50°C for 5 seconds, and 60°C for 30 seconds. The reaction products were then treated with 1 unit of SAP at 37°C for 1 hour and 72°C 15 minutes to remove excess fluorescent dye terminators. One microliter of the final reaction samples containing the extension products was then added to 9 microliters of Hi-Di formamide (ABI, Foster City, CA, USA). The mixture was incubated at 95°C for 5 min, followed by 5 min on ice, and then analyzed by electrophoresis in an ABI Prism 3730xl DNA analyzer. Analysis was carried out using Gene Mapper software (version 4.0; Applied Biosystems, Foster City, CA, USA). [fig_ref] Table 3: Baseline Characteristics of the Subjects CTX, carboxy-terminal collagen crosslinks [/fig_ref] shows the primer sets and Tm used for the SNaPshot assay.
# Statistical analysis
Statistical analysis was performed using statistical package for social science (SPSS) software version 11.5 for Windows (SPSS, Chicago, IL, USA). Data are expressed as the mean±standard deviation. Groups were compared using ttest and analysis of variance (ANOVA), whenever appro- [fig_ref] Table 4: Frequency of Genetic Variations in Control and Scoliosis Patients SNP, single nucleotide... [/fig_ref]. No variations of statistical importance were observed in genotype frequencies and allele frequencies of the eight promoter SNPs among the lower risk group, high risk group and controls in the Korean population, when the SNPs were studied independently [fig_ref] Table 4: Frequency of Genetic Variations in Control and Scoliosis Patients SNP, single nucleotide... [/fig_ref]. The lysosomal-associated transmembrane protein 4 beta (LAPTM4B) polymorphism (rs2449539) was found to be significantly different among the controls and lower risk and high risk groups. In comparison of the three groups individually, the frequency of TT genotype was most common in the high risk group, followed by the low risk group and control group, respectively (p=0.014). In contrast, the frequency of the TC genotype was most common in the control group, followed by the low risk group and high risk group, in that order (p=0.003). In comparison between two groups (controls versus AIS patient group), the frequency of TT genotype was most common in the AIS patients group (p=0.008). Similarly, the frequency of TC genotype was most common in the control group as compared to the AIS patients group (p=0.004). These results are summarized in [fig_ref] Figure 2: Fig [/fig_ref].
Additionally, IGF1 polymorphism (rs5742612) was also found to be significantly different among the controls and the lower risk and high risk groups. In comparison of the three groups individually, the frequency of GG genotype was most common in the control group, followed by the low risk group and high risk group, in that order (p=0.010). group and 35 patients in the high risk group [fig_ref] Table 3: Baseline Characteristics of the Subjects CTX, carboxy-terminal collagen crosslinks [/fig_ref]. The mean Cobb's angle for lower risk group patients was 25.8° (SD=7.6°) and 58.8° (SD=21.0°) for high risk group patients. Age, sex, height and weight had no statistical significant difference among the three groups. BMI was 20.1 (SD=3.5) in the control group, 19.3 (SD=2.0) in the low risk group and 19.1 (SD=2.8) in the high risk group, and this mathematical variation was not statistically significant bone formation marker alkaline phosphatase (ALP) levels were 174.7 IU (SD=98.1) in the control group, 77.0 IU (SD=24.5) in the low risk group and 108.5 (SD=16.4) the in high risk group. Again, despite variation, its statistical value was questionable. Also, differences in CTX, representative of the degree of bone destruction, and osteocalcin, a bone formation marker, were found to be statistically inconclusive. Mean lumbar spine and femur neck bone mineral density (FNB-MD) in AIS patients tended to be lower than that in controls, yet the difference failed to reach statistical significance. Vitamin 25(OH)D3 levels were 19.0 ng/mL (SD=9.0) in the high risk group, 23.1 ng/mL (SD=8.2) in the lower risk group and 26.2 ng/mL (SD=8.2) in the control group, and vitamin D levels were shown to be significantly lower in the high risk group than those in the lower risk and control groups (p=0.019).
Genotypes were shown to be in Hardy-Weinberg equilibrium. The distribution of the genotypes and alleles of the cases and controls for the ten promoter SNPs are presented (rs2449539) (p=0.002).
# Discussion
In Korea, AIS patients make about 100000 visits to physicians annually, thereby incurring an expenditure of millions of dollars or even more, and this expenditure has increased steeply over the past five years. Clinicians and patients need to be aware of the risk of curve progression as Between the two groups (controls versus AIS patient group), the frequency of GG genotype was most common in the control group (p=0.006). However, there was no significant difference in allele frequencies for one SNP (rs5742612) (p=0.059).
As shown in [fig_ref] Table 4: Frequency of Genetic Variations in Control and Scoliosis Patients SNP, single nucleotide... [/fig_ref] , two SNPs at rs2449539 (p=0.003) and rs5742612 (p=0.006) were found to exhibit significant associations with AIS in. The other 8 SNPs demonstrated no such significant associations. There was a significant difference in allele frequencies for one SNP kit has been validated for whites only. Thus, Korean specific SNP tests should be developed to personalize the treatment of AIS therein.
In this study, the authors selected a control group that was age-and sex-matched, so that age and sex did not differ among the three groups compared. BMI was 20.1 (SD=3.5) in the control group, 19.3 (SD=2.0) in the lower risk group, 19.1 (SD=2.8) in the high risk group and arithmetically low in the AIS patient group (lower+high risk group), but this difference was not statistically significant. Several authors reported that scoliotic girls had lower mean weight and a lower bone mineral status compared to those in the control population. [bib_ref] Anthropometry and body composition profile of girls with nonsurgically treated adolescent idiopathic..., Barrios [/bib_ref] [bib_ref] Abnormal leptin bioavailability in adolescent idiopathic scoliosis: an important new finding, Liu [/bib_ref] Leptin along with soluble leptin receptor was shown to play an important role in the regulation of bone and energy metabolism in children. Liu, et al. [bib_ref] Abnormal leptin bioavailability in adolescent idiopathic scoliosis: an important new finding, Liu [/bib_ref] insisted that leptin and soluble leptin receptor were abnormal and associated with deranged growth and anthropometric phenotypes in AIS girls. According to Liu's study, abnormality of leptin receptor leads to lower BMI, and osteoporosis is accompanied by the possibility of scoliosis. Although our study did not show any variation of significance, increasing the sample size of the study population may help in establishing any correlation that may exist between decreased BMI and AIS.
In the present study, vitamin 25(OH)D3 level was 26.2 (SD=8.2) in the control group, 23.1 (SD=8.2) in the lower risk group and 19.0 (SD=9.0) in the high risk group, the difference of which was statistically significant in the surgical-they make treatment decisions. Peterson and Nachemson's 9 progression equation might be of some help to clinicians and patients who want to base their treatment on the risk of progression. But some patients do not follow this equation, and to solve this problem, a more accurate diagnosis and diagnostic tool that can predict whether or not deterioration will occur is needed.
Though AIS is thought to be multifactorial disorder, differing degrees of interaction between multiple factors depend on linear and summation causality. Although AIS has been linked to multifactorial causes, genetic factors are considered the most important cause.
There are many reports about the supportive role of genetic inheritance in the development of AIS. A meta-analysis of different twin studies has been performed and found a concordance rate for AIS of 73% in monozygous twins versus 36% in dizygous twins.With recent advances of genetic analysis techniques, many different gene loci linked to AIS is reported by family-linkage studies. Miller, et al. [bib_ref] Identification of candidate regions for familial idiopathic scoliosis, Miller [/bib_ref] reported that gene loci in the primary regions on chromosomes 6, 9, 16, and 17 and in the secondary regions on chromosomes 1, 3, 5, 7, 8, 11, 12, and 19, after scanning 202 families and 1198 individuals, were linked to AIS in 2005. Yet, other studies failed to achieve the same results, and instead, found other candidate genes. Therefore, it is hard to establish whether the etiology of AIS is due to single gene or not. [bib_ref] Polygenic inheritance of adolescent idiopathic scoliosis: a study of extended families in..., Ward [/bib_ref] Recently, many genetic association studies with the candidate gene approach have been performed with the help of information gathered from the international HapMap project. In previous studies, the selection of candidate gene selection followed to prior knowledge of the expression of observed phenotypes in AIS and different SNPs studied with the case-only and case-control analysis. More recently, a more comprehensive explanation on the possible genes involved in the etiopathogenesis of AIS has been presented by GWASs of all SNPs in the genome. In the recent report of Ward, et al.,the comparison of a GWAS of 1.8 million genetic markers was performed between 1200 AIS patients and 1500 controls. Their study found 202 markers associated to curve progression, and after further refinement, 30 markers were reported as the most useful prognostic markers for curve progression. After verifying the results of this study, they launched the development of commercially available kit with a NPV greater than 99% for AIS-PT. However, there was an important limitation to their work; genotype frequencies for several of the SNP markers used in the AIS-PT kit vary by race. Accordingly, their AIS-PT Reviewing the relation between rs2449539 SNP and the Cobb's angle, genotype TT was compared with the CC, TC type and a significant difference (p=0.0028) was found. Comparison between CC type and TT type also showed a significant difference (p=0.0071). SNP, single nucleotide polymorphism. frequencies of the remaining 4 SNPs between AIS patients and controls in the Korean population when the SNPs were studied independently. Similarly, the allele frequencies were comparable between cases and controls [fig_ref] Table 4: Frequency of Genetic Variations in Control and Scoliosis Patients SNP, single nucleotide... [/fig_ref]. Thus, rs2449539 polymorphism of the LAPTM4B gene may be important to the complex genetic etiopathogenesis of AIS. Comparing Ward's study and our study, there are lot of genetic differences between Caucasians and Asians. 5 Two Chinese papers reported the associations of rs1149048 near MATN1, rs10488682 near MTNR1B and rs4753426 near insulin-IGF1 with AIS in Chinese. [bib_ref] Promoter polymorphism of matrilin-1 gene predisposes to adolescent idiopathic scoliosis in a..., Chen [/bib_ref] However, these associations were not shown to exist in a Japanese population. [bib_ref] Lack of association between adolescent idiopathic scoliosis and previously reported single nucleotide..., Takahashi [/bib_ref] With exception of rs5742612 SNP upstream IGF1, genotype and allele frequency of two SNPs between the three groups did not differ (p from 0.145 to 0.999). We found that allele A of rs5742612 was not significantly related with a predisposition to AIS (p=0.059), but individuals with genotype GG were shown to be at a lower risk of AIS in comparison to AA and AG (p=0.006). In our study, the SNPs of rs1149048 and rs4753426 exhibited no association with AIS predisposition in Koreans, nor did we find any association for these two SNPs with curve severity. However, the rs5742612 SNP in the upstream IGF1 gene was shown to be closely related with AIS in Koreans. Our result fell somewhere between those for Chinese and Japanese populations.
There were some limitations to this study. The number of samples tested was relatively small, which diminishes the statistical power of the study and the possibility of detecting correlations. Further studies with a larger patient population are recommended. The association with other factors, such as the markers of bone metabolism and other candidate genes, should be tested.
In conclusion, this study provides evidence that the SNP rs2449539 in LAPTM4B and rs5742612 in upstream IGF1 genes were associated with both susceptibility to and curve severity in AIS. Our results suggest that LAPTM4B and IGF1 may both be important to AIS predisposition and progression. Nevertheless, expanding our database by increasing the study size so that more SNPs or candidate genes can be studied in the Korean population would prove immensely beneficial in developing a SNP marker for AIS specifically designed for the Korean population.
corresponding national agency for this indication.
Funding from the College of Medicine in Yonsei University (topic number: 6-2010-0039) was received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.
[fig] Figure 1: (A) A female patient (8 years 1 month) when first discovered in had a [/fig]
[fig] Figure 2: Fig. 2. Reviewing the relation between rs2449539 SNP and the Cobb's angle, genotype TT was compared with the CC, TC type and a significant difference (p=0.0028) was found. Comparison between CC type and TT type also showed a significant difference (p=0.0071). SNP, single nucleotide polymorphism. [/fig]
[table] Table 1: Selected SNP Markers in This Study CHL1, close homolog of L1; DSCAM, Down syndrome cell adhesion molecule; LAPTM4B, lysosomal-associated transmembrane protein 4B; FOXB1, forkhead box protein B1; CBLN4, cerebellin 4 precursor; RRAGC, ras-related GTP binding C; BRIP1, fanconi anemia group J protein; MATN1, matrilin 1, cartilage matrix protein, MTNR1B, melatonin receptor 1B; IGF1, insulin-like growth factor 1; SNP, single nucleotide polymorphism. [/table]
[table] Table 2: Primer Sets and Tm for the SNaPshot Assay [/table]
[table] Table 3: Baseline Characteristics of the Subjects CTX, carboxy-terminal collagen crosslinks; BMD, bone mineral density. * χ 2 test or t-test were used where appropriate. † Three groups: control group vs. high risk group vs. lower risk group. ‡ Two groups: control group vs. scoliosis group. § Corrected height using Bjure's formula. [/table]
[table] Table 4: Frequency of Genetic Variations in Control and Scoliosis Patients SNP, single nucleotide polymorphism; CHL1, close homolog of L1; DSCAM, Down syndrome cell adhesion molecule; LAPTM4B, lysosomal-associated transmembrane protein 4 beta; FOXB1, forkhead box protein B1; CBLN4, cerebellin 4 precursor; RRAGC, ras-related GTP binding C; BRIP1, fanconi anemia group J protein; MATN1, matrilin 1, cartilage matrix protein, MTNR1B, melatonin receptor 1B; IGF1, insulin-like growth factor 1. *Three groups: control group vs. high risk group vs. lower risk group. † Two groups: control group vs. scoliosis group. [/table]
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Drug Delivery Systems for the Oral Administration of Antimicrobial Peptides: Promising Tools to Treat Infectious Diseases
Antimicrobial peptides (AMPs) have a great potential to face the global expansion of antimicrobial resistance (AMR) associated to the development of multidrug-resistant (MDR) pathogens. AMPs are usually composed of 10-50 amino acids with a broad structural diversity and present a range of antimicrobial activities. Unfortunately, even if the oral route is the most convenient one, currently approved therapeutic AMPs are mostly administrated by the intravenous route. Thus, the development of novel drug delivery systems (DDSs) represents a promising opportunity to protect AMPs from chemical and enzymatic degradation through the gastrointestinal tract and to increase intestinal permeability leading to high bioavailability. In this review, the classification and properties as well as mechanisms of the AMPs used in infectiology are first described. Then, the different pharmaceutical forms existing in the market for oral administration are presented. Finally, the formulation technologies, including microparticle-and nanoparticle-based DDSs, used to improve the oral bioavailability of AMPs are reviewed.
# Introduction
Almost 100 years after the discovery of antibiotics, an increasing number of multidrug-resistant (MDR) pathogens are alarming. The overuse and misuse of antibiotics have led to antimicrobial resistance (AMR), which is one of the most important public health threats worldwide [bib_ref] Antimicrobial resistance: implications and costs, Dadgostar [/bib_ref] [bib_ref] Antibiotic resistance in pediatric infections: global emerging threats, predicting the near future, Romandini [/bib_ref] [bib_ref] Antimicrobial resistance is a global health emergency, Toner [/bib_ref]. AMR occurs when bacteria, viruses, or fungi develop resistance to antibiotics, antiviral, or antifungal drugs, respectively [bib_ref] Comparison of antiviral resistance across acute and chronic viral infections, Mason [/bib_ref]. At least 700,000 people die annually worldwide due to drug-resistant diseases. Worst-case projections estimate that AMR could cause 10 million deaths each year by 2050, leading to a global economic output of 100 trillion USD. AMR is a complex issue of global concern with potentially dramatic health and economic consequences. In 2015, WHO launched the Global Action Plan on AMR and the Global AMR and Use Surveillance System (GLASS). In this context, antimicrobial peptides (AMPs) provide a great potential as an alternative strategy to fight against MDR microorganisms [bib_ref] The value of antimicrobial peptides in the age of resistance, Magana [/bib_ref] [bib_ref] Antimicrobial peptides as therapeutic agents: opportunities and challenges, Mahlapuu [/bib_ref] [bib_ref] Antimicrobial peptides: new hope in the war against multidrug resistance, Mwangi [/bib_ref]. AMPs are synthetic or naturally conserved molecules found in organisms ranging from prokaryotes to humans and are part of the body's first line of defense against pathogens (bacteria, viruses, fungi, and parasites). Most of them are positively charged with hydrophobic residues with a broad spectrum of antimicrobial activities. For decades, AMPs have shown a growing interest as potential therapeutic agents. Several hundreds of AMPs are under preclinical and clinical development, nevertheless, only a few AMPs are currently approved [bib_ref] Clinical applications of antimicrobial peptides (AMPs): where do we stand now?, Divyashree [/bib_ref]. They are used for the treatment of infectious diseases like bacterial skin infections, Clostridium difficile pseudomembranous colitis, HIV infection, or Candida infections. Thus, AMPs are a promising class of molecules active against bacteria [bib_ref] Antimicrobial peptides: the Achilles' heel of antibiotic resistance?, Lewies [/bib_ref] , viruses [bib_ref] Antiviral peptides as promising therapeutic drugs, Vilas Boas [/bib_ref] , or fungi. Furthermore, they could be effective against MDR pathogens and have a potent activity against intracellular bacteria and also against the biofilms that are involved in antibiotic resistance [bib_ref] Antimicrobial peptides: new hope in the war against multidrug resistance, Mwangi [/bib_ref] [bib_ref] Antimicrobial Peptides and Cell-Penetrating Peptides for Treating Intracellular Bacterial Infections, Buccini [/bib_ref] [bib_ref] Antimicrobial and antibiofilm peptides, Somma [/bib_ref] [bib_ref] The antimicrobial peptides and their potential clinical applications, Lei [/bib_ref] [bib_ref] Antimicrobial peptides and their therapeutic potential for bacterial skin infections and wounds, Pfalzgraff [/bib_ref]. Now, for several reasons, the AMPs that are used as therapeutic agents are mainly administrated by the intravenous route rather than by the oral route. Indeed, the gastrointestinal environment affects the drug stability, solubility, and permeability across the mucosal barriers. In addition, due to their nature, AMPs have a low bioavailability and solubility, are easily degraded by proteases, and potentially present toxicity and immunogenicity, which limit their use as antimicrobial agents. However, the oral route has several advantages: easy to use, non-invasiveness, and convenience for self-administration [bib_ref] Advances in oral drug delivery for regional targeting in the gastrointestinal tract..., Hua [/bib_ref]. To minimize the drawbacks of AMPs and the oral route, the development of new drug delivery systems (DDSs) is required [bib_ref] The value of antimicrobial peptides in the age of resistance, Magana [/bib_ref] [bib_ref] Nanosystems as vehicles for the delivery of antimicrobial peptides (AMPs), Martin-Serrano [/bib_ref] [bib_ref] Nanomedicines for the delivery of antimicrobial peptides (AMPs), Teixeira [/bib_ref]. This review describes (i) the properties and activities of the AMPs that are used against microbial pathogens, (ii) their indications for oral administration, and (iii) the micro-and nano-DDSs to improve oral bioavailability for local and systemic drug delivery.
## Amps: classification and properties
The history of AMPs began in the early 1920s with the discovery by Alexander Fleming of a bacteriolytic substance in the tissues and secretions of human and animals, and in some vegetable tissues [bib_ref] Observations on a bacteriolytic substance ("lysozyme") found in secretions and tissues, Fleming [/bib_ref] [bib_ref] On a remarkable bacteriolytic element found in tissues and secretions, Fleming [/bib_ref]. The name "lysozyme" has been applied to this enzyme, and the peptidic nature of this salivary antiseptic was subsequently established. Since this discovery nearly a century ago, the interest in AMPs has continued to grow, as evidenced by the number of articles published on this topic with an average of 15,000 articles per year in the last decade. Due to the pleiotropic functions not only killing microbes but also controlling host physiological functions such as inflammation, angiogenesis, and wound healing, alternative terms for AMPs have also appeared like "host defense peptides, " "alarmins, " and even "defensins." AMPs are synthesized by virtually all living organisms, from bacteria to humans via plants. AMPs represent the first line of defense against invading pathogens, being a key part of the innate immune system. AMPs are either ribosomally synthesized oligopeptides or non-ribosomally synthesized peptides. In the latter case, peptides are assembled by multimodular enzymes designated as non-ribosomal peptide synthetases (NRPS). Several regularly updated databases such as APD3 [bib_ref] APD3: the antimicrobial peptide database as a tool for research and education, Wang [/bib_ref] , the Collection of AMPs (CAMP) [bib_ref] Collection of antimicrobial peptides database and its derivatives: applications and beyond, Waghu [/bib_ref] , the Database of Antimicrobial Activity and Structure of Peptides (DBAASP) [bib_ref] v3: database of antimicrobial/cytotoxic activity and structure of peptides as a resource..., Pirtskhalava [/bib_ref] , or the Data Repository of AMPs (DRAMP)provide information on sequences, structures, activities, or clinical status of thousands of AMPs identified so far.
Antimicrobial peptides are usually 10-50 amino acids long and lower than 10 kDa, and they contain a composition rich in cationic and hydrophobic amino acids. However, AMPs lack any consensus amino acid sequence and present a broad structural variety and range of antimicrobial activities. The diversity of AMPs causes difficulty in their classification. AMPs can be classified in numerous ways: biological sources, peptide properties, structure, or activity.
## Classification based on biological sources
The biological sources of AMPs are wide. Indeed, natural peptides have been identified in all kingdoms of life, from bacteria, fungi, plants to animals [bib_ref] Antimicrobial peptides: classification, design, application and research progress in multiple fields, Huan [/bib_ref] [bib_ref] Antimicrobial peptides (AMPs): ancient compounds that represent novel weapons in the fight..., Ageitos [/bib_ref]. Animal AMPs can be further classified into insect AMPs, amphibian AMPs, fish AMPs, reptile AMPs, and mammal AMPs (32-34) [fig_ref] FIGURE 1 |: Structural diversity of antimicrobial peptides [/fig_ref].
## Classification based on peptide properties
Antimicrobial peptides can be classified based on peptide properties such as charge, amino acid composition, hydrophobicity, and length. AMPs are oligopeptides containing a varying number of amino acids (usually 10-50 amino acids, ranging in size from 2 to 10 kDa). They can be sorted by size: short (10-24 aa), medium , and long (50-100 aa). Based on net charge, there can be cationic, neutral, and anionic peptides although most of them are cationic and display a net positive charge ranging from +2 to +13 and may contain a specific cationic domain [bib_ref] Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity..., Kumar [/bib_ref]. The cationic nature can be attributed to the presence of lysine and arginine (and sometimes histidine) residues, which allow them to interact with negatively charged bacterial cell membranes, causing the direct destabilization of the surface of membranes with pore formation and subsequent cell lysis. Based on amino acid composition, AMPs can be predominantly rich in specific amino acids such as proline (e.g., apidaecin and pyrrhocoricin), tryptophan, arginine, glycine, histidine (e.g., Histatin-5), or rare modified amino acids (e.g., nisin) [bib_ref] Antimicrobial peptides: classification, design, application and research progress in multiple fields, Huan [/bib_ref] [bib_ref] Tryptophan-rich and proline-rich antimicrobial peptides, Mishra [/bib_ref]. Based on hydrophobicity, hydrophobic, amphipathic, and hydrophilic peptides exist. Hydrophobicity governs the extent to which AMPs will be able to be partitioned into the membrane lipid bilayer. It is required for membrane permeabilization, whereas amphipathicity determines whether AMPs can be inserted into the bacterial cell membranes to form hydrophobic channels or pores.
## Classification based on structure
Antimicrobial peptides are classically divided into four categories based on their structures, including linear α-helical peptides, βsheet peptides, both α-helix and β-sheet peptides, and a linear extension structure. In addition to these four categories, a fifth referred to as the topologically complex AMPs, including various post-translational modifications (PTMs), has recently been added [bib_ref] The vast structural diversity of antimicrobial peptides, Koehbach [/bib_ref] [fig_ref] FIGURE 1 |: Structural diversity of antimicrobial peptides [/fig_ref].
The AMPs that adopt a linear α-helix structure represent the largest and best-studied group. They are predominantly found in the extracellular matrix of insects and amphibians like magainin from Xenopus laevis although some other wellknown examples are produced by mammals like the human peptide LL-37, a member of cathelicidins [bib_ref] Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity..., Kumar [/bib_ref] [bib_ref] The vast structural diversity of antimicrobial peptides, Koehbach [/bib_ref]. Some of these AMPs are unstructured in solution and undergo conformational changes upon interactions with target membranes. Amidation at the C-terminus has been shown to increase its antimicrobial activity by stabilizing α-helical conformation and by eliminating the negative charge of the carboxyl group, therefore enhancing peptide binding to negatively charged target membranes [bib_ref] The effect of amidation on the behaviour of antimicrobial peptides, Mura [/bib_ref]. AMPs in the β-family are characterized by at least a pair of two β-strands in the structure. Almost all of these AMPs contain cysteine residues forming one or more disulfide bonds, which stabilize the structure [bib_ref] The expanding scope of antimicrobial peptide structures and their modes of action, Nguyen [/bib_ref]. These peptides are therefore more structured in solution and do not undergo major structural changes in a membrane environment. The β-sheet peptides include, for example, bovine lactoferricin or human defensins. Some AMPs adopt a structure with both α-helix and β-sheet elements, such as the cis defensins superfamily [bib_ref] Convergent evolution of defensin sequence, structure and function, Shafee [/bib_ref]. The fourth category represents AMPs with a linear extension structure, which do not fold into a particular 3D structure. They often contain a high proportion of certain amino acids such as arginine, tryptophan, or proline like indolicidin [bib_ref] The vast structural diversity of antimicrobial peptides, Koehbach [/bib_ref]. A recent fifth group of AMPs has been proposed to gather AMPs with cyclic and complex topologies [bib_ref] The vast structural diversity of antimicrobial peptides, Koehbach [/bib_ref]. These peptides do not adopt a linear structure unlike AMPs belonging to the first four classes. In this group, two types of cyclic AMPs are found: "head to tail" and "head to side chain" cyclic topologies. To stabilize their structure, most of the cyclic AMPs contain disulfide bonds or thioether bridges. Plant cyclotides represent a family of backbone-cyclic AMPs with three stabilizing disulfide bonds. Lasso peptides like bacterial microcin are part of head to side chain AMPs and consist of a macrolactam ring formed between the N-terminal α-amino group and an aspartate or glutamate side chain and a linear C-terminal peptide tail [bib_ref] Lasso peptides: an intriguing class of bacterial natural products, Hegemann [/bib_ref].
## Amps for the treatment of infectious diseases
Antimicrobial peptides exhibit several mechanisms of action for an interaction with bacteria and other microorganisms such as viruses and fungi. In general, AMPs kill microorganisms by disturbing membrane integrity or by interacting with the synthesis of intracellular components such as DNA, RNA, and proteins. They can also exert a broad range of immunomodulatory activities [fig_ref] FIGURE 1 |: Structural diversity of antimicrobial peptides [/fig_ref].
## Mechanisms of action of amps
## Antibacterial activities
Membrane interaction is a key factor for the direct activity of AMPs. Many AMPs cause disruption of the physical activity of the microbial membrane and/or translocation across the membrane into the cytoplasm of bacteria to act on an intracellular target. Some of them are presented in [fig_ref] TABLE 1 |: The antimicrobial peptide [/fig_ref]. AMPs kill bacteria by disturbing membrane integrity through membrane lysis (i.e., colistin, bacitracin, daptomycin, and polymyxin B), membrane poration (i.e., gramicidin D and tyrothricin), or the inhibition of cell wall synthesis (i.e., teicoplanin and vancomycin) [fig_ref] TABLE 1 |: The antimicrobial peptide [/fig_ref]. Gram-negative and Gram-positive bacteria have molecules at the surface that confer a negative charge, allowing an electrostatic interaction with cationic peptides. Thus, teichoic acids in the cell wall of Grampositive bacteria and lipopolysaccharides in the outer membrane of Gram-negative bacteria provide additional electronegative charge to the bacterial surface. Several models have been proposed to explain the interaction of AMPs with the bacterial membrane. Thus, three possible pathways to disrupt the inner membrane have been described: (i) in the barrel-stave model, peptides can perpendicularly insert into the membrane, promoting peptide-peptide interactions thanks to the AMP's amphipathic structure and resulting in the formation of a peptide-lined transmembrane pore, (ii) concerning the toroidalpore model, the insertion of the peptides is induced by a curvature in the lipid layer, and a pore is generated by both the peptide and the phospholipid head group, and (iii) in the carpetmodel, the peptide is absorbed onto the membrane, covering the entire surface leading to the loss of the membrane integrity and the formation of micelles [fig_ref] FIGURE 1 |: Structural diversity of antimicrobial peptides [/fig_ref]. Moreover, peptides can translocate into the cytoplasm and directly inhibit the cell wall and protein synthesis, bacterial cell division, or DNA replication by interacting with specific proteins involved in the biological process [bib_ref] Antimicrobial and antibiofilm peptides, Somma [/bib_ref] [bib_ref] The antimicrobial peptides and their potential clinical applications, Lei [/bib_ref] [bib_ref] Development and challenges of antimicrobial peptides for therapeutic applications, Chen [/bib_ref] [bib_ref] Antimicrobial peptides: an emerging category of therapeutic agents, Mahlapuu [/bib_ref].
## Antiviral activities
Some AMPs may present their activities against viruses. Antiviral peptides (AVPs) can cause membrane instability by integrating into viral envelopes. Both enveloped RNA and DNA viruses can be targeted by AVPs. AVPs can (i) interact with different glycoaminoglycans present on the cell surface competing with the virus for cellular binding sites, (ii) block the viral entry into the cell, (iii) suppress the cell fusion by interfering with the activity of ATPase protein, (iv) suppress viral gene expression, or (v) interfere with the assembly process of the viruses [bib_ref] Clinical applications of antimicrobial peptides (AMPs): where do we stand now?, Divyashree [/bib_ref] [bib_ref] Antimicrobial and antibiofilm peptides, Somma [/bib_ref] [bib_ref] The antimicrobial peptides and their potential clinical applications, Lei [/bib_ref]. As examples among the AVPs approved by the FDA, the polypeptide enfuvirtide that is a membrane fusion inhibitor block virus (HIV-1) from entering the host cells and azatanavir, an inhibitor of HIV-1 proteases, preventing the maturation of the proteins needed to assemble the viral capsid, can be cited [fig_ref] TABLE 1 |: The antimicrobial peptide [/fig_ref].
## Antifungal activities
Some AMPs pass through the fungal membrane by pore formation or act on beta-glucan or chitin synthesis and others interact with the membrane and cause cell lysis of fungi. They can lead to fungi death by (i) the inhibition of DNA, RNA, or protein synthesis, (ii) induction of apoptosis, (iii) permeabilization of membrane, and (iv) inhibition of cell wall synthesis and enzyme activity [bib_ref] Antimicrobial and antibiofilm peptides, Somma [/bib_ref]. Thus, the cyclic lipopeptides (anidulafungin and caspofungin) that are used as antifungal drugs are the inhibitors of the beta-(1,3)-D-glucan synthase [fig_ref] TABLE 1 |: The antimicrobial peptide [/fig_ref].
## Immunomodulatory activities
In addition to a broad spectrum of antimicrobial activities, AMPs have anti-inflammatory and immunomodulatory properties. They are described as the effective modulators of inflammation and neutralizers of toxins. AMPs can indirectly promote pathogen clearance of the host by stimulating chemotaxis (by recruiting/activating immunocytes), immune cell differentiation, and the initiation of adaptive immunity, while also preventing harmful inflammation and sepsis by the inhibition of cytokine release and direct scavenging of bacterial endotoxins [bib_ref] Antimicrobial peptides and their therapeutic potential for bacterial skin infections and wounds, Pfalzgraff [/bib_ref] [bib_ref] Antimicrobial peptides: an emerging category of therapeutic agents, Mahlapuu [/bib_ref]. Indeed, some AMPs modulate host immunity by influencing Toll-like receptor (TLR) recognition of microbial products (i.e., the neutralization of bacterial products such as lipopolysaccharide and lipoteichoic acid to suppress inflammation) and nucleic acids released upon tissue damage to promote auto-inflammation (51) [fig_ref] FIGURE 1 |: Structural diversity of antimicrobial peptides [/fig_ref]. AMPs having immunomodulatory activities belong to the two major families: defensins and cathelicidins. Human defensins exhibit chemotactic properties and are produced by multiple cell types (i.e., neutrophils, macrophages, lymphocytes, and intestinal epithelial cells). The human cathelicidin membrane disrupting peptide LL-37 acts as a chemoattractant for monocytes, neutrophils, mast cells, and T cells [bib_ref] Antimicrobial peptides as therapeutic agents: opportunities and challenges, Mahlapuu [/bib_ref] [bib_ref] new era of antibiotics: the clinical potential of antimicrobial peptides, Browne [/bib_ref] [bib_ref] Antimicrobial peptides, Zhang [/bib_ref]. In addition, the immunomodulatory properties of several marine-derived AMPs have been demonstrated. Thus, defensins from oyster or mytilus are the AMPs acting as host defense peptides that disrupt the membrane of microbial pathogens and play a major role in immunomodulation by acting on the innate and adaptive immune response. Among the approved AMPs that are used as antibacterial agents, the glycopeptide vancomycin exhibits pharmacological activity against Gram-positive bacteria and immunomodulatory activity affecting tumor necrosis factoralpha (TNF-α) pathways. Thus, Arbabanel et al. showed that oral vancomycin can be used as an effective treatment for concomitant primary sclerosing cholangitis and inflammatory bowel disease in pediatric. Indeed, oral vancomycin-mediated disease resolution is associated with elevated peripheral TGF-β levels without alterations in Th1 or Th2 cytokine production patterns and increased regulatory T-cell levels [bib_ref] Immunomodulatory effect of vancomycin on Treg in pediatric inflammatory bowel disease and..., Abarbanel [/bib_ref].
## Advantages and limitations of amps
Antimicrobial peptides have a broad spectrum of antimicrobial activities (antibacterial, antiviral, and antifungal) and are a promising class of drugs to face the development of MDR pathogens. They have advantages over conventional antibiotics or antifungals, which include slower emergence of resistance, antibiofilm activity, and an ability to modulate the host immune response. AMPs are less immunogenic than recombinant proteins and antibodies. In addition, they are in general considered to have a safety profile because their metabolites are natural amino acids and there are having short half-life, few peptides accumulate in tissues [bib_ref] The value of antimicrobial peptides in the age of resistance, Magana [/bib_ref] [bib_ref] Clinical applications of antimicrobial peptides (AMPs): where do we stand now?, Divyashree [/bib_ref]. However, despite the beneficial properties of AMPs, they present some limitations such as: (i) short half-life because of a rapid degradation by proteolytic enzymes, both in the bloodstream and in the gastrointestinal system; (ii) plasma protein binding, which leads to their inactivation; (iii) low metabolic stability and low oral bioavailability; (iv) rapid excretion through the kidneys and liver; (v) high toxicity (i.e., nephrotoxicity) and immunogenicity; (vi) a poor correlation between in vitro antimicrobial activity and their efficacy in vivo; and (vii) high costs of production [bib_ref] Nanosystems as vehicles for the delivery of antimicrobial peptides (AMPs), Martin-Serrano [/bib_ref] [bib_ref] Nanostructured antimicrobial peptides: the last push towards clinics, Carratalá [/bib_ref] [bib_ref] Antimicrobial peptides: an emerging category of therapeutic agents, Mahlapuu [/bib_ref] [fig_ref] FIGURE 1 |: Structural diversity of antimicrobial peptides [/fig_ref]. For these reasons, their use for in vivo applications has not been fully satisfactory and to date, only a few AMPs are approved by the FDA for clinical use [fig_ref] TABLE 1 |: The antimicrobial peptide [/fig_ref]. Most of the commercialized AMPs (except colistin and polymyxin B) are used for treating Gram-positive bacterial infections, and the development of AMPs to treat Gram-negative bacterial infections is needed. Moreover, concerning the route of administration, therapeutic peptides are mostly restricted to topical administration (skin and eye infections) or to parenteral administration, while the oral route (p.o) is mostly convenient for medication adherence. Indeed, p.o provides treatment acceptability for patients and facilitates the administration with non-invasive and ambulatory treatments; it is also the main route for local treatment of the gastrointestinal tract. Accordingly, it constitutes the first investigated administration route during the pharmaceutical development of a new drug. Unfortunately, this oral administration route is most of the time not suitable for the emerging therapeutic peptides, and significant efforts to knock down the locks of peptide administration by p.o are conducted. Indeed, after oral administration, peptides are exposed to the aggressive biological environment (pH, enzymes, and gut microbiota) and a complex structure (mucus, epithelial barrier, and hepatic first pass) of the gastrointestinal tract before they have local action or reach the systemic circulation [fig_ref] FIGURE 2 |: Barriers of AMP absorption and interest of drug delivery systems by oral... [/fig_ref]. Generally, gastrointestinal degradation and low permeability lead to oral peptide bioavailability <1-2% (53). Nevertheless, this work demonstrated some limitations notably due to the administration of this AMP in drinking water that, for example, may not reproduce the pharmacokinetic of human dosing. LFF571, a new investigational thiopeptide, was synthesized to treat C. difficile infections. The aqueous solubility of this analog was enhanced in comparison with 4-aminothiazolyl analogs of the antibiotic natural product GE2270 A. Pharmacokinetic studies of the infected hamster were performed, and LFF571 solution (prepared with PEG-400 or Cremophor R EL, two solubilizing agents) was administrated at a dosage of 20 mg.kg −1 [bib_ref] Discovery of LFF571: an investigational agent for Clostridium difficile infection, Lamarche [/bib_ref]. A very low bioavailability was observed (as expecting to treat local C. difficile infection) and no detectable levels of C. difficile toxins A and B were measured in cecal content (animals were successfully treated). A randomized clinical study (phase II exploratory study in USA and Canada) was performed to compare LFF571 and vancomycin safety and efficacy [bib_ref] randomized clinical trial to compare the safety and efficacy of lff571 and..., Mullane [/bib_ref]. Moreover, a study was conducted to evaluate the safety, tolerability, and pharmacokinetic of LFF571 in healthy human volunteers [bib_ref] first-in-human, randomized, double-blind, placebo-controlled, singleand multiple-ascending oral dose study to assess the..., Ting [/bib_ref]. In both studies, LFF571 was well-tolerated in patients and in comparison with vancomycin administration, and the frequencies of serious adverse events were similar. Unfortunately, this phase II study was discontinued in 2019.
## Indication of amps after the oral administration of conventional dosage forms
Moreover, to date, a few small peptides are on the market: peptides with good stability and high potency. These peptides are administrated with conventional forms [composed of active pharmaceutical ingredient (AMPs) and excipient(s)]. They include solid dosage forms such as tablets, capsules, powders, granules, lozenges, and liquid dosage forms such as solutions, emulsions, suspensions, and syrups. For example, vancomycin is administered by the oral route for both local and systemic delivery [fig_ref] TABLE 1 |: The antimicrobial peptide [/fig_ref]. For local delivery, vancomycin is formulated in 125-and 250-mg capsules or in powder for oral solution to treat the infection of the intestinal mucosa (pseudomembranous colitis induced by C. difficile). Indeed, after oral administration, vancomycin is not usually absorbed into the blood and is excreted almost exclusively in the feces. However, absorption is reported in patients with inflammatory disorders of the intestinal mucosa. Nisin containing pectin/hydroxypropyl methyl cellulose (HPMC) compression-coated tablets was formulated for the treatment of local colon diseases such as irritable bowel syndrome, inflammatory bowel disease, and ulcerative colitis [bib_ref] Colonic delivery of compression coated nisin tablets using pectin/HPMC polymer mixture, Ugurlu [/bib_ref]. Authors propose to coat a tablet with pectin and HPMC (two hydrophilic polymers, which swell to form a hydrogel layer upon contact with aqueous media) to form an enzymatically controlled delivery system. Adding pectin limits the hydration and swelling of HPMC that allow to maintain tablet integrity and thus nisin stability. Furthermore, in vivo studies with pectin/HPMC-coated tablet formulation conducted in healthy volunteers have demonstrated an interesting interplay between the tablet position in the gut, the hydration of the matrix, and the subsequent release pattern. Thus, HPMC maintains tablet integrity until the colon [bib_ref] Scintigraphic evaluation of colon targeting pectin-HPMC tablets in healthy volunteers, Hodges [/bib_ref]. For surotomycin (CB-315), peptides were proposed to treat diarrhea with severe C. difficile infection, tablet, and age-appropriate oral solid formulation that can be dispersed or dissolved. During clinical trials phase I, an insignificant absorption of surotomycin was observed and the primary route of elimination was in the feces following oral administration [bib_ref] Pharmacokinetics of surotomycin from phase 1 single and multiple ascending dose studies..., Chandorkar [/bib_ref]. Throat lozenge pharmaceutical forms were also developed to administer tyrothricin by the oral route in the treatment of patients with acute pharyngitis. Finally, liquid forms prepared from powders were used. Indeed, teicoplanin, to treat diarrhea with severe C. difficile infection, is presented as powder for oral solution at the dosages of 100, 200, and 400 mg. During pharmacokinetic studies, when teicoplanin is administered by the oral route at a 250-or 500-mg single dosage to healthy subjects, it was demonstrated that teicoplanin was not detected in the serum or urine but only recovered in feces (about 45% of the administered dosage) as unchanged medicinal product. Colistin sulfate was administered orally as a suspension via a nasogastric tube in the gastrointestinal tract for selective digestive tract decontamination in intensive care units [bib_ref] Emergence of colistin resistance in Enterobacteriaceae after the introduction of selective digestive..., Halaby [/bib_ref].
## Systemic treatment after the oral administration of amps
Despite limited oral absorption of peptides, some of them are still administered orally and indicated for systemic bacterial or viral infections. For example, capsules (200 mg) of a HIV protease inhibitor, atazanavir (ATV), are on the market. ATV has a low oral bioavailability, which can be enhanced clinically by a co-administration with ritonavir and food [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref]. To avoid food and ritonavir co-administration uses, amorphous solid dispersion system of ATV was also prepared with sodium lauryl sulfate as a carrier and polyoxylglycerides (Gelucire R 50/13) as an absorption enhancer [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref]. ATV solid dispersion showed a 4.7-fold increase in bioavailability compared with ATV alone [bib_ref] Pharmaceutical approach to HIV protease inhibitor atazanavir for bioavailability enhancement based on..., Fukushima [/bib_ref].
Currently, vancomycin was administered by the oral route only for local C. difficile treatment. Indeed, after oral
N.D., no data; AUC, Area under the curve; SNEDDS, Self-nanoemulsifying drug delivery system; SEDDS, Self-emulsifying drug delivery system; GI, Gastrointestinal.
administration, during pharmacokinetic studies in human adults and during multiple dosing of vancomycin hydrochloride capsule at 250 mg every 8 h for seven dosages, no blood concentration was detected. So, to extend the use of vancomycin, pharmaceutical forms have been studied to improve its oral bioavailability. For example, multiple emulsion (water-in-oil-in-water) was proposed to administer vancomycin (as the model drug). The emulsion incorporating C18 unsaturated fatty acid oil (linoleic acid or linolenic acid) has improved the bioavailability more than 40% after emulsion administration into the rat colon loop (at a dosage of 5 mg.kg -1 of vancomycin) (69) [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref]. On the other hand, for emulsion incorporating C18 unsaturated fatty acid oil (oleic acid) or docosahexaenoic acid, enteral bioavailability was lower (more than 25%) [bib_ref] Enhanced enteral bioavailability of vancomycin using water-in-oil-in-water multiple emulsion incorporating highly purified..., Kajita [/bib_ref]. Similarly, the formulation of a solution with a co-administration of the surfactant as an absorption promoter [PEG-8 caprylic/capric glycerides (Labrasol R ) and D-α-tocopheryl polyethylene glycol 1,000 succinate (TPGS)] was studied [bib_ref] Enhanced intestinal absorption of vancomycin with Labrasol and Dα-tocopheryl PEG 1000 succinate..., Prasad [/bib_ref]. Vancomycin solutions were administered at a dosage of 20 mg.kg -1 into the rat ileum. Formulation containing 50% of Labrasol R and 12.5% of TPGS increased the AUC 0−6h value of the vancomycin about 2.4 times in comparison with the formulation with 50% of Labrasol R without TPGS [bib_ref] Enhanced intestinal absorption of vancomycin with Labrasol and Dα-tocopheryl PEG 1000 succinate..., Prasad [/bib_ref]. Thus, the excipients that are used in pharmaceutical forms have demonstrated their importance to improve the absorption of AMPs. In addition, the stability, biocompatibility, safety, and efficacy of AMPs can be further improved through novel formulation strategies and design of new DDSs.
## Ddss for the oral administration of amps
After oral ingestion peptides can be easily affected by an biological environment; pepsin, protease (trypsin and chymotrypsin), acidic, or neutral pH, temperature resulting in the loss of their bioactivity in the gastrointestinal tract. Moreover, after oral administration, to obtain systemic delivery, besides potential gastrointestinal degradation, peptides have also to be transport across mucosal, epithelial, and endothelial barriers [bib_ref] How to design the surface of peptide-loaded nanoparticles for efficient oral bioavailability?, Malhaire [/bib_ref]. Broadly, different approaches to update the inconvenience of conventional dosage forms were proposed to protect and/or to improve intestinal absorption of peptides. In this context, a number of approaches such as chemical modification of the peptide structure, a co-administration of absorption enhancer and/or protease inhibitor and/or mucolytic agents, or peptide encapsulation in well-adapted DDSs are proposed [bib_ref] Systemic delivery of peptides by the oral route: formulation and medicinal chemistry..., Brayden [/bib_ref] [bib_ref] Oral drug delivery systems using chemical conjugates or physical complexes, Al-Hilal [/bib_ref].
Drug delivery systems exhibit different chemical or physical properties; they can be a protected drug, modified drug pharmacokinetic, controlled drug release, and improved therapeutic efficacy with less side effects of drugs [bib_ref] Advances in oral peptide therapeutics, Drucker [/bib_ref]. Thus, these systems emerge as pioneering and promising forms to enhance therapeutic effectiveness. DDSs are particulate pharmaceutical forms such as microparticles and nanoparticles (including liposomes, polymer-and lipid-based nanoparticles, and micelles) that allow drug encapsulation [fig_ref] FIGURE 2 |: Barriers of AMP absorption and interest of drug delivery systems by oral... [/fig_ref].
## Ddss for gastrointestinal diseases
The loading of AMPs into DDS could protect peptides from enzymatic degradation after oral administration for local treatment of the gastrointestinal tract. For example, the loading of nisin into the DDS is interesting due to its loss of bioactivity after an interaction with food (inactivation by enzymatic degradation, inactivity at alkaline pH). To demonstrate the interest of encapsulation, different nanoparticles as well as microparticle system were developed [bib_ref] Dynamic encapsulation of hydrophilic nisin in hydrophobic poly (lactic acid) particles with..., Ji [/bib_ref] [bib_ref] Sustainable inhibition efficacy of liposome-encapsulated nisin on insoluble glucan-biofilm synthesis by Streptococcus..., Yamakami [/bib_ref] [bib_ref] Cationic lipid content in liposome-encapsulated nisin improves sustainable bactericidal activity against Streptococcus..., Yamakami [/bib_ref] [bib_ref] Nisin-loaded alginate-high methoxy pectin microparticles: preparation and physicochemical characterization, Khaksar [/bib_ref] [bib_ref] Production of nisin-loaded solid lipid nanoparticles for sustained antimicrobial activity, Prombutara [/bib_ref] [bib_ref] In vitro characterization and evaluation of the cytotoxicity effects of nisin and..., Goudarzi [/bib_ref] [bib_ref] Comparative study between the antibacterial effect of nisin and nisinloaded chitosan/alginate nanoparticles..., Zohri [/bib_ref] , but even if encapsulation improves the stability of nisin at alkaline pH or in the presence of enzymes, no pharmaceutical indications were described after oral administration. Thus, these different works demonstrated the interest of DDS to protect the peptide from gastrointestinal media. Similarly, the encapsulation of microcin J25 (the peptide with a bactericidal activity against a range of pathogenic enteric bacteria such as Escherichia coli and Salmonella) into the liposome coated with whey protein and pectin has protected significantly the peptide during in vitro digestion study. Finally, the efficacy of orally administered encapsulated cryptdin-2 onto chitosan nanoparticles (more than 105 nm particle size) was also demonstrated against Salmonella Typhimurium infection in mice [bib_ref] Improved oral therapeutic potential of nanoencapsulated cryptdin formulation against Salmonella infection, Rishi [/bib_ref]. The property of chitosan can modulate the intestinal behavior of nanoparticles, which increase stability and protect cryptdin-2 against gastrointestinal conditions.
## Ddss for systemic delivery
To enhance the absorption of AMPs following oral administration, microparticulate and more specifically, nanoparticulate systems introduced advanced features. Indeed, due to their size, surface charge, and/or targeting moieties on the surface, nanoparticles are expected to diffuse through the mucus layer and to transport peptides across the intestinal barrier to reach blood circulation by different pathways (mainly transcellular pathways) [bib_ref] Biopharmaceutical parameters to consider in order to alter the fate of nanocarriers..., Roger [/bib_ref]. As a consequence, a lot of nanoparticulate delivery systems are described in the literature to improve the oral bioavailability of AMPs [fig_ref] FIGURE 2 |: Barriers of AMP absorption and interest of drug delivery systems by oral... [/fig_ref].
## Self-emulsifying ddss
Self-emulsifying DDS (SEDDS) and self-nanoemulsifying DDS (SNEDDS) are defined as the isotropic mixtures of oil, surfactants, and co-solvents. They present advantages for oral drug delivery like the protection against enzymatic degradation, reduced first pass metabolism, exhibiting mucus permeating properties, and enhanced absorption [bib_ref] A game changing approach for the oral administration of hydrophilic macromolecular drugs, Mahmood [/bib_ref]. Accordingly, daptomycin was incorporated into SEDDS that was prepared with tricaprylin (Dermofeel R MCT) and mono-diglyceride of medium chain fatty acids (mainly caprylic and capric) (Capmul R MCM) as oil and PEG-40 hydrogenated castor oil (Cremophor R 40) as the surfactant. Daptomycin was studied for the treatment of complicated skin infections, bacteremia, and right-side endocarditis caused by multiresistant Gram-positive bacteria.
In vitro mucus permeation study demonstrated that SEDDS formulation improved daptomycin permeation by a factor 2 in comparison to pure daptomycin and protected daptomycin against enzymatic degradation [bib_ref] Bernkop-Schnürch A. Development and in vitro characterisation of an oral self-emulsifying delivery..., Zupančič [/bib_ref]. Similarly, SEDDS containing vancomycin, 25% of glycerol monocaprylate (Capmul R 808G), 37.5% of PEG-40 (Cremophor R R40), 13.6% of diethylene glycol monoethyl ether (Transcutol R HP), and 26.5% of dimethyl sulfoxide (DMSO) was developed. SEDDS formulation improved intestinal in vitro mucosa permeating properties, nearly 45% of vancomycin permeated the mucus in SEDDS formulation within 4 h, whereas <5% permeated with free vancomycin [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref]. Ex vivo permeation across porcine small intestinal mucosa confirmed these results (30 vs. 5% with SEDDS formulation and free vancomycin, respectively) [bib_ref] Development and in vitro evaluation of a self-emulsifying drug delivery system (SEDDS)..., Zaichik [/bib_ref]. SNEDDS formulation of ATV was also developed. Optimized formulation was prepared with glyceryl monolinoleate (Maisin TM 35-1) as oil and diethylene glycol monoethyl ether (Transcutol R P) as the surfactant. After oral administration into rats at a dosage of 7.2 mg.kg -1 of ATV, the area under the curve (AUC) of ATV was improved 2.57-fold with SNEDDS compared to pure ATV (administered in the form of 0.3% carboxymethyl cellulose suspension) (62) [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref].
## Microparticle delivery system
Microparticles (microspheres or microcapsules) are defined as particles with size larger than 1 µm. They are promising encapsulation systems for protecting peptides from degradation, enhancing peptide stability, and providing an increased surface to volume ratio for peptide release and gastrointestinal absorption [bib_ref] Microencapsulation: a promising technique for controlled drug delivery, Singh [/bib_ref]. In this way, oral polymeric microparticulate as a carrier of polymyxin B was developed. Polymyxin B, which has a potent bactericidal activity against a broad range of Gramnegative bacteria (e.g., Pseudomonas aeruginosa), is not absorbed orally. After encapsulation into crosslinked alginate/chitosan microparticles, the biological activity of polymyxin B was conserved due to microparticle stability in a gastric environment [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref] [bib_ref] Toxicity and gut associated lymphoid tissue translocation of polymyxin B orally administered..., Coppi [/bib_ref] [bib_ref] Ex-vivo evaluation of alginate microparticles for Polymyxin B oral administration, Coppi [/bib_ref] [bib_ref] Alginate microparticles for Polymyxin B Peyer's patches uptake: microparticles for antibiotic oral..., Coppi [/bib_ref]. Moreover, these microparticles demonstrated their ability to target the gut-associated lymphoid tissue (GALT) by Peyer's patch uptake, but more experiments have to be performed to demonstrate an eventual improvement of polymyxin B absorption [bib_ref] Alginate microparticles for Polymyxin B Peyer's patches uptake: microparticles for antibiotic oral..., Coppi [/bib_ref].
## Liposome delivery system
Liposomes (vesicles in which an aqueous volume is entirely surrounded by a bilayer phospholipid membrane) are the first nanoparticulate delivery systems that reached the market [bib_ref] Doxil R -The first FDA-approved nano-drug: lessons learned, Barenholz [/bib_ref]. Conventional formulations of liposomes were instable in the gastrointestinal environment, but the modification of their composition by incorporating a specific phospholipid (e.g., DSPC, DPPC, and tetraether lipids) [bib_ref] Doxil R -The first FDA-approved nano-drug: lessons learned, Barenholz [/bib_ref] [bib_ref] Stability of SUV liposomes in the presence of cholate salts and pancreatic..., Kokkona [/bib_ref] or by polymer coating (e.g., chitosan and PEG) (93, 94) enhanced their stability. Recent research on oral delivery systems has shown that liposomes can be employed to improve the bioavailability of encapsulated materials by protecting them against the chemical or enzymatic degradation environment and by improving their intestinal absorption [bib_ref] Recent advances in liposome surface modification for oral drug delivery, Nguyen [/bib_ref]. in Sprague-Dawley rats, the absolute bioavailability was 1.74, 6.7, and 21.8% from vancomycin solution, uncoated liposome, and folic acid-coated liposome, respectively. Thus, the folic acid and the liposomal formulation increase by a 12.5-fold the vancomycin bioavailability compared to vancomycin solution. In the same way, Arregui et al. [bib_ref] Daptomycin proliposomes for oral delivery: formulation, characterization, and in vivo pharmacokinetics, Arregui [/bib_ref] prepared a proliposome formulation [defined as dry, free-flowing particles coated with phospholipids, which can immediately form a liposomal suspension when in contact with water (96)] containing diacetyl phosphate and stearylamine to increase drug loading and to enhance the oral absorption of daptomycin. Pharmacokinetic studies in rats demonstrated that, after the oral administration of daptomycin at a dosage of 40 mg.kg -1 , a greater AUC 0−t (46.39 ± 5.69 µg/ml/h), and higher C max (8.35 ± 0.64 µg/ml) were observed with proliposomal compared to free drug (AUC 0−t and C max were less than the limit of quantification) [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref].
## Nanoparticle delivery system
Nanoparticles (including nanospheres and nanocapsules) are lipid-and polymer-based nanocarriers. These nanoparticle approaches have some advantages for oral administration by protecting the encapsulated drug from enzymatic degradation, facilitating mucus diffusion, and membrane permeation [bib_ref] Current status of selected oral peptide technologies in advanced preclinical development and..., Aguirre [/bib_ref]. All these systems have already demonstrated their ability to improve peptide bioavailability [bib_ref] review of advanced oral drug delivery technologies facilitating the protection and absorption..., Choonara [/bib_ref] [bib_ref] Oral delivery of diabetes peptides-comparing standard formulations incorporating functional excipients and nanotechnologies..., Lakkireddy [/bib_ref] [bib_ref] Nanoparticles: oral delivery for protein and peptide drugs, Jun [/bib_ref].
Concerning AMPs, niosomes were developed. Niosomes (lipid-based nanoparticles) are similar to liposomes, but a bilayer is formed by a non-ionic surfactant and stabilized by the addition of cholesterol. These particles show a high stability in the gastric environment and a high permeability across the intestine (100). Polymyxin B niosomes were prepared using sorbitan monostearate (Span R 60) and cholesterol. Vesicles were stable in simulated gastric and intestinal fluids, and about 86.2 and 78.5% of polymyxin B were retained, respectively [fig_ref] TABLE 2 |: Oral AMP formulations to improve bioavailability [/fig_ref]. Pharmacokinetic studies (at 2.0 mg.kg -1 dosage of polymyxin B in rats) demonstrated that polymyxin B niosome administrated orally present pharmacokinetic parameters (AUC 0−48 , t 1/2 ) similar to polymyxin B sulfate administrated intravenously [bib_ref] Bioavailability enhancement of polymyxin B with novel drug delivery: development and optimization..., Chauhan [/bib_ref].
Polymeric nanoparticles provide also the possibility to enhance oral absorption. Eudragit R RL 100 [a copolymer of poly (ethylacrylate and methyl-metacrylate)] nanoparticles were prepared by nanoprecipitation to encapsulate ATV. During in vivo pharmacokinetic studies in rats (at 7.2 mg.kg −1 dosage of ATV), the values of C max and AUC 0−24 increased to 1.1-and 2.91-fold, with pure drug and optimized nanoparticle formulation, respectively. These results demonstrate the considerable performance of the nanoparticulate DDS in enhancing the bioavailability of ATV [bib_ref] Atazanavir-loaded eudragit RL 100 nanoparticles to improve oral bioavailability: optimization and in..., Singh [/bib_ref].
# Conclusion
To date, there have been numerous studies investigating the interest of AMPs. Indeed, they display an antibiotic alternative and have already demonstrated their great potential to treat infectious diseases, including those caused by MDR strains. To be approved as therapeutic agents, AMPs must overcome their disadvantages, which limit their administration. Thus, in terms of future perspectives, one of the biggest challenges will be the development of pharmaceutical forms of the AMPs for all routes of administration and especially, oral administration, the main convenient administration route. Nevertheless, formulated with conventional forms, AMPs are always subjected to their limitations (enzymatic degradation, low bioavailability, rapid metabolism, opsonization, etc.). By this way, nano-and micro-DDSs emerge to play a critical role in determining the success of current and future products. Indeed, nanotechnology is an emerging field that offers a unique potential in comparison with conventional forms, including, for example, the protection against biological environment, the transport through barrier and cells, the improvement of bioavailability, or release modification. In this review, we have described the significance and high potential of AMPs to treat both systemic and local gastrointestinal infectious diseases as well as the oral AMP delivery systems available as innovative formulations. These new systems have to overcome the lack of specific regulatory guidelines notably for their characterizations (the absence of harmonized standard protocols), the complexity of their process of formulation and scale-up, and the uncertainties of their toxicity. They also require additional in vivo pharmacokinetic studies. In future, the encapsulation of AMPs into the DDS and their use in synergy with conventional antimicrobial drugs could be promising to obtain more effective oral treatments especially to treat MDR pathogen infections.
# Author contributions
[fig] FIGURE 1 |: Structural diversity of antimicrobial peptides (AMPs) and their activities against bacteria, viruses, or fungi. A wide variety of biological sources, including microbes, insects, amphibians, reptiles, mammals, or plants, produce AMPs, which are classified into five structural classes. Representative examples of these five classes are shown as a cartoon representation and colored by hydrophobicity [sourced from the RCSB Protein Data Bank (https://www.rcsb.org/)]: (A) α-helical structure of human LL-37 (PDB entry: 2K6O); (B) β-sheet structure of bovine lactoferricin (PDB entry: 1LFC); (C) α-helix and β-sheet structure of human beta-defensin-1 (PDB entry: 1IJV); (D) Linear extension structure of bovine indolicidin (PDB entry: 1G89); (E) Cyclic structure of Bacillus subtilis Subtilisin A (PDB entry: 1PXQ). Direct pathogen killing and immunomodulatory activities of AMPs lead to antibacterial, antiviral, and antifungal activities. AMPs' advantages and limitations to treat infectious diseases are listed. [/fig]
[fig] FIGURE 2 |: Barriers of AMP absorption and interest of drug delivery systems by oral route. Current drug delivery systems (DDS), including microparticles, nanoparticles, liposomes, and self-emulsifying drug delivery systems (SEDDS), are assessed for oral antimicrobial peptides (AMPs) administration. The encapsulation of AMPs in DDS presents advantages to avoid gastrointestinal barriers. [/fig]
[fig] FUNDING: MA was funded by NANOMED EMJMD supported by the European Union and the Erasmus+ Program by the European Union in the Framework Agreement Number 2016-2057/001-001 EMJMD, No. 574439-EPP-1-FR-EPPKA1-JMD-MOB. [/fig]
[table] TABLE 1 |: The antimicrobial peptide (AMP) drugs approved by the Food and Drug Administration (FDA). [/table]
[table] TABLE 2 |: Oral AMP formulations to improve bioavailability. [/table]
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The problem with Big Data in Translational Medicine. A review of where we've been and the possibilities ahead
a b s t r a c tUnderstanding the parallel evolutions of Big Data and Translational Medicine, and the types of disruptive technology that bring them together, requires a look back at their evolution and a discussion of the hindrances in applying big data techniques to translational medicine. We will then take a look into the future, at the concept of the "Complete Health Record" and how that may change the very nature of translational medicine.
# Introduction
It has been 15 years since the Human Genome project announced that it had sequenced 90% of the human genome. It took another three years until they announced the successful completion of the project, having sequenced 99% of the human genome . Compare that to today, where an individual's genome can be sequenced in a matter of three days [bib_ref] Illumina Expands World's Most Comprehensive Next-Generation Sequencing Portfolio, Corp [/bib_ref]. With those advances, it is now technically possible to create a patient registry enabled by a patient's Complete Health Record, combining genomic data, the vast amount of data available through Connected Medical Devices, the ever increasing number of personal fitness devices (through the Internet of Things), along with data from the electronic medical record (EMR) and other types of available patient data. One would think that the insights gained from the Complete Health Record would solve a wide range of health problems (see [fig_ref] Figure 1: The Complete Health Record -Includes all the health information about a patient,... [/fig_ref]. But here is where the problem of improving patient care through translational medicine and the problems of big data intersect: the problem is not (necessarily) in the integration of the data: as with all big data problems, the problem is one of availability and analysis, in the finding and understanding of signals, trends, causal (and not corollary) relationships, and then turning those insights into actionable information.
To understand how to overcome these issues, and the truly disruptive technology that I believe lies on the horizon, we'll approach this review in three parts: a look at the development of the concept of "big data", a discussion of the hindrances in applying big data techniques in our environment today and then a look into the future, the concept of the "Complete Health Record" and how that may change the very nature of translational medicine.
## The evolution of big data
When I started working in this industry in the late 1980s, running statistical analyses against large data sets on massively parallel
## Contents lists available at sciencedirect
Applied & Translational Genomics j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a t g computers, the concern was as much about processor speed (and number of available processors) as it was about the sheer volume of data. How long would the computing job run? When would we need to switch tapes or disks to load in more data? Consider the CrayY-MP C90 supercomputer, state of the art for crunching data in 1991, had 16 CPUs, with 2 Gb of central memory, ran at 16 Gigaflops and had 16 Gb of SSD storage (Cray. My current laptop has more raw processing power, central memory and storage than that Cray! In the late 1990s came the availability of multiple networked PC processors, referred to as "High Performance Computing (HPC)" with around the same amount of processing power of a Cray, but with hundreds of terabytes of storage and a considerably lower cost. And while organizations were busy implementing HPC clusters and networking vast amounts of data, vendors started offering Software as a Service (SaaS), which itself evolved into storing vast amounts of data in the "Cloud". The issue evolved from technical limitations of physical storage or processing power to such questions as: "How much compute power and storage space can be purchased?" and "How quickly can the system be spun up?"
And the big question: how to integrate the data. In the early 2000s, while SaaS and the Cloud were evolving, came a focus on data interoperability and integration standards. HL7. CDISC. Continuaa Alliance. SAFE BioPharma. IHE. All focused on enabling interoperability and integration. In 2006, I was evangelizing the concept of standards in conference presentations focused on encouraging organizations to embrace standards, citing the benefit "Do you have your next blockbuster drug in your discarded portfolio and don't know it?" The implication of that statement is by using standards you can have access to data, and views across data, which were previously hidden, stored in individual silos. And that once this data was made available to a wider range of researchers, additional insights could be gained, simply through the mindshare of additional researchers and the possibility of new ways of looking at data.
When it comes to these standards, we've largely arrived at a place where it is possible to realize that vision. By the time of this article in 2015, the industry has largely moved from evangelizing the need for developing standards to implementing mature standards that have been around for years, easing integration to a degree not possible just a decade ago.
## Big data and translational medicine today
And while we now have the ability to process and store massive amounts of data, and the standards and technologies that enable the integration of that data, there are still problems in Big Data in general and in applying Big Data techniques to Translational Medicine more specifically that hinder the vision of finding those critical insights.
Critics of Big Data use the oft cited phrase common to many information technology initiatives: the problem with Big Data is about the right data, delivered to the right people at the right time (Forte Wares, 2014). I've personally used this phrase for years, mostly when speaking about user experience and user interfaces, but it is equally applicable to Big Data, and especially when applied to Translational Medicine.
Let's use that paradigm to test a common application of translational medicine: the patient registry.
## Right data
Not only questions the veracity of the data, but the applicability of the data and (perhaps most importantly) the accessibility of the data.
- If the registry includes data from fitness trackers, was the data really generated by that patient? - For registries driven by electronic medical records, is the information up to date? - Is the right information captured for the type of study you are trying to recruit? - Do you have access? While you may have good EMR data sets, have you formed agreements with fitness data providers like FitBit or patient focused data aggregators such as 23AndMe or PatientsLikeMe? Have you solved the problem of correlating patients from those services with the EMR data you already have?
## Right person
When thinking about the right person, we need to think not only about the analysts themselves, but also consider the analysis technology that the analyst can use to provide insights.
- Do you have medical specialists on hand who understand all the sources of data in the registry? - Do you have technology that can pose questions in the right way, interrogating data across domains, utilizing the Complete Medical Record, and can look at relationships between genomic, fitness, medical records, and patient adherence? Across patients? Across populations?
## Right time
One of the tenets of Big Data is an acknowledgement that the underlying data is rapidly changing.
- Can you find patients to recruit for your trial, at precisely the right point in that patient's health timeline, defined by the interaction between their recent fitness, current adherence pattern, and current medical record?
o Was there a life-event that negatively impacted a patient's adherence pattern years ago, but not currently? o Have you controlled for the patient's current adherence history in the inclusion/exclusion criteria of a clinical trial?
Those are tough questions, not only showing how necessary it is to use the Complete Health Record and Big Data analysis techniques to drive insights, but also showing how difficult it is to use patient registries if the concept of the Complete Health Record is not taken into account.
## Art of the possible
Yet there are emerging capabilities and trends which show using Big Data analysis techniques against the Complete Health Record, or at least real-world data, can drive insights not possible a few years ago.
Let's consider the art of the possible, from some real world use cases that I've personally witnessed:
- A biopharmaceutical company has a drug going off patent in five years. By interrogating large EMR data sets, across geographies, with tens of millions of patient lives, other co-indications for that drug are detected through improvement in symptomology. These coindications are in heretofore unstudied therapeutic areas, completely unrelated to the original FDA approval. The improvements in symptomology are detected by the analysis, not necessarily looking for a specific symptom, but using signal detection to look across indications, across symptoms. - When planning large Phase III studies, or groups of studies, it is possible to take patient registry data, EMR data sets, and operational data from previous studies to better plan the protocol, understanding the profile of the patient AND the profile of the investigational site .In the end, this develops a better protocol, greatly enhances patient recruitment and leads to fewer protocol amendments. The end effect is saving time and money in the execution of the trial while ending up with better data for your submission. The best part: it is all done graphically, enabling the user to focus on the questions to ask the data, and not on how to format the questions or the underlying data set.
## Emerging technologies
There are examples of emerging technologies and services that indicate there may well be solutions to some of the analysis issues. Companies such as Tamr and Mark Logic are taking novel approaches to solving the right data, right person, right time problems.
Tamr (http://www.tamr.com) utilizes a curated workflow along with machine learning. A curation workflow identifies and maps data the system doesn't understand. The system then learns from that curation, applying machine learning algorithms, reducing curation intervention in subsequent data sets. Tamr offers an adapter for CDISC, enabling the conversion, validation and packaging of clinical study data into CDISC format.
Mark Logic (http://www.marklogic.com) is paving the way in Enterprise NoSQL solutions, which enable integration, storage, analysis and search across multiple, complex data-types, including both structured and unstructured data.This enables researchers to ask new questions as opposed to simply testing hypothesis.
## The drive to the complete health record
While the technology to drive insights from disparate sources and types of information, to drive the right data to (and for) the right person at the right time is certainly evolving in a positive direction, perhaps the greatest problem faced in driving insights using Big Data methodologies is the problem of availability, specifically the availability of the Complete Health Record.
Consider the different types of data enumerated in [fig_ref] Figure 1: The Complete Health Record -Includes all the health information about a patient,... [/fig_ref]. Many of those data types are available for the purposes of building patient registries: Provider Focused Electronic Medical Records (EMR), Connected Medical Devices, Genomic Information. But a vast amount of data is not available in a single place for the patient, let alone for research purposes. Nor is it certain that the patient has access to 'all' their information, even though it is "theirs". Indeed, this raises the question of ownership (which we won't deal with in this article): is the patient's data theirs or is it the physician/providers data?
From the researcher standpoint, where can they get data sets that include not just the EMR or Genomic information commonly in a patient registry, but also gain insights from patient created data, or data created from the increasing number of Connected Medical Devices, not just the devices provided by the provider or physician?
Is the notion of using the Complete Health Record in patient registries, to drive a complete view of the patient, even possible?
The possibility does exist to build patient registries and other research data sets from the Complete Health Record of a patient, but as an industry we have work to do to get us there. Many countries have created centralized medical records. In the US, we have been moving towards connecting health records, even if only regionally. But even those efforts only connect Provider Focused data and completely ignore Connected Medical Devices and patient generated data.
There are existing technologies which allow the collection of patient focused as well as provider focused data. enables patients to pull together their Complete Health Record, including data not just from provider-focused EMR and medical devices, but also pharmacy information, patient focused Connected Medical Devices, fitness information and a host of other patient focused health data (Full Disclosure: the author served as CTO for .
When provider organizations adopt this technology, integrating it with their EMR, it opens a door for greater availability of data, with the possibility of access to patient focused health data and not just provider focused health data. But that is also the downside of this approach: the requirement for the provider to enable access to their EMR, even if only through the download of an HL7 CCD (Continuity of Care Document) and the requirement for the patient to grant access to that data. Microsoft places a heavy (but necessary) emphasis on privacy, requiring the patient to opt-in to the use of their data. If the patient is unaware of the ability to use their data for research purposes, if the apps don't exist to take advantage of that patient data, then that Complete Health Record becomes useful only for the patient.
A different approach has been taken by Apple with their ResearchKit (Apple. While the details and uses of the Research Kit are emerging at the time of this article, early uses show promise. The approach taken by Apple with their ResearchKit is to enable the creation of apps specifically for collecting research data. Participating research organizations can contribute to the open source framework, create apps to gather data from patients and then publicize those apps within their target patient communities. The more patients that contribute, the richer the data. This approach has the downside of not capturing provider-focused data, nor the inability to integrate EMR data (at least as of this writing), but has the upside of gathering data from increasing numbers of patients.
What's needed is a combination of these approaches. An approach where research organizations can create apps, focused on collecting not just the individual data types they create or utilize, but apps that can access all the different types of data available to the patient, including provider-focused health data. Apps that take into account not just the immediate data-points researchers THINK are of interest, but apps that have access to the vast array of patient data, have access to the patient's Complete Health Record, the intersection of EMR, PHR, Connected Medical Devices, pharmacy information, fitness information and patient behavioral data.
These combined approaches enable the "holy grail" of Big Data in Translational Medicine: the ability to comb through data, to identify insights, enable signal detection, and find patterns that can create new and novel research questions rather than the traditional method of creating hypothesis and then generating the questions to test those hypotheses.
# Conclusion
The standards exist for integrating both provider focused and patient focused health data, for the most part. The technology exists, and more advanced technology is emerging, that enable researchers to ask out-ofthe-box questions of the data, questions that weren't envisioned when they created their initial data-sets. Platforms exist for the capturing of patient focused data and storing that data alongside provider focused EMR data.
What's needed to solve the problems with Big Data in Translational Medicine is a combination of approaches: research focused apps combined with access to a population of patients Complete Health Records, enabling greater numbers of patients to contribute and allowing researchers to have access to complete, all-encompassing health data, which will drive insights and the ability to detect patterns, rather than simply testing hypothesis, ultimately driving more timely and targeted therapies to market.
[fig] Figure 1: The Complete Health Record -Includes all the health information about a patient, not just what is in the EMR. [/fig]
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Structural, Functional and Neurochemical Cortical Brain Changes Associated with Chronic Low Back Pain
# Background
Low back pain (LBP) is defined as the musculoskeletal syndrome characterized by a set of symptoms (being the presence of pain focused on the final segment of the spine the main one) in the lumbar area, located between the lower ribs and the sacral region [bib_ref] Low back pain, Knezevic [/bib_ref]. Clinically, it can be presented as acute pain episodes if <4 weeks of duration, subacute if 4-12 weeks of duration or chronic if >3 months of duration [bib_ref] Acute and chronic low back pain, Patrick [/bib_ref]. Chronic low back pain (CLBP) has a more complex nature compared with acute episodes since cognitive, emotional, behavioral and social factors directly affect the CLBP experience [bib_ref] Risk factors for low back pain and sciatica: An umbrella review, Parreira [/bib_ref]. In fact, evidence is consistent regarding the role of anxiety, depression, catastrophism, kinesiophobia and somatization as risk factors of LBP chronification [bib_ref] Epidemiology of low back pain in adults, Manchikanti [/bib_ref].
LBP is the main cause of disability worldwide [bib_ref] Predicting transition to chronic pain, Apkarian [/bib_ref] , with 80% of the population suffering from it at some point in their lives. Most of the people can expect to be recovered in 4-6 weeks (50-80%), but more than half have the possibility of relapse in the following 12 months. In addition, up to 10-15% the pain can become chronic [bib_ref] Predicting transition to chronic pain, Apkarian [/bib_ref]. Thus, a high percentage of these patients do not respond to treatment, causing limitation in functional capacity, suffering emotional disturbances derived from the constant state of pain.
Resources dedicated to LBP management are not negligible. For instance, in Spain, +1.2 M patients seek medical attention in a 6-month period, involving an economic burden estimated at EUR 16,000 M a year (comparable with other conditions, including cancer or cardiovascular diseases) and social impact (up to 40% of working absenteeism is caused by LBP) [bib_ref] Calidad de vida en pacientes con dolor lumbar crónico, Castellamo-Tejedor [/bib_ref].
Approximately in 80-90% of the patients suffering LBP, pain is not associated with a specific injury, such as fracture, trauma, systemic diseases or root compression [bib_ref] Non-specific low back pain, Balagué [/bib_ref]. This clinical picture is defined as non-specific low back pain. The 10-20% left is associated with red flags. Red flags are characteristics of the patient's medical history and physical examination which are considered to be associated with a higher risk of serious pathology associated with patients' complaints, needing referrals for medical attention [bib_ref] The epidemiology, economic burden, and pharmacological treatment of chronic low back pain..., Juniper [/bib_ref].
It should be noted that the probability of X-ray to identify the cause of back pain is less than 1%. In a previous study conducted with LBP patients [bib_ref] Evidence against the use of lumbar spine radiography for low back pain, Bosch [/bib_ref] , no abnormalities were found in 65% of the radiographs. The prevalence of degenerative changes was high (>62%) in patients aged 55 years or older, and the prevalence of possible tumor was low (<1%). This leads us to suspect that the use of radiography in LBP results in a substantial increase in cost and a risk due to the increase in radiation to patients in relation to the benefits [bib_ref] Structural Changes of Lumbar Muscles in Non-specific Low Back Pain: A Systematic..., Goubert [/bib_ref].
Therefore, since LBP is considered the musculoskeletal condition with the highest prevalence (14.8% acute LBP and 7.7% chronic LBP), which causes a significant decrease in the patient's and family's quality of life [bib_ref] cronificación y tratamiento del dolor lumbar, Morales [/bib_ref] , the aim of this narrative review is to provide an overview of neurochemical, structural and functional changes in the brain cortex associated with LBP.
## Imaging methods for assessing cortical brain changes
Neuroplasticity (defined as "the ability of the nervous system to change its activity in response to intrinsic or extrinsic stimuli by reorganizing its structure, functions, or connections") has positive characteristics (known as adaptive), which may also become negative (maladaptive) [bib_ref] The adaptive neuroplasticity hypothesis of behavioral maintenance, Peterson [/bib_ref]. Because of these maladaptive neuroplastic changes in chronic LBP, several methods for assessing brain properties have been used and developed.
-Transcranial magnetic stimulation (TMS): Represents a painless and non-invasive technique for investigating the integrity and function of the corticospinal pathway and the primary motor cortex (M1). A magnetic body on M1 can depolarize the corticospinal cells by which, at a sufficient intensity, the stimulus produces a muscular response which is called motor evoked potential (MEP), which is recorded by electromyography electrodes. The PEM latency and amplitude are considered primary outcomes for corticospinal function testing. -Voxel-based morphometry: This brain morphological imaging method uses 3D magnetic resonance and allows the measurement of gray matter (GM) volume and morphological and structural brain changes detection. The intensity of these changes is shown to be negatively associated with the duration of pain, and the tendency in chronic LBP is toward a decrease in gray matter [bib_ref] Association between brain and low back pain, Konno [/bib_ref]. -Functional magnetic resonance imaging (fMRI): It is a non-invasive medical examination. It is based on a powerful magnetic field for observing small changes which occur in brain (particularly, changes in the cerebral blood flow). The BOLD signal is an indirect marker of neural activity. It is used to examine the functional anatomy and perform a brain mapping (determining which part of the brain is controlling essential functions). The most evaluated areas using fMRI in patients with CLBP are the primary and secondary somatosensory cortex (S1 and S2), the anterior cingulate cortex (ACC), the prefrontal cortices and the thalamus. -Arterial spin labeling: This is the fMRI technique based on measuring cerebral perfusion (uses water in arterial blood as a free diffusion marker) non-invasively. It allows the absolute quantification of regional cerebral blood flow, whose image may be more accurate than BOLD and seems to be one of the most appropriate tools for studying chronic pain experience features [bib_ref] Neural correlates of chronic low back pain measured by arterial spin labeling, Wasan [/bib_ref]. -Magnetic resonance spectroscopy: It is a non-invasive brain imaging method used for exploring metabolic concentrations in certain regions of the brain. It detects radiofrequency signals generated by the magnetic nuclear spins of magnetically active nuclei such as protons, phosphorus, carbon, and fluorine, which are excited by external magnetic fields. Some changes in the concentration of metabolites (N-acetyl-aspartate (NAA), creatine or glutamate) that have been found in several patients with chronic pain are represented, suggesting biochemical brain alterations in chronic pain. -FreeSurfer: A set of tools for neuroimaging analysis through algorithms to quantify the functional and structural properties of the brain. It automatically creates models of the macroscopically visible structures in the human brain, given a reasonable T1-weighted input image. -Diffusion-weighted (DWI) and diffusion tensor imaging (DTI): This magnetic resonance base allows the exploration of the micro and macro architecture of the brain, allowing the understanding of tissue injury in vivo, the development of white matter tracts and functional connectivity within the brain [bib_ref] Diffusion-weighted and diffusion tensor imaging of the brain, made easy, Huisman [/bib_ref]. DWI can produce multiple informative metrics at each voxel, including fractional anisotropy (the degree of diffusion restriction to reflect white matter integrity), apparent diffusion coefficient (a measure of the magnitude of diffusion of water within a tissue, and can be used in monitoring brain infarctions), axial diffusivity and radial diffusivity (diffusion rates along the main and transverse diffusion directions respectively). Among the different signal models, DTI is one of the most popular [bib_ref] Estimating Brain Connectivity with Diffusion-Weighted Magnetic Resonance Imaging: Promise and Peril, Grier [/bib_ref]. DTI provides information about the covariance structure of the molecule diffusion displacement distribution, which is related to the directionality of the water diffusion process. Therefore, this method is characterized by high sensitivity to small macro and microstructural changes in white matter tissue. The trace images provide information about the magnitude of diffusion, and the shape of the diffusion tensor may change independently from the overall size or magnitude of the diffusion tensor [bib_ref] Diffusion-weighted and diffusion tensor imaging of the brain, made easy, Huisman [/bib_ref].
## Cortical changes in chronic low back pain
Multiple mechanisms contributing to the transition from acute to chronic pain have been proposed, involving both the peripheral nervous system and the central nervous system (CNS). Although the state of the brain in chronic pain has a role yet to be clarified, it is widely accepted that we cannot think of chronic pain as an input of nociceptive stimuli into a brain that is functioning correctly. Therefore, neuroplastic remodeling can lead to the maintenance of pain over time, even in the absence of nociceptive input [bib_ref] Chronic Pain: Where the Body Meets the Brain, Crofford [/bib_ref].
Neuroimaging studies have revealed numerous structural and functional changes in the brain of those with chronic musculoskeletal pain. These changes can be broadly classified as neurochemical, structural, or functional.
## Neurochemical changes
The detection of neurochemical brain alterations by magnetic resonance spectroscopy in the presence of persistent pain allows the scientific community to detect changes associated with specific pain situations for better understanding of the underlying mechanisms, developing potential therapies and aids in the diagnosis of painful disorders. Significant changes (either increased or decreased depending on the marker) have been observed at the thalamus, dorsolateral prefrontal cand orbitofrontal cortices, being a differentiator factor between healthy subjects and patients with chronic LBP [bib_ref] Chronic Back Pain Is Associated with Decreased Prefrontal and Thalamic Gray Matter..., Apkarian [/bib_ref]. In fact, the magnitude of the changes found were directly proportional to pain intensity and duration. Although it is not possible to prove whether neurochemical changes induce chronic LBP, evidence seems consistent regarding the neurochemical profile variations induced by this condition [bib_ref] Cortical changes in chronic low back pain: Current state of the art..., Moseley [/bib_ref].
A systematic review conducted by Zhao et al. [bib_ref] Neurochemical changes in patients with chronic low back pain detected by proton..., Zhao [/bib_ref] summarized the biochemical changes in brain regions of interest and the hypothesized possible mechanisms explaining those findings. Nine studies meeting the inclusion criteria were included, recruiting a total of 135 subjects with chronic LBP and 137 healthy controls. Brain metabolites were studied through magnetic resonance spectroscopy (i.e., N-acetyl-aspartate (NAA), choline, creatine, glutamate, glutamine, gamma-aminobutyric acid (GABA) and glucose), finding significant differences between cases and controls in specific brain regions, such as the thalamus, insula, S1, dorsolateral prefrontal cortex (DLPFC), ACC and primary motor cortex (M1).
NAA is present in very high concentrations in brain neurons and has been recognized as a brain marker. This metabolite is reduced in various brain regions in patients with chronic LBP as in neurodegenerative diseases, demonstrating the correlation between neuronal loss and degeneration [bib_ref] N-acetyl aspartate: A marker for neuronal loss or mitochondrial dysfunction, Clark [/bib_ref]. As suggested by Apkarian et al. [bib_ref] Chronic Back Pain Is Associated with Decreased Prefrontal and Thalamic Gray Matter..., Apkarian [/bib_ref] , a cerebral atrophy in the thalamus, S1 and DLPFC could be related to the NAA decrease. In addition, myo-inositol (which is present in glial cells) is significantly reduced in the ACC and thalamus in patients with chronic LBP [bib_ref] 1H-MR spectroscopic detection of metabolic changes in pain processing brain regions in..., Gussew [/bib_ref].
Regarding glutamate, which is the most abundant excitatory neurotransmitter in the brain, one study showed a decrease in the ACC in patients with chronic LBP [bib_ref] 1H-MR spectroscopic detection of metabolic changes in pain processing brain regions in..., Gussew [/bib_ref]. This finding is controversial with the results reported by other studies [bib_ref] Cerebral metabolic changes and chronic back pain: Study taking into consideration clinical..., Janetzki [/bib_ref] , which through proton magnetic resonance spectroscopy, detected an increase in glutamate (which in high concentrations has an excitotoxic effect that can lead to cell death of glutamatergic neurons), which play a key role in pain processing [bib_ref] Imaging central neurochemical alterations in chronic pain with proton magnetic resonance spectroscopy, Harris [/bib_ref].
Finally, GABA is the main inhibitory neurotransmitter, and its receptors are found in the thalamus, spinal cord and cortex [bib_ref] The role of spinal cord extrasynaptic α 5 GABA A receptors in..., Delgado-Lezama [/bib_ref]. A decrease in GABA levels by selectively activating GABA(B)-receptor-bearing rostral agranaular insular cortex neurons at the insula increases pain through projections to the amygdala, while preventing its degradation by using an enzyme inhibitor or gene transfer mediated by a viral vector relieves it by enhancing the descending inhibition of spinal nociceptive neurons, suggesting its key role in the pathophysiology of some chronic pain conditions [bib_ref] Imaging central neurochemical alterations in chronic pain with proton magnetic resonance spectroscopy, Harris [/bib_ref]. In contrast, the study by Zhao et al. [bib_ref] Neurochemical changes in patients with chronic low back pain detected by proton..., Zhao [/bib_ref] found no significant changes in the GABA neurotransmitter in patients with chronic LBP compared to healthy subjects, which may be due to extremely low concentrations and the overlapping of signals from other brain metabolites.
## Structural changes
Current evidence suggests a gray matter reduction in the DLPFC, right anterior thalamus, S1, S2, posterior parietal cortex, left temporoparietal junction, superior frontal gyrus, right frontal pole and middle cingulate cortex in people with chronic LBP since results demonstrated a strong correlation between the density changes magnitude with pain intensity [bib_ref] Chronic Back Pain Is Associated with Decreased Prefrontal and Thalamic Gray Matter..., Apkarian [/bib_ref] [bib_ref] Brain structural and psychometric alterations in chronic low back pain, Ivo [/bib_ref] [bib_ref] S1 is associated with chronic low back pain: A functional and structural..., Kong [/bib_ref] [bib_ref] Multivariate classification of structural MRI data detects chronic low back pain, Ung [/bib_ref] [bib_ref] Changes in brain gray matter due to repetitive painful stimulation, Teutsch [/bib_ref] [bib_ref] Brain structural alterations associated with dysfunctional cognitive control of pain in patients..., Chehadi [/bib_ref] [bib_ref] Phenotype matters: The absence of a positive association between cortical thinning and..., Dolman [/bib_ref] [bib_ref] The brain's default mode network, Raichle [/bib_ref].
LBP cannot be understood just as an altered functional state. Chronic LBP is also a consequence of neuroplastic changes and structural brain reorganization, altering the processing of nociceptive sensory information. Apkarian et al. [bib_ref] Chronic Pain: Where the Body Meets the Brain, Crofford [/bib_ref] examined brain morphometry (through voxel-based morphometry) under chronic pain conditions and concluded that subjects with chronic LBP suffer a greater neocortical gray matter atrophy than the normal atrophy attributable to age in healthy populations.
Regarding the volume of cortical gray matter, the difference found between patients with chronic LBP compared with healthy subjects was 5.4% smaller (which is equivalent to 30 cm 3 ). It should be noted that a decrease of 0.5% per year (2.8 cm 3 ) was attributable to aging in both groups, and controlling for affect, age and medications could reduce or eliminate these GM volume alterations found in CLBP [bib_ref] Phenotype matters: The absence of a positive association between cortical thinning and..., Dolman [/bib_ref]. In addition, a local decrease in gray matter density was observed bilaterally at the DLPFC in subjects with chronic LBP compared with controls. However, there were no differences within the three regions of the DLPFC assessed in the study. This decrease in density was significantly correlated with pain-related measures (pain intensity, pain duration, sensory and negative-affective dimensions of chronic LBP), age and gender, but not with anxiety or depression [bib_ref] Chronic Back Pain Is Associated with Decreased Prefrontal and Thalamic Gray Matter..., Apkarian [/bib_ref]. Similarly, although Ivo et al. [bib_ref] Brain structural and psychometric alterations in chronic low back pain, Ivo [/bib_ref] found significant decreases in GM density at the DLPFC, thalamus and middle cingulate cortex, no correlations between structural data with anxiety or depression were found.
On the other hand, the thalamus revealed decreased gray matter in the right anterior area (which is responsible for mediating nociceptive inputs to the cortex, hypothesizing that thalamocortical processes may play an important role in the pathophysiology of chronic pain) [bib_ref] Brain structural and psychometric alterations in chronic low back pain, Ivo [/bib_ref].
The regional pattern of atrophy appears to be specific to chronic pain, as it affects regions involved in pain perception and may explain the transition from acute to chronic stage [bib_ref] Chronic Pain: Where the Body Meets the Brain, Crofford [/bib_ref]. Chehadi et al. [bib_ref] Brain structural alterations associated with dysfunctional cognitive control of pain in patients..., Chehadi [/bib_ref] using Voxel-based morphometry revealed the negative correlation between though suppression (which is a common type of cognitive control of pain) and GM volume in the left superior and left middle temporal gyri (both associated with pain intensity).
Apkarian et al. [bib_ref] Chronic Back Pain Is Associated with Decreased Prefrontal and Thalamic Gray Matter..., Apkarian [/bib_ref] also analyzed chronic LBP patients with neuropathic pain components (significant radiculopathy in the leg) and patients without neuropathic pain. The DLPFC gray matter density decrease was significantly greater in subjects with neuropathic pain (27%) compared with those subjects without the neuropathic component (14%). This may be related to the more negative and debilitating effect of neuropathic pain in this subgroup of patients with chronic LBP.
Kong et al. [bib_ref] S1 is associated with chronic low back pain: A functional and structural..., Kong [/bib_ref] performed a neuroimaging study in healthy subjects and subjects with chronic LBP seeking for morphometric and volumetric differences through T1-FreeSurfer sequence exploration. The study focused on thickness differences located at S1 (specifically, the upper third, which, according to Penfield's sensory homunculus, captures the cortical representation of the lower back). The comparison between patients with chronic LBP and the control group showed that the cortical thickness measurement of the postcentral gyrus (S1 area) was bilaterally greater in those subjects with chronic LBP. Thus, volume increase was observed in the upper third of S1 and not in the middle or lower third, which highlights the specificity of the changes in the representation of the lower back. In addition, these changes can be related to hypersensitivity of the CNS in patients with chronic LBP (characterized by lower pain threshold and lower pain tolerance values) compared with healthy subjects.
Finally, Ung et al. [bib_ref] Multivariate classification of structural MRI data detects chronic low back pain, Ung [/bib_ref] carried out one study aiming to differentiate patients with chronic LBP based on structural changes in the brain and to investigate pathological changes in certain regions of the cortex. They found a pattern of regional gray matter density that distinguished, with 76% accuracy, patients with chronic LBP from healthy controls. The most notable changes were an increase in gray matter in the left S1 and S2 cortices, left M1, and premotor cortex. Teutsch et al. [bib_ref] Changes in brain gray matter due to repetitive painful stimulation, Teutsch [/bib_ref] found a gray matter increase in regions processing pain modulation (especially in S1), transiently in healthy subjects subjected to a repetitive noxious stimulus. This adaptation may be related to the compromise of a normal antinociceptive system, but in patients with LBP, the increase (especially in S1), may be the result of a defective antinociceptive system due to a noxious stimulus sustained over time, indicating that the pain no longer depends on peripheral afferent input but on central processing.
## Functional changes
Assessing functional connectivity in the brain refers to the temporal dependence of neuronal activity between anatomically separated regions, it being essential to carry out cognitive processes by integrating information. The brain's default mode network (BDMN) is the first network to show functional connectivity in a non-task resting state and is believed to play a fundamental role in the synchronization of all brain regions [bib_ref] The brain's default mode network, Raichle [/bib_ref].
Kregel et al. [bib_ref] Structural and functional brain abnormalities in chronic low back pain: A systematic..., Kregel [/bib_ref] described altered functional connectivity in patients with CLBP during rest and increased activity in pain related areas following painful stimulation in several brain regions, especially those associated with pain dimensions (known as the "pain matrix" formed by the prefrontal cortex, cingulate cortex, insula, thalamus, S1 and S2 cortices). Wasan et al. [bib_ref] Neural correlates of chronic low back pain measured by arterial spin labeling, Wasan [/bib_ref] experimentally induced pain in chronic LBP patients and healthy subjects, finding regional blood flow changes in certain brain areas, especially those referenced in the pain matrix. Pain exacerbation was performed in 16 patients with chronic LBP and 16 healthy subjects, using two methods: heat and clinical maneuvers. Only the clinical maneuvers, leg elevation or pelvic tilt, accounted for a significant increase in pain from the clinical point of view, being greater than 30% of the initial pain. This increase in endogenous pain was associated with changes in cerebral blood flow in certain cortical areas such as the medial and DLPFC, S1 and S2 cortices, right insula, and superior parietal lobes in patients with chronic LBP. Although the latter are not part of the pain matrix, they have a high functional connectivity with the rest of the areas in pain states.
On the other hand, Matsuo et al. [bib_ref] Attenuation of cortical activity triggering descending pain inhibition in chronic low back..., Matsuo [/bib_ref] aimed to examine the dysfunction of descending inhibitory paths mechanisms in subjects with chronic LBP through fMRI in a 3-Tesla MRI scanner. They recruited 11 patients and 13 healthy subjects who discontinued pharmacological treatment 24 h before the study participated. A mechanical stimulus of 500 kPa was applied for 30 s in 3 blocks, with 30 s rest, reaching a painful level for the participants who were asked to remember. The most notable findings were that chronic LBP patients lacked activation in the ACC and DLPFC after pain stimuli, while clear activation of these regions appeared in healthy controls. ACC and DLPFC mediate the affective and cognitive components of pain perception and are also believed to be the cortical origin of the descending inhibitory system.
Baliki et al. [bib_ref] Chronic pain and the emotional brain: Specific brain activity associated with spontaneous..., Baliki [/bib_ref] studied brain activity associated with spontaneous pain and acute thermal pain in patients with chronic LBP (which is one of the main complaints of these patients). They analyzed 13 patients with chronic LBP suffering spontaneous pain through fMRI differentiating two periods; one in which the spontaneous pain is maintained at a high intensity and another in which the spontaneous pain increases transiently, and they were able to determine which different regions of the brain are responsive in each of the phases. In high but sustained spontaneous pain, they observed activity at the medial prefrontal cortex (MPFC) and ACC with projections to areas of the brain involved in emotions, cognition, and motivation (i.e., posterior thalamus and the amygdala). In contrast, when spontaneous pain is exacerbated, the brain activation pattern observed corresponds to that observed in acute pain, encompassing regions involved in sensory and affective dimensions of pain (i.e., right insula, S1, S2 and cerebellum).
In addition, they found a significant relationship between increased brain activity in MPFC in situations of high intensity pain in phase 1 (high but sustained pain), and in the right insula with a longer duration of pain in phase 2 (transient increase in spontaneous pain). These findings explain whether the activity in the insula reflects the chronicity of LBP and the activity in the MPFC reflects its intensity. The response to thermal stimuli activated, in both the controls and patients, the insula bilaterally, S2, cingulate cortex and DLPFC, which made it clear that spontaneous pain does not activate the same brain regions as an external stimulus. It was observed a dissociation between the emotional and sensory regions in terms of encoding pain intensity in acute pain, compared to sustained spontaneous pain. Whereas the sensory region (insula) encodes the perceived magnitude of acute pain, the emotional region (MPFC) encodes the magnitude of spontaneous pain in chronic LBP.
According to Apkarian et al. [bib_ref] Chronic Back Pain Is Associated with Decreased Prefrontal and Thalamic Gray Matter..., Apkarian [/bib_ref] and Baliki et al. [bib_ref] Chronic pain and the emotional brain: Specific brain activity associated with spontaneous..., Baliki [/bib_ref] , a decrease in gray matter can cause its dysfunction and consequently an increase in the activity of the MPFC, which reflects a negative emotional state caused by chronic pain. This provides a link between brain atrophy and continued suffering from chronic low back pain. A subsequent study by Hashmi et al. [bib_ref] Shape shifting pain: Chronification of back pain shifts brain representation from nociceptive..., Hashmi [/bib_ref] hypothesized that the representation of LBP over time moves away from sensory areas and gradually becomes involved in emotional and limbic regions. Patients with subacute low back pain were observed and divided into one of two groups over a year: (1) those who had recovered and (2) those who had progressed to chronic pain. They were also compared with patients with chronic LBP of longer duration. The results obtained in fMRI determined that the perception of low back pain in patients with chronic LBP over 10 years did not activate the same brain circuits as in those with LNP of 2 months of duration. The latter activated circuits more related to acute pain and reward, (i.e., insula, thalamus, S1, and ACC), whereas in subjects with chronic LBP, emotional circuits were activated (i.e., amygdala, MPFC, orbitofrontal cortex and hippocampus).
Changes observed in subjects with persistent pain at 1 year compared to baseline activity were a significant increase in functional connectivity between the MPFC and the nucleus accumbens. This argues that brain activity in the transition from acute to chronic pain shifts away from sensory circuits to increase activity in emotion circuits (mesolimbic circuits). The first year may be critical for this pain chronification since it was observed that patients with persistent subacute pain (1 year) had similar activity to patients with chronic LBP lasting more than 10 years, which proposed a time window for the stabilization of chronic pain from 6 to 12 months, which suggests that the best form of intervention to prevent the chronification of low back pain is before the first year [bib_ref] Shape shifting pain: Chronification of back pain shifts brain representation from nociceptive..., Hashmi [/bib_ref].
Li et al. [bib_ref] Suppressed descending pain modulatory and enhanced sensorimotor networks in patients with chronic..., Li [/bib_ref] assessed the association between gray matter volume changes and functional changes in the cerebral cortex in patients with chronic LBP. They recruited 16 chronic LBP patients and 16 healthy subjects, inducing a mechanical painful stimulus at the left lower back, and analyzed the brain structure and functional connectivity through voxelbased morphometry and fMRI, respectively. The results obtained were consistent with previous findings, observing a decrease in gray matter in the DLPFC, thalamus, ACC and MPFC, areas involved in nociceptive and affective/cognitive processing, and an increase in gray matter in areas related to sensory information (S1 and M1). They also found disrupted default neural network functional connectivity and increased connectivity after painful stimuli in S1, S2, cerebellum, and insula. The increase in gray matter in S1 and M1 corresponded to the somatotopic representation of the trunk, which shows causality with the pain located in the lower back. On the other hand, connectivity between bilateral sensorimotor areas and the superior parietal lobe (which is the area of sensory integration) was increased in subjects with chronic LBP compared with healthy subjects, and was positively correlated with pain intensity. This increase in the activity of S1 and M1 may lead to an increase in gray matter in the region of the trunk of S1 and M1, which can be considered a sign of chronic low back pain. These current results show an insight into the influence of the interaction between the structural reorganization of the brain and the functional changes that may be the basis of the chronification of low back pain.
Regarding the motor cortex and muscular activation changes found, a large number of studies reported that people with chronic LBP plan movement differently, and especially have post-activation of trunk muscle contraction during rapid upper extremity movement (i.e., a delayed early postural adjustment). Due to the participation of cortical structures, such as M1, the supplementary motor area, cerebellum or basal ganglia, in the execution and planning of the anticipated postural adjustment, and since the muscles of the spine are controlled by corticospinal pathways, it is logical to think of the relationship that exists between motor alteration and plastic changes in M1 and other cortical motor areas.
Tsao et al. [bib_ref] Reorganization of the motor cortex is associated with postural control deficits in..., Tsao [/bib_ref] observed the reorganization and excitability of the motor cortex present in the changes of the postural activation of the transversus abdominis (TrA) muscle in 11 patients with chronic LBP and 11 healthy subjects. They asked study subjects to perform rapid arm movements during a trunk-altering task to assess postural activation of TrA. The results associated the reorganization of motor cortex networks with delays in TrA activation, as well as a change in its cortical representation, as we have already mentioned. The magnitude of the representation change in the motor cortex was related to the magnitude of the delay in postural activation of TrA. Activation of the deep abdominal and trunk muscles (especially TrA) occurs prior to deltoid activation in voluntary limb movements, and that prior contraction is controlled by the CNS. The problem lies in the fact that patients with chronic LBP have a delay in the activation of TrA, and those other lumbar muscles, such as the multifidus, are also affected. It is suggested that this reorganization in the motor cortical map of TrA and the rest of the muscles can distort the coordination between the trunk muscles in subjects with chronic LBP, increasing their pain and disability.
A review of the literature carried out by Massé-Alaire et al. [bib_ref] Chronic pain and the emotional brain: Specific brain activity associated with spontaneous..., Baliki [/bib_ref] highlighted the link between pain-related brain reorganization and altered TrA control. They observed that the ACC, thalamus and MPFC in patients with chronic LBP present biochemical changes and hyperexcitability. The ACC participates in motor planning and is directly connected to M1 and the supplementary motor area, both of which are involved in automatic postural adjustment, which is impaired in subjects with chronic LD. They also observed a reorganization of the sensory maps with medial displacement of S1. All of this neuroplasticity may be related to proprioception and tactile acuity deficiency underlying observed motor changes. Chronic LBP causes a decrease in intracortical inhibition of the pain area in M1, which means an alteration in neuronal homeostasis with changes in the cortical maps between neighboring neuronal networks. Specifically, in the LBP, corticospinal excitability corresponding to the painful region, the low back, decreases with increasing pain and disability, and this can be explained by a change in the motor maps of M1, the posterolateral deviation of the representation of TrA, related in amplitude to the delay of TrA activation.
As Massé-Alaire described in another study, changes in the representation of S1 may have a fundamental role in the relationship between pain and impaired motor control of movement, given its substantial role in both sensory encoding and aspects sensory-discriminative of pain. S1 and M1 have reciprocal connectivity, which implies that a displacement in the representation of the trunk in S1 can affect the connectivity with M1 and, consequently, a lower performance in motor control of the spine in patients with chronic LD. Additionally, altered neural processing, supplementary motor area connectivity, and cerebellar connectivity and density changes are involved in postural control and anticipatory postural adjustment through transcortical and cerebellar-cortical connections with M1 areas. They also stated that the plasticity of M1 is greater in patients with severe chronic LBP than in those with moderate or mild chronic LBP.
Ceko et al. [bib_ref] Partial recovery of abnormal insula and dorsolateral prefrontal connectivity to cognitive networks..., Čeko [/bib_ref] examined whether CLBP affects connectivity of brain networks supporting cognitive functioning and changes after treatment. They found bilateral insula as the region of aberrant cognitive resting-state connectivity comparing patients and controls with fMRI and structural changes in white matter with DTI. In addition, previous studies [bib_ref] Changes in Empathy in Patients with Chronic Low Back Pain: A Structural-Functional..., Ma [/bib_ref] [bib_ref] Abnormal Anatomical and Functional Connectivity of the Thalamo-sensorimotor Circuit in Chronic Low..., Mao [/bib_ref] also observed reduced fractional anisotropy values in the corpus callosum, bilateral anterior thalamic radiation, right posterior thalamic radiation, right superior longitudinal fasciculus and left anterior corona radiate associated with the abnormal functional connectivity, damaged white matter tracts, altered resting-state functional connectivity of bilateral thalamo-motor/somatosensory pathways and impaired empathic abilities in patients with CLBP. However, brain function seems to restore partially after treatment [bib_ref] Changes in Empathy in Patients with Chronic Low Back Pain: A Structural-Functional..., Ma [/bib_ref].
Therefore, these brain plasticity findings contribute to a better understanding of the pathophysiological mechanisms in patients with CLBP and clarify whether the induction of positive plasticity changes promoting function and reducing pain should be considered during CLBP rehabilitation. Clinicians should consider therapeutic strategies for normalizing S1 and M1 maps. Sensorimotor integration training will favor the activation of sensory recreations and the required motor planning to reinforce synaptic efficiency and cause cortical changes, such as synaptogenesis and the multiplication of dendritic connections [bib_ref] Partial recovery of abnormal insula and dorsolateral prefrontal connectivity to cognitive networks..., Čeko [/bib_ref] [bib_ref] Changes in Empathy in Patients with Chronic Low Back Pain: A Structural-Functional..., Ma [/bib_ref] [bib_ref] Abnormal Anatomical and Functional Connectivity of the Thalamo-sensorimotor Circuit in Chronic Low..., Mao [/bib_ref] [bib_ref] Cerebral reorganization in chronic low back pain and neurostimulation to improve motor..., Massé-Alarie [/bib_ref].
Finally, there are some limitations to be acknowledged. Firstly, this is a narrative review and no systematic procedures were followed. Therefore, since relevant studies may not appear in this study, there is a possibility of biased conclusions in this study. In addition, it is important to emphasize the need to pay attention to the removal of the influence of systematic errors in the measurements, which can be large in the case of diffusion-weighted measurements, where the size of systematic errors and their impact on DTI parameters and the possibility of eliminating their impact can be assessed. For instance, although DTI scalar maps emerged as a measure sensitive to tissue structure, they fail to characterize the highly complex diffusion topology, as in the presence of white matter crossing fibers, where the diffusion displacement distribution is multimodal. Thus, DTI suffers from limited biological specificity concerning several microstructural tissue properties within a given voxel (i.e., myelination, axonal packing, axonal orientation dispersion) and is also influenced by multiple non-biological factors, such as the scanner parameters, data quality or head motion.
# Conclusions
Chronic LBP should not be considered as a single musculoskeletal pathology. Advances in neuroscience demonstrated that it encompasses CNS, spinal, and muscular neuroplastic changes. For better pain understanding (especially from the patients' point of view), it is necessary to understand that cortical changes lead a perpetuation of pain, despite having recovered from the initial injury. Current evidence suggests that the passage from acute-subacute to chronic pain is propitiated by the mesolimbic pathways (emotional and motivational) of the brain. Therefore, a clinical correlation between the radiological findings of the lumbar spine with referred pain cannot be expected in chronic LBP.
The cortical brain changes reported in the literature include structural changes (e.g., decrease in gray matter at the DLPFC), functional changes (e.g., modification of the functional connectivity at regions forming the "pain matrix") and neurochemical changes (e.g., lack or overplus of neurotransmitter in specific areas of the brain). There is also a clear affectation of the M1 area activity, resulting in the function and muscular coordination alteration of the trunk and the abdominal muscles. Therefore, treatment programs should be focused on reversing these changes in order to achieve proper function of the abdominal muscles and the trunk.
[fig] Author: Contributions: Conceptualization, Y.M.-E., G.P.-M. and J.A.V.-C.; methodology, C.F.-d.-l.-P., G.P.-M. and J.A.V.-C.; software, G.P.-M. and J.A.V.-C.; validation, G.P.-M. and J.A.V.-C.; formal analysis, G.P.-M. and J.A.V.-C.; investigation, C.F.-d.-l.-P., G.P.-M. and J.A.V.-C.; resources, G.P.-M. and J.A.V.-C.; data curation, G.P.-M. and J.A.V.-C.; writing-original draft preparation, Y.M.-E., G.P.-M. and J.A.V.-C.; writing-review and editing, C.F.-d.-l.-P., G.P.-M. and J.A.V.-C.; visualization, G.P.-M. and J.A.V.-C.; supervision, G.P.-M.; project administration, J.A.V.-C. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. [/fig]
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Respiration modulates oscillatory neural network activity at rest
AU : Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly: Despite recent advances in understanding how respiration affects neural signalling to influence perception, cognition, and behaviour, it is yet unclear to what extent breathing modulates brain oscillations at rest. We acquired respiration and resting state magnetoencephalography (MEG) data from human participants to investigate if, where, and how respiration cyclically modulates oscillatory amplitudes (2 to 150 Hz). Using measures of phase-amplitude coupling, we show respiration-modulated brain oscillations (RMBOs) across all major frequency bands. Sources of these modulations spanned a widespread network of cortical and subcortical brain areas with distinct spectrotemporal modulation profiles. Globally, delta and gamma band modulations varied with distance to the head centre, with stronger modulations at distal (versus central) cortical sites. Overall, we provide the first comprehensive mapping of RMBOs across the entire brain, highlighting respiration-brain coupling as a fundamental mechanism to shape neural processing within canonical resting state and respiratory control networks (RCNs).
# Introduction
We all breathe. Human respiration at rest comes naturally and comprises active (but automatic) inspiration and passive expiration. The rhythmicity of each breath is initiated and coordinated by coupled oscillators periodically driving respiration, most prominently the preBötzinger complex located in the medulla. This microcircuit typically controls respiration autonomously, making the act of breathing seem effortless. Importantly, however, respiration is also under top-down control, as evident from adaptive breathing during, e.g., speaking, laughing, and crying. Hence, there is a bidirectional interplay of the cortex and rhythmic pattern generators of respiration: Efferent respiratory signals from the preBötzinger complex project to suprapontine nuclei (via locus coeruleus and olfactory nucleias well as to the central medial thalamus, which is directly connected to limbic and sensorimotor cortical areas. In turn, cortical areas evoke changes in the primary respiratory network, e.g., to initiate specific motor acts (e.g., swallowing or singing) or transitions between brain states (e.g., during panic attacks).
As neural oscillations have been established as sensitive markers of brain states in general, the question arises to what extent rhythmic brain activity is modulated by the rhythmic act of breathing. Indeed, studies of respiration-brain coupling have recently attracted increased attention, reporting a range of cognitive and motor processes to be influenced by respiration phase. Human participants were found to spontaneously inhale at onsets of cognitive tasksand respiration phase modulated neural responses in sensoryand face processingtasks as well as during oculomotor controland isometric contraction. Parallel to this body of work, animal studies have conclusively shown respiration to entrain brain oscillations not only in olfactory regions, but also in rodent whisker barrel cortexand even hippocampus. In other words, brain rhythms previously attributed to cognitive processes such as memory were demonstrated to at least in part reflect processes closely linked to respiration. Despite significant advances in the animal literature, these links are still critically understudied in humans. Notable exceptions include intracranial EEG (iEEG) work in epilepsy patients corroborating that oscillations at various frequencies can be locked to the respiration cycle even in nonolfactory brain regions. Moreover, 2 noninvasive studies recently linked respiration phase to changes in task-related oscillatory activity. Overall, both animal and human studies all lead to 3 fundamental questions that recognise respiration as a vital, continuous rhythm persisting during all tasks and activities as well as at rest: (i) to what extent does breathing modulate rhythmic brain oscillations at rest; (ii) where are these modulatory effects localised in the brain; and (iii) how does modulation unfold over the course of the respiration cycle. Therefore, what is needed is a comprehensive account integrating recent findings of respiration-brain coupling against the anatomical backdrop of canonical resting state and respiratory control networks (RCNs). A variety of neural networks have extensively been described to organise the brain's intrinsic or ongoing activity, among which the default mode network (DMN), the dorsal attention network (DAN), and the salience network (SN) have received particular attention. Previous studies have demonstrated intriguing anticorrelated dynamics of activity between these large-scale networks (i.e., increases in one network lead to decreases in another. Such fluctuating relationships between cortical networks could conceivably be modulated by changes in bodily states such as respiration. The full picture is complemented by 2 distinct pathways responsible for the feedforward generation of the respiratory rhythm and the neural processing of respiration-related signals, respectively: In addition to pattern generators like the preBötzinger complex in the medulla, deeper sites known to be involved in respiratory control comprise further subregions within the brain stemand cerebellar nuclei. On the other hand, nasal respiration evokes feedback signalling in response to mechanical, thermal, or odour stimulation within olfactory areas in the forebrain, most prominently the olfactory bulb (OB) and piriform cortex. These (orbito-)frontal feedback signals are a central contributor for respiration-brain coupling, as animal studies have demonstrated that respiratory rhythms (i.e., air-driven mechanoreceptor signals within the OB) are translated into neural oscillations and propagated to upstream areas. Interestingly, the RCN also includes directly connected cortical sites like primary and supplementary motor areas (SMAs)and even shows anatomical overlap with resting state networks, namely within medial prefrontal cortex (mPFC), insula, and anterior cingulate cortex (ACC). We thus aimed to investigate respiration-related modulations of oscillatory brain activity and its spectrotemporal characteristics at rest, relating their anatomical sources to canonical networks of both resting state activity and respiratory control.
To this end, we simultaneously recorded spontaneous respiration and eyes-open resting state magnetoencephalography (MEG) data from healthy human participants. Using the modulation index (MI) as a measure of cross-frequency phase-amplitude coupling, we first assessed respiration-induced modulation of brain oscillations globally across the entire brain. We then extracted single-voxel time series to localise the anatomical sources of these global modulation effects using beamforming. We employed nonnegative matrix factorisation (NMF) for dimensionality reduction, effectively yielding a spatially constrained network of cortical and subcortical sources of respiration phase-dependent changes in rhythmic brain activity. Finally, we identified distinct spectrotemporal profiles of network components, highlighting an intriguing organisational pattern behind respiration-induced modulation of neural oscillations across the brain.
# Results
## Respiration phase modulates global field power
To assess the fundamental question of whether respiration modulates oscillatory brain activity at rest, we first computed the MI in sensor space (using all 268 channels) for whole-brain MI quantifies to what extent the amplitude envelopes of frequency-specific brain oscillations (top right, red) were modulated by respiration (centre right, blue). This way, we computed modulation indices for each sensor, frequency, and participant before localising voxelwise time series in source space (see. (B) Mean normalised MI (± SEM) over the entire frequency spectrum (right) and corresponding t-values from the cluster permutation test (left). Random shifts of respiration phase were employed to correct for low-frequency bias and to express MI in units of SD of a surrogate distribution (leading to normalised MI; see Materials and methods). (C) Mean PTAs across the respiratory cycle over the entire frequency spectrum. PTAs were computed by averaging frequency-specific amplitude envelopes (panel A) time locked to peak inhalation. Note that PTAs were standardised for illustration purposes. Therefore, they show relative changes over the respiration cycle within each frequency band and do not directly correspond to absolute MI amplitudes from panel B. Also note that SNR decreases towards the edges of the panel (i.e., approaching ± π) due to increased variation of underlying single respiratory cycles that were used for phase-locked analysis. Underlying data are provided in the folder "on the OSF directory. MAU : AbbreviationlistshavebeencompiledforthoseusedthroughoutFigs1 À 4:Pleaseverifythatallentriesarecorrect: I, modulation index; PTA, phase-triggered average; SD, standard deviation; SNR, signal-to-noise ratio.
https://doi.org/10.1371/journal.pbio.3001457.g001 global field power ranging from 2 to 150 Hz. This analysis quantifies to what extent the amplitude of global brain oscillations is modulated by the phase of respiration. Our cluster permutation analysis revealed significant respiration-locked modulation of global field power indicated by the high normalised MI across the entire frequency spectrum (all p < 0.001, cluster corrected at α = 0.05; see. Local peaks with strongest modulation occurred at about 2, 30, 75, and 130 Hz (with strongest absolute modulation effects in the beta band), indicating differential modulation of specific brain oscillations (see S1 Fig for range and distribution of subject-level MI spectra). Next, we computed the phase-triggered average (PTA) to characterise these global modulation effects over a respiratory cycle. PTA is computed as the average of oscillatory amplitude across windows centred on all time points of peak inhalation. We found respiration phase to differentially modulate oscillations of various frequencies with distinct time courses. In particular, whereas most frequency bands showed strongest modulation effects around the inspiration peak, beta oscillations were visibly coupled to a different phase of respiration around inspiration onsetThis first analysis therefore revealed that the amplitude of global oscillatory brain activity was significantly modulated by respiration in a broad frequency range from 2 to 150 Hz with a temporal modulation that differs across frequencies. To gain a deeper understanding of how respiration modulates rhythmic activity across the brain, 2 questions immediately ensued, namely to localise the anatomical sources of such modulation effects and to explore their spectrotemporal profiles in more detail.
## Modulatory effects of respiration phase can be traced to cortical and subcortical networks
To identify the anatomical sources of these global modulations, we quantified how strongly respiration modulated the amplitude of brain oscillations within each voxel in the brain of each participant at each frequency between 2 and 150 Hz by computing the MI. Next, we used sparse NMF to reduce the dimensionality of the three-dimensional data set (participants × voxels × frequency; see Materials and methods). This resulted in an optimal low-dimensional representation consisting of 18 components. Each component reflected respiration-modulated brain oscillations (RMBOs) across the frequency spectrum, quantified as NMF weights for each participant, voxel, and frequency. For spatial specificity of NMF components, each component's spatial map was thresholded at the 99th percentile, yielding the n = 202 voxels with the strongest modulation. For all 18 components, we show the spatial location of the network on an inflated brain as well as the full MI spectrum with shading corresponding to frequency bands of significant modulation (all p < 0.002, cluster corrected across frequencies and components at α = 0.05; see. For individual spatial maps for all 18 components, see S3 and S4 Figs. Phase-dependent modulations of all 18 components are shown in S5 , this provides a comprehensive spatiotemporal spectral account of respiration-modulated networks in the resting brain.the network's cortical sources to be localised along the midline (ACC, SMA, posterior cingulate cortex (PCC), cuneus, and lingual sulcus) as well as in lateralised frontal (frontal eye field (FEF) and insula), temporal, and parietal cortices (angular gyrus and intraparietal sulcus (IPS)). The network's deeper, subcortical sites included several lateralised (crus 1, lobules 7b/8) and midline (vermis 9/10) subsections within the cerebellum, left parahippocampal cortex, and medial sources in the orbitofrontal cortex (OFC; extending onto the gyrus rectus) and brain stem. In order to quantify modulation effects between components, we compared each component's MI at a given frequency with the average MI across all other components. Components #3 and #14 (both located within the left cerebellum) showed above average modulation in the high gamma band, whereas components #6 (r. STG/r. temporal pole) and #11 (brain stem) were more strongly modulated in the delta and alpha band, respectively. Component #10 (bil. SMA) showed above average modulation in the beta and low gamma range. Finally, component #12 (bil. ACC) was less strongly modulated at low gamma frequencies than the grand average across components (see S6 . These results provide several important insights. First, respiration significantly modulates oscillatory brain activity within a specific, but widely distributed cortical and subcortical brain network. Second, across these areas, significant RMBOs can be found across almost the entire frequency range from 2 to 150 Hz. Third, the temporal modulation pattern of RMBOs is by no means uniform across frequencies and brain areas.
## Distinct spectrotemporal profiles of rmbo sites
Having localised the anatomical network underlying RMBOs, we next attempted to map distinct modulation patterns to anatomical subnetworks, with similarly modulated sites being grouped together. To this end, we employed hierarchical clustering of all 18 network components based on their MI across the frequency spectrum (as shown in. This data-driven approach yielded a total of 7 clusters comprising between 1 and 5 components Similarly, cluster C comprised 2 components within bilateral ACC and right FEF with significant modulation from alpha up to high gamma oscillations. Cluster D was formed by a total of 6 components spanning inferior, medial, and superior temporal gyrus (ITG, MTG, and STG), parietal cortices (anterior intraparietal sulcus (aIPS)/temporoparietal junction (TPJ), and angular gyrus) as well as deep cerebellar areas showing RMBOs. Due to its widespread topography, at least 1 cluster component showed significant modulation across the entire frequency spectrum. Cluster E again consisted of a single component (spanning bilateral (pre-)cuneus/lingual sulcus) with significant modulation in the theta, beta, and both gamma bands. Finally, 2 clusters were formed exclusively by deep sources: Cluster F comprised 2 components within the left cerebellum where oscillations across the whole frequency spectrum were significantly modulated by respiration. Cluster G consisted of 4 components within the left parahippocampal cortex, brain stem, cerebellum, and gyrus rectus/medial OFC and showed significant modulation from theta up to the high gamma band.
In order to investigate how MI spectra varied with anatomical location, we conducted a linear mixed effect model (LMEM) analysis modelling oscillatory modulations as a function of components' distance to the head centre (considering x, y, and z planes). This analysis revealed that the fixed effect of distance to the head centre significantly influenced modulations within the delta (t(502) = 3.55, p < 0.001) as well as the low (t(502) = 2.49, p = 0.013) and high gamma bands (t(502) = 3.85, p < 0.001), with stronger modulations for more distal (compared to central) components. Further, frequency-specific analyses were conducted to characterise to what extent this overall distance effect was driven by sagittal, frontal, and transversal location, respectively (see S1 Text and S1 .
Intriguingly, not only were different frequency bands modulated within a network of cortical and subcortical sites, but the time courses of these modulatory effects were equally frequency specific. Polar plots inshow the temporal modulation of RMBOs across the respiratory cycle for each cluster. Respiration phase was differentially coupled with amplitudes of low-frequency oscillations (such as delta and theta) compared to high-frequency oscillations (e.g., within the gamma band). Low frequencies consistently showed higher amplitudes during the beginning and end of a respiration cycle (with lowest amplitudes around peak inspiration), whereas the pattern appeared reversed for higher frequencies (see. While specific spatiotemporal interactions of respiration-brain coupling exceeded the conceptual scope of this study, our findings are the first to suggest such spatiospectral gradients and thus warrant detailed examination in future work.
## Rmbos within nodes of resting state and rcns
Extending the distinction of deep versus more superficial components, cortical RMBOs were predominantly found in brain areas that have previously been established as nodes within the DMN (PCC, angular gyrus, and precuneus), DAN (FEF and aIPS), or saliency network (SN: insula and ACC; see. Moreover, all deep and cerebellar modulation sites corresponded to a mostly subcortical network of brain areas controlling respiratory function, including bilateral cerebellum, gyrus rectus/OFC, brain stem, and SMA. Finally, as a potential link for future studies,to suggest that, although RMBOs of different frequencies had distinct temporal modulation profiles in general, there could also be certain sequential modulation patterns across clusters within a particular frequency. For example, while significant modulation of beta oscillations showed a general peak around expiration onset (distinct from e.g., high gamma modulation), this peak appeared to occur earlier and less pronounced in cluster B (PCC and SMA) than in cluster D (cerebellum and temporoparietal cortex). Future work could aim to verify such latency effects between nodes of the RMBO network and their potential functional significance. At present, our results provide a unique perspective on the general link between respiration phase and changes in oscillatory activity, showing how the sources of these modulatory effects correspond to nodes of canonical networks in control of resting state activity and respiratory function.
# Discussion
Using noninvasive MEG recordings of human participants at rest, we performed the first spatially and spectrally comprehensive analysis of brain activity that is modulated by respiration. We identified RMBOs across the entire spectrum between 2 and 150 Hz within a widespread network of cortical and subcortical brain areas. The voxel-based analysis employed adaptive beamforming for source localisation. Adaptive beamforming optimally combines MEG recordings from all sensors to estimate the time series of neural activation at a given voxel while maximally suppressing interferences from other voxels. Although spatial resolution decreases with distance from sensors, it is generally suitable for cortical and subcortical areas. Intriguingly, instead of a uniform modulation pattern across brain areas and frequencies, our analysis revealed respiratory modulation signatures that differed between brain areas in frequency and the temporal modulation profile. Our results demonstrate that respiration significantly modulates oscillatory brain activity in a manner that is precisely orchestrated across frequency bands and networks of resting state activity and respiratory control. In what follows, we will integrate our novel results with the existing animal and human literature, characterise the functionality of neural oscillations within distinct networks, and attempt to provide an overview of potential multilevel mechanisms behind RMBOs.
## Subcortical and cortical sites of respiration-brain coupling
Gamma oscillations within the OB were the first to be described in detailand reflect local computations within the OB. In a next step, slower (e.g., beta band) oscillations are thought to organise such local activity across brain areasand appear to be the most coherent within the OB. Similarly, even slower theta oscillations play a crucial role in the temporal organisation of neural activity within the hippocampal network and, consequently, its coordination with the mPFC. Our findings substantially advance these notions by showing that respiration phase modulates both low and high oscillatory frequencies within a spatial array comprising OB/OFC, brain stem, and cerebellum. As described earlier, the preBötzinger complex is widely regarded as the main pattern generator of respiratory rhythms within the brain stem, where ascending catecholaminergic neurons receive projections from the cerebellar vermis. In addition to brain stem projections, the vermis regulates autonomic responses including cardiovascular tone and respiration through connections to the spinal cord and hypothalamus. These cerebellothalamic pathways affect cortical gamma activity, in that cerebellar projections to the "motor" ventral anterior lateral (VAL) nucleus of the thalamus, the (higher order) posterior thalamic nucleus (VP), and intralaminar nuclei are used to coordinate and synchronise gamma oscillations within sensorimotor areasand across the neocortex.
The cerebellum itself projects to motor and nonmotor cortical areas, including prefrontal and posterior parietal cortex. In turn, it receives input from a wide range of higher-order, nonmotor areas within the extrastriate cortex, posterior parietal cortex, cingulate cortex, and the parahippocampal gyrus, which is monosynaptically connected to the OB. As the first olfactory relay station in the brain, the OB receives direct projections from receptors within the nasal cavity. These feedback signals not only encode odour information in olfactory receptor cells, but also mechanical stimulation of mechanoreceptors triggered by nasal airflow. As outlined above, subsequent neural activity patterns are then transmitted to upstream sites including OFC, hippocampus, and insula, which we fittingly found to be part of the RMBO network.
In sum, our findings integrate and extend a variety of individual results in 2 ways: First, cortical nodes within the RMBO network precisely reflect bidirectional projection areas of the deep and subcortical nodes (OB, brain stem, and cerebellum) via medullar and thalamic pathways. Second, the cortical nodes markedly resemble "sensorimotor distributions" shown in multiple functional magnetic resonance imaging (fAU : PleasenotethatfMRIhasbeendefinedasfunctiona MRI) studies of respiratory control, raising the question as to how different cortical areas-motor areas, ACC, and insular cortex -are involved in the act of breathing. As both premotor and supplementary motor cortices contain representations of respiratory muscles, they have long been implicated in respiratory control. Similarly, ACC has been identified in studies of air hungerand CO 2 -stimulated breathing. Finally, insular cortex activation is a consistent feature of many neuroimaging studies of dyspnoea. The close mapping of frontal, cingulate, and parietal areas to canonical resting state networks (seesuggests a general involvement of respiration in human brain processing irrespective of particular task demands. In this context, it is noteworthy that nodes of resting state networks exhibit amplitude correlations predominantly in the beta frequency band. In our data, this frequency band shows strongest global modulation by respirationand features prominently in the coupling of specific resting state networks to respiration, suggesting that these amplitude correlations within resting state networks are at least partially related to respiration.
## Active sensing, respiration, and behaviour
The widespread extent and spectral diversity of the RMBO network critically corroborate previous suggestions of respiration as an overarching "clock" mechanism organising neural excitability throughout the brain. Excitability adapts neural responses to current behavioural demands, which is why respiratory adaptation to such demands in animalsand humanshave accordingly been interpreted as functional body-brain coupling. Indeed, animals as well as humans appear to actively align their breathing to time points of particular behavioural relevance for the sake of efficiency through optimised neural processing. Consequently, human respiration has fittingly been cast as active sensing, adopting key premises from predictive brain processing accountsto explain how respiration synchronises time frames of increased cortical excitability with the sampling of sensory information. Our results provide first insights into how established mechanisms like cross-frequency phase-amplitude coupling (in this case, coupling peripheral to neural rhythms) are implemented on a global scale to translate respiratory rhythms into neural oscillations of various frequencies and how the resulting anatomical pattern of RMBOs reflects spectral specificity.
## Potential mechanisms behind rmbos
Cross-frequency coupling is widely regarded as the core mechanism of translating slow rhythms into faster oscillations and has conclusively been shown to be driven by respiratory rhythms within the OB in mice: During nasal inspiration, air enters the respiratory tract and triggers mechanoreceptors connected to the OB, thereby initiating infraslow neural oscillations closely following the respiratory rhythm (pAU : PleasenotethatasperPLOSstyle; italicsshouldnotb hase-phase coupling). The phase of these slow oscillations then drives the amplitude of faster oscillations (phase-amplitude coupling) and propagates to upstream areas both within and beyond the olfactory system. With reference to the concept of active sensing introduced above, we argue that a similar case can be made for the cerebellum: There is broad consensus that the cerebellum is involved in computations attributed to internal forward models, predominantly in motor control. These forward models are just as crucial for perception and cognition as they are for motor performance, leading to the suggestion that cerebellar processing may help to align and adaptively modify cognitive representations for skilled and error-free cognitive performance. The prominent role of cerebellar sources in our present findings suggests that respiration modulates these functional connections by means of cross-frequency coupling, linking respiration to motor and cognitive function.
Strong support for this hypothesis comes from a recent studyshowing that the cortical readiness potential, originating within premotor areas, fluctuates with respiration. Notably, the authors suggest cross-frequency coupling to involve neural interactions between premotor areas and both insular and cingulate cortex as well as the medulla, which is precisely the pathway we propose to connect deep and cortical nodes within the RMBO network. A simple graph model of excitatory and inhibitory cells has been shown as proof of principle for cortical gamma modulation through respiration (modelled as sinusoidal input). The authors later concluded that respiration-locked brain activity has 2 driving sources: On the one hand, respiration entrains OB activity via mechanoreceptors, as seen in local field potentials. On the other hand, Heck and colleagues propose extrabulbar sources within the brain stem, which are of critical importance for the generation of the respiratory rhythm itself. Functionally, respiration thus appears to modulate higher oscillatory frequencies (e.g., gamma) for the purpose of integrating locally generated assemblies across the brain. Our data now show that respiration-brain coupling (i) spans an even more extensive network including deep cerebellothalamic pathways; and (ii) involves a wider variety of oscillatory modulation than previously assumed. Importantly, our analyses demonstrate that the spectral specificity of respirationrelated modulations within the RMBO network cannot be explained by mere changes in CO 2 alone (see S3 Text).
While the RMBO network presented here provides the most comprehensive account of human respiration-brain coupling to date, central research questions emerge as objectives for future work. First, having established the sources of respiration-related changes to neural oscillations, the transition from resting state to task context will illuminate the relevance of RMBOs for behaviour. Cognitive, perceptive, and motor performance have been shown to be modulated by respiration, warranting a closer assessment of the where (i.e., which site) and when (i.e., at which phase) of task-related RMBOs. Second, we have outlined functional pathways connecting the cerebellum to cerebral cortex via medullar and thalamic projections as well as the close link between OB and parahippocampal as well as prefrontal cortices. These putative hierarchies should be tested with directional measures of functional connectivity in order to reveal organisational relations within the RMBO network. Similarly, directed connectivity analysis can disambiguate bottom-up from top-down signals within the wider RMBO network and potentially illuminate the notably lateralised effects within it: Although lateralisation is not uncommon in well-established functional networks (e.g., related to attention; see, it will be instructive for future work to validate whether RMBOs reliably prove stronger in one hemisphere (as was the case for insula and FEF) or whether there is a separate dynamic underlying the involvement of individual nodes.
In summary, our comprehensive investigation of respiration-brain coupling emphasises respiration as a powerful predictor for amplitude modulations of rhythmic brain activity across diverse brain networks. These modulations are mediated by cross-frequency coupling (linking respiratory to neural rhythms) and encompass all major frequency bands that are thought to differentially support cognitive brain functions. Furthermore, respiration-brain coupling extends beyond the core RCN to well-known resting-state networks such as default mode and attention networks. Our findings therefore identify respiration-brain coupling as a pervasive phenomenon and underline the fact that body and brain functions are intimately linked and, together, shape cognition.
# Materials and methods
## Participants
A total of 28 volunteers (14 female, age 24.8 ± 2.87 years [mean ± SD]) participated in the study. All participants denied having any respiratory or neurological disease and gave written informed consent prior to all experimental procedures. The study was approved by the local ethics committee of the University of Muenster (approval ID 2018-068-f-S) and complied with the Declaration of Helsinki.
## Procedure
Participants were seated upright in a magnetically shielded room while we simultaneously recorded respiration and MEG data. MEG data were acquired using a 275 channel whole-head system (OMEGA 275, VSM Medtech, Vancouver, Canada) and continuously recorded at a sampling frequency of 600 Hz. During recording, participants were to keep their eyes on a fixation cross centred on a projector screen placed in front of them. To minimise head movement, the participant's head was stabilised with cotton pads inside the MEG helmet.
Data were acquired in 2 runs of 5-minute duration with an intermediate self-paced break. Participants were to breathe automatically through their nose while tidal volume was measured as thoracic circumference by means of a respiration belt transducer (BIOPAC SystemsAU : Please , Goleta, United States of America) placed around their chest. Continuous monitoring via video ensured participants were breathing through their nose instead of their mouth. Individual respiration time courses were visually inspected for irregular breathing patterns such as breath holds or unusual breathing frequencies, but no artefacts were detected.
For MEG source localisation, we obtained high-resolution structural magnetic resonance imaging (MRI) scans in a 3T Magnetom Prisma scanner (Siemens, Erlangen, Germany). Anatomical images were acquired using a standard Siemens 3D T1-weighted whole brain MPRAGE imaging sequence (1 × 1 × 1 mm voxel size, TR = 2,130 ms, TE = 3.51 ms, 256 × 256 mm field of view, 192 sagittal slices). MRI measurement was conducted in supine position to reduce head movements, and gadolinium markers were placed at the nasion as well as left and right distal outer ear canal positions for landmark-based co-registration of MEG and MRI coordinate systems. Data preprocessing was performed using Fieldtriprunning in MATLAB R2018b (The Mathworks, Natick, USA). Individual raw MEG data were visually inspected for jump artefacts and bad channels, but neither were detected. Both MEG and respiration data were resampled to 300 Hz prior to further analyses.
## Mri co-registration
Co-registration of structural MRIs to the MEG coordinate system was done individually by initial identification of 3 anatomical landmarks (nasion, left and right pre-auricular points) in the participant's MRI. Using the implemented segmentation algorithms in Fieldtrip and SPM12, individual head models were constructed from anatomical MRIs. A solution of the forward model was computed using the realistically shaped single-shell volume conductor modelwith a 5-mm grid defined in the Montreal Neurological Institute (MNI) template brain (MNI, Montreal, Canada) after linear transformation to the individual MRI.
## Computation of global field power
For the computation of global field power, the time courses of each channel of each participant were individually subjected to a continuous wavelet transform using a Morlet wavelet for 36 frequencies (from 2 Hz to 20 Hz in steps of 2 Hz and then in steps of 5 Hz up to 150 Hz). Next, we computed the absolute value of this complex-valued data and averaged these amplitude values across channels.
## Head movement correction
Based on previous respiration-related work from our lab, it was reasonable to assume that there would be respiration-induced changes in head position and/or rotation. Therefore, we computed individual Spearman correlations between the normalised respiration time course and head movement traces of translation and rotation (in x, y, and z direction, respectively) using the accurate online head movement tracking that is performed by our acquisition system during MEG recordings. Correlation coefficients were Fisher z-transformed and averaged across runs (for each participant) and across participants to yield group-level average correlation coefficients for all 6 head movement time courses. A series of t tests revealed significant correlations between the respiration signal and translation in the x planeAs some correlation between respiration and head movement was to be expected, it was critical to rule out that our results were confounded by these head movements. To this end, we used a correction method established by Stolk and colleagues. This method again used the head movement tracking information (described above), i.e., 6 continuous signals (temporally aligned to the MEG signal) that represent the x, y, and z coordinates of the head centre (H x , H y , and H z ) and the 3 rotation angles (H ψ , H ϑ , and Hφ) that together fully describe the head movement. We constructed a regression model comprising these 6 "raw" signals as well as their derivatives and, from these 12 signals, the first-, second-, and third-order nonlinear regressors to compute a total of 36 head movement-related regression weights (using a thirdorder polynomial fit to remove slow drifts). This regression analysis was performed on the power spectra of single-sensor (and single-voxel) time courses for analyses in sensor and source space, respectively, removing signal components that can be explained by translation or rotation of the head with respect to the MEG sensors.
In addition to controlling potential artefacts caused by head movement, we report a related control analysis for high-frequency muscle artefacts in S2 Text.
## Computation of mi and pta
The MI quantifies cross-frequency coupling and specifically phase-amplitude coupling. Here, it was used to study to what extent the amplitude of brain oscillations at different frequencies is modulated by the phase of respiration. To this end, the instantaneous phase of the respiration time course was computed with the Hilbert transform. Next, the time series at each sensor location were sequentially subjected to a continuous Morlet wavelet transformation at frequencies ranging from 2 to 150 Hz (with 2 Hz spacing below 20 Hz and 5 Hz spacing above 20 Hz) using the cwtft function in MATLAB with default settings. This function computes a continuous Morlet wavelet transform using a Fourier transform-based algorithm. The Fourier transform of our wavelet is defined as
[formula] C f ð Þ ¼ p À 1 4 e À ðf À f 0Þ2 2 H f ð Þ; ð1Þ [/formula]
where H(f) is the Heaviside function, and f 0 is the centre frequency in radians/sample. We then computed the amplitude envelope and smoothed it with a 300-ms moving average. MI computation was then based on the average amplitude at 20 different phases of the respiratory cycle. Any significant modulation (i.e., deviation from a uniform distribution) is quantified by the entropy of this distribution. To account for frequency-dependent biases, we followed previously validated approachesand computed 200 surrogate MIs using random shifts of respiratory phase time series with concatenation across the edges. The normalised MI was computed by subtracting, for each frequency, the mean of all surrogate MIs and dividing by their standard deviation leading to MI values in units of standard deviation of the surrogate distribution (see. Visual inspection confirmed that this removed the frequency bias in raw MI values (stronger MI for low frequencies compared to high frequencies). The computation resulted in normalised MI values for each channel, frequency, and participant. Following the established approach by Maris and colleagues, significance of these normalised MI values on the group level was determined by means of cluster-based permutation testing using ft_freqstatistics in Fieldtrip. This test controls for multiple testing and involves different steps. Specifically, we conducted a series of 1-tailed t tests of individual MI values at each frequency against the 95th percentile of the null distribution from the 200 surrogate MI values. t-Values were then thresholded at p = 0.05 and spectrally adjacent significant data points were defined as clusters. For each cluster, we then defined the cluster-level statistics as the sum of the t-values within every cluster. Each cluster was then tested for significance using Monte Carlo approximation. For this approximation, single subject MI spectra were randomly interchanged with the previously used 95th percentile spectra, and the t test was recomputed followed by clustering and computation of the cluster-level summed t-values. After repeating the randomisation procedure 5,000 times, the original cluster statistics were compared to the histogram of the randomised null statistics. Clusters in the original data were deemed significant when they yielded a larger test statistic than 95% of the randomised null data. To assess oscillatory modulation over time, the PTA was computed from the smoothed, band-specific amplitude envelopes averaged across all sensors. Time points of peak inhalation were detected from the respiration phase angle time series using MATLAB's findpeaks function. For each time point of peak inhalation, global field power across all 36 frequencies was averaged within a time window of ± 1,000 samples centred around peak inhalation. The resulting 36 frequencies × 2,000 samples matrix was finally normalised across the time dimension, leading to z-scores of whole-head oscillatory power phase locked to the respiration signal. This analysis is equivalent to a wavelet-based time-frequency analysis. Computations were done separately for both MEG runs, normalised across the time domain, and finally averaged across runs and participants.
## Extraction of time series in source space
Source reconstruction was performed using the linearly constrained minimum variance (LCMV) beamformer approach, where the lambda regularisation parameter was set to 0%. This approach estimates a spatial filter for each location of the 5-mm grid along the direction yielding maximum power. A single broadband (2 to 150 Hz) LCMV beamformer was used to estimate the voxel-level activities across all frequencies.
## Rank optimisation and nmf
In our efforts to anatomically localise respiration phase-dependent modulation effects, we employed a spatially sparse variant of NMF to reduce the high (voxelwise) dimensionality in our data. Sparse NMF allows us to describe modulation indices across the brain as a lowdimensional combination of locally constrained network components, each of which provides a spectral profile for each participant. NMF has previously been applied for topological analyses of M/EEG data during tasks, at rest, and in decoding approaches. As the MI is inherently nonnegative and the interpretation of NMF output matrices is rather straightforward, nonnegative factorisation in general was well suited to meet our demands. The sparse factorisation approach in particular has 2 key advantages over a regular NMF approach: First, regarding network identification, the sparsity constraints are highly beneficial in obtaining spatially specific rather than broad topologies, which was central for the next steps of our analyses. Second, these spatially specific topologies greatly enhance the precision with which time × frequency modulation characteristics can be displayed within one network component -the more distant voxels are included, the more component-specific modulations are diluted. In order to balance baseline differences between participants in preparation of the NMF, MI matrices of all 28 participants (20,173 voxels × 36 frequencies) were first normalised by their standard deviation. These matrices were then averaged across both runs to yield one average matrix per participant. Individual matrices were transposed and concatenated to form one group-level input matrix (1,008 [frequencies × participants] × 20,137 voxels) for the NMF. To determine the number of main components to be extracted from NMF, we used the choosingR MATLAB functionthat chooses the optimal rank based on singular value decomposition. Specifically, the function extracts the singular values of a data matrix (in our case, participants' normalised MI matrices; size 36 frequencies × 20,173 voxels) and computes the sum of all nonzero elements of its diagonal. The optimal rank is then determined as the number of singular values that accounts for 90% of all diagonal entries. Applying this procedure to participants' individual normalised MI matrices (36 frequencies × 20,173 voxels) returned a dimensionality of 18 as the optimal desired number of network components for the subsequent NMF analysis.
Subsequently, we initialised the sparsenmfnnls algorithm from the NMF toolbox for MATLAB. The algorithm factorises the concatenated input matrix X as
[formula] X � AY;ð2Þ [/formula]
with the nonnegative matrices A and Y aiming to minimise the following quantity:
[formula] X À AY 2 F þ ZAY 2 F þ l X N i¼1 y 2 i1 ;ð3Þ [/formula]
where η and λ are sparsity parameters. As NMF solutions vary as a function of their random starting position, we repeated this procedure 100 times and selected the best sparse solution based on its residuals. Two matrices were generated as the output of this procedure: First, the basis matrix A (1,008 [frequencies × participants] × 18 components) represents the participant-specific spectral profile, effectively quantifying each participant's relative contribution to the network components separately for each frequency. The basis matrix was reshaped to a 36 × 28 × 18 (frequencies × participants × network components) matrix for all further analyses. As the second NMF output, the coefficient matrix Y (18 components × 20,173 voxels) represents the spatial profile of the network components, quantifying each voxel's relative contribution to the components.
## Component-level statistical analyses
While most components represented a single focal location due to the sparsity constraints embedded in the NMF algorithm, 4 components comprised distinct subnetworks of 2 or 3 anatomical sites. Spatial maps of all 18 network components are shown inThese maps were generated by thresholding full-brain maps (with a total of n = 20,173 voxels) at the 99th percentile, yielding spatially specific maps with an extent of n = 202 voxels. To determine the frequency range(s) for which the MI within a particular component was significant on the group level, we used the same cluster-based permutation approach described in detail in the section "Computation of MI and PTA." Here, this approach was used on all components together to correct for multiple comparisons across all 36 frequencies and 18 components.
## Modulation differences across nmf components
In order to compare modulation spectra across the RMBO network, we conducted a control analysis that compares frequency-specific effects across theThis yielded a matrix of 18 components × 36 frequencies quantifying the difference between each component's MI spectrum relative to the grand average across all components. S10 Fig visualises this matrix thresholded at z = ± 2.33 (corresponding to p = 0.01). A component's MI values were considered significantly different from the mean of all other components when the difference exceeded the critical z value. Components #3 and #14 (both located within the left cerebellum) showed greater than average modulation in the high gamma band, whereas components #6 (r. STG/r. temporal pole) and #11 (brain stem) were more strongly modulated in the delta and alpha band, respectively. Component #10 (bil. SMA) showed above average modulation in the beta and low gamma range. Finally, component #12 (bil. ACC) was less strongly modulated at low gamma frequencies than the grand average across components.
## Lmems
We employed LMEM to investigate the relationship between the spatial organisation and spectral characteristics within the network of modulated components. LMEM models a response variable (in our case, modulation indices within a particular frequency band) as a linear combination of fixed effects shared across the population (i.e., anatomical coordinates of network components) and participant-specific random effects (i.e., modulatory variation between participants). To assess potential links between spatial and spectral component properties, we first computed each component's anatomical distance to the head centre as the vector norm of MNI coordinates in the x, y, and z plane: r ¼ ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi x2 þ y 2 þ z 2
[formula] p :ð4Þ [/formula]
We then specified an LMEM to predict modulation indices of a particular frequency band within each component as a function of its distance to the head centre:
[formula] MI j ¼ b 0 þ ðb 1 þ S 1j Þ � r þ e j :ð5Þ [/formula]
For participant j, the MI is expressed as a combination of the intercept (β 0 ), the fixed effect of the component's distance to the head centre (β 1 ), and an error term (e j~N (0,σ 2 )). We accounted for between-participant variation by specifying a random slope (S 1j ). An analogous approach was used to predict modulation indices within each component separately for each plane (see S1 Text and S1 .
## Hierarchical clustering
Having localised the sources of global field power modulations within a constrained subset of anatomical sites, our next aim was to characterise these sources in terms of their spectrotemporal fingerprints. This way, we hoped to reveal systematic patterns of phase-locked oscillatory modulations over time and/or frequencies within the cortical and subcortical network. To this end, we first computed the group-level average matrix of modulation indices for 20,173 voxels × 36 frequencies × 20 time bins. We used the anatomical distribution of each network component (thresholded at the 99th percentile) to reduce this matrix to a component-specific spatial map and aggregated 36 single frequencies into frequency bands as follows: delta (2 to 4 Hz), theta (4 to 8 Hz), alpha (8 to 2 Hz), beta (12 to 30 Hz), low gamma (30 to 70 Hz), and high gamma (70 to 150 Hz). This yielded one matrix (6 frequency bands × 20 time bins) per network component, all of which were concatenated to construct a distance matrix for the hierarchical clustering using the hcluster function within the Icasso toolbox for MATLAB. This data-driven approach was employed to detect similarities of and differences between network components with regard to how oscillatory activity was modulated over the course of a respiration cycle. Following the suggested approach, we used visual inspection of the dendrogram to evaluate the clustering solutions. Based on a local maximum of the resulting silhouette value distribution, we settled on a total of 7 clusters (see. We computed the average course of modulation indices over frequency bands within each cluster based on z-transformed spectral profiles of the contributing network components (as described above).
## Sau : abbreviationlistshavebeencompiledforthoseusedthroughoutsupportinginformat
upporting information S1 LMEM results for MI values as a function of x, y, and z planes. Frequency bands were defined as described in the main text. Underlying data are provided in the folder "Supple- whole-brain NMF components were thresholded at the 99th percentile, resulting in anatomical locations with an extent of n = 202 voxels. Crosshairs are positioned at the peak voxel of each location, and atlas labels are provided (corresponding to the nomenclature in. Underlying data are provided in the folder " . Clusters of NMF components are shown in the same order as inRight panel bar graphs show the number of participants whose modulation within the respective component was strongest for the depicted frequency band (versus all other frequency bands). Coloured bars and circular segments mark NMF components for which the respective frequency band was significantly modulated by respiration phase. Underlying data are provided in the folder "Supplementary Information" on the OSF directory. NMF, nonnegative matrix factorisation. (TIF) |
Renal function and clinical outcome of patients with cancer-associated venous thromboembolism randomized to receive apixaban or dalteparin. Results from the Caravaggio trial
## Supplementary data inclusion and exclusion criteria
Any type of cancer (other than basal-cell or squamous-cell carcinoma of the skin, primary brain tumor or known intra-cerebral metastases and acute leukemia) that met at least one of the following criteria were included: i) active cancer defined as a diagnosis of cancer within six months before the study inclusion, or treatment for cancer at the time of inclusion or during 6 months before randomization, or recurrent locally advanced or metastatic cancer; ii) cancer diagnosed within 2 years before the study inclusion (history of cancer).
## Main exclusion criteria included: i) eastern cooperative oncology group (ecog) performance status iii or iv
or life expectancy of less than 6 months; ii) administration of therapeutic doses of LMWH, fondaparinux, or unfractionated heparin for more than 72 hours or three or more doses of vitamin K antagonist before randomization; iii) active or high risk of bleeding contraindicating anticoagulant treatment or concomitant thienopyridine therapy (clopidogrel, prasugrel, or ticagrelor) or aspirin over 165 mg daily or dual antiplatelet therapy; iv) hemoglobin level lower than 8 g/dL or platelet count < 75x109/L or history of heparin-induced thrombocytopenia or liver failure.
Randomization was centrally performed through an interactive online system and stratified according to the type of VTE (symptomatic or incidental) and timing of the cancer diagnosis (active or history of cancer).
Patients underwent scheduled visits at four weeks, three, six and seven months after randomization, and anytime during the study if required by intervening clinical events.
## Definition of study outcome events
The primary outcome was objectively confirmed recurrent VTE, which included proximal DVT of the lower limbs (symptomatic or incidental), symptomatic DVT of the upper limbs, and PE (symptomatic, incidental, or fatal) occurring during the 6-month trial period.
The principal safety outcome of the Caravaggio study was major bleeding defined as acute clinically overt bleeding associated with one or more of the following: a decrease in the hemoglobin level of at least two grams per deciliter, a transfusion of two or more units of red cells, bleeding occurring at a critical site (intracranial, intraspinal, intraocular, pericardial, intraarticular, intramuscular with compartment syndrome, or retroperitoneal), bleeding resulting in surgical intervention, or fatal bleeding. Secondary safety outcomes included: clinically relevant non-major bleeding event defined as acute clinically overt bleeding that does not meet the criteria for major; clinically relevant bleeding defined as the composite of major and clinically relevant non-major bleeding.
## Renal function assessment
Creatinine was locally measured at the study centers, mainly by the use of calibrated enzymatic assays. All available assessments of renal function (starting from study treatment initiation) and on study outcome events were collected at each visit and anytime during the study period if necessary. Phone contact was planned for those patients not returning for follow-up visit at seven months from inclusion in Caravaggio. In this equation, k is 0·7 for females and 0·9 for males; α is -0·329 for females and -0·411 for males; min indicates the minimum of (serum creatinine / k) or 1; max indicates the maximum of (serum creatinine / k) or 1; the equation does not require weight as the results are reported normalized to 1·73 m 2 body surface area, which is an accepted average adult surface area.
[formula] -MDRD: eGFR = 186 x (serum creatinine) -1·154 x (age) -0·203 x (0·742 if female) x (1·210 if black). 3 [/formula]
# Statistical analysis
Patients included in the Caravaggio study who received at least one dose of study treatment (modified intention to treat population) were included in this analysis. Study patients were censored at the time of death or permanent discontinuation or 180 days from randomization. Patients who experienced non-major bleeding remained in the study unless anticoagulant treatment was permanently discontinued.
Differences in patient characteristics between the apixaban and dalteparin groups, between patients with vs. without study outcome events, and between patients with eGFR above and below predefined cut off values were analyzed with descriptive statistics. Values were presented as mean ± SD or median, respectively.
To assess the effect of RI in the risk for study outcome events, two different analyses were performed:
i.) comparison of event rates in subgroups of patients randomized to apixaban or dalteparin identified based on a specific cut-off level for eGFR (60 or 50 ml per minute) at inclusion in the study;
ii.) proportional hazards model for the time to study outcome events with eGFR (according to the Cockroft-Gault formula) as a time-varying covariate. In these analyses, missing values in eGFR post-baseline measurements were replaced using LOCF method.
Cumulative incidences were presented either as proportion or per patient-year. Notes: Percentages are calculated relative to the total number of subjects in the mITT analysis set in each group.
Appendix Patient 11123 (eGFR >= 60), reported one DVT and one PE event at the same day. One MB/CRNMB can have more than one site of bleeding.
For the overall data: n = number of patients with event. N = total number of subjects. For bleeding sites: n = number of patients with the specific site of bleeding. N = number of subjects with major bleeding. For VTE type: n = number of patients with specific type of VTE. N = number of subjects with VTE event.
ICH=intracranial hemorrhage; GI=gastrointestinal; GU= genitourinary.
[table] Table 3: Event rates by CKD stage according to different formulasClinically relevant non-major bleeding, overall [/table]
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Clinicopathologic study of succinate-dehydrogenase-deficient gastrointestinal stromal tumors
Gastrointestinal stromal tumors (GISTs) that are not driven by kinase mutations, as are most GISTs, often show loss of function of the succinate dehydrogenase (SDH) complex and are considered SDH-deficient GISTs. SDH-deficient GISTs share many distinct characteristics compared with conventional GISTs. However, data regarding these characteristics, particularly among Asian people, are relatively limited. The objective of this study was to characterize the clinicopathologic characteristics, treatment, and prognosis of these uncommon GISTs.This retrospective observational study enrolled 12 patients with SDH-deficient GISTs, who were selected from 335 patients with GIST diagnosed at our institution between October 31, 2013 and October 31, 2016 by succinate dehydrogenase subunit B staining.There were 8 male and 4 female patients, with a median age of 57 years (range, 21-73 years). Ten patients (83.3%) were diagnosed at or after the age of 40 years and represented 7.2% (10/138) of the entire population of elderly patients with gastric GISTs. The tumor size ranged from 3 to 19 cm (median, 7 cm); the primary tumor was multifocal in 6 cases (50%), and tumors had a multinodular or plexiform architecture in 10 cases (83.3%). Ten cases (83.3%) showed pure epithelioid morphology, with the remaining 2 cases (16.7%) showing mixed histologic subtype. Lymph node metastasis was found at the time of primary resection in 50% (3/6) of patients. Four cases (33.3%) had distant metastasis at presentation. Four patients (33.3%) developed disease progression during imatinib treatment after initial resection, but all of these patients regained disease control when the treatment was altered to sunitinib targeted therapy.SDH-deficient GISTs arise exclusively in the stomach and account for approximately 7.4% (12/162) of gastric GISTs. Moreover, those affecting people older than 40 years are not uncommon and sunitinib may work well for cases showing treatment failure with imatinib.Abbreviations: aKG = a-ketoglutarate, BVI = blood vessel invasion, CSS = Carney-Stratakis syndrome, CTr = Carney triad, GI = gastrointestinal, GISTs = gastrointestinal stromal tumors, HIF-1a = hypoxia-inducible factor 1 alpha, HPFs = high-power fields, IGF = insulin-like growth factor, IHC = immunohistochemistry, NIH = National Institutes of Health, PAGFRA = platelet-derived growth factor alpha, SDH = succinate dehydrogenase, SDHB = succinate dehydrogenase subunit B, TET = ten-eleven translocation, VEGF = vascular endothelial growth factor, WT = wild type.
# Introduction
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal (GI) tract, with an estimated annual incidence of 14 to 20 per million people. [bib_ref] Gastrointestinal stromal tumor: recent advances in pathology and genetics, Yamamoto [/bib_ref] [bib_ref] Gastrointestinal stromal tumours: from KIT to succinate dehydrogenase, Doyle [/bib_ref] The great majority of GISTs harbor activating mutations of KIT (75-80%) or platelet-derived growth factor receptor alpha (PDGFRA) (5-8%). [bib_ref] Gastrointestinal stromal tumours: from KIT to succinate dehydrogenase, Doyle [/bib_ref] However, approximately 15% of GISTs in adults and more than 90% of pediatric GISTs lack these tyrosine kinase mutations and are generally classified as "wildtype" (WT) GISTs. [bib_ref] Succinate dehydrogenase-deficient gastrointestinal stromal tumors, Wang [/bib_ref] Recent studies have shown that WT GISTs are quite heterogeneous in terms of clinical phenotype, genetic etiology, and molecular pathways. [bib_ref] Immunohistochemistry for SDHB divides gastrointestinal stromal tumors (GISTs) into 2 distinct types, Gill [/bib_ref] [bib_ref] SDHA mutations in adult and pediatric wild-type gastrointestinal stromal tumors, Oudijk [/bib_ref] Among them, succinate dehydrogenase (SDH)-deficient GISTs, which are associated with SDH deficiency by immunohistochemistry (IHC), are the largest group.
SDH, which is located in the inner mitochondrial membrane, is involved in the fundamental processes of energy production by participating in the electron transport chain (complex II) and catalyzing the oxidative dehydrogenation of succinate to fumarate in the Krebs cycle. [bib_ref] Succinate dehydrogenase deficient gastrointestinal stromal tumors (GISTs)-a review, Miettinen [/bib_ref] This complex consists of 4 subunit proteins (SDHA, succinate dehydrogenase subunit B , SDHC, and SDHD), all of which are entirely encoded by chromosomal DNA. Mutational inactivation or loss of any SDH component (A, B, C, or D) leads to the loss of SDHB expression due to a destabilization of and subsequent loss of function of the SDH complex, and therefore, SDHB IHC can be used as a surrogate to identify these GISTs. [bib_ref] Loss of expression of SDHA predicts SDHA mutations in gastrointestinal stromal tumors, Wagner [/bib_ref] SDH-deficiency causes the truncation of the Krebs cycle, which leads to metabolic reprogramming of mitochondrial respiration, and sustained malignant proliferation of glucose and fatty acids. [bib_ref] Metabolic reprogramming for producing energy and reducing power in fumarate hydratase null..., Yang [/bib_ref] In addition, SDH-deficiency contributes to succinate accumulation, and its pathologic elevation creates a "pseudohypoxic" state, which then triggers the hypoxia-inducible factor 1 alpha (HIF-1a)-mediated hypoxia response that supports tumor formation by activating angiogenesis. [bib_ref] Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA..., Janeway [/bib_ref] Furthermore, due to the structural similarities between succinate and a-ketoglutarate (aKG), succinate accumulation is thought to inhibit aKG-dependent dioxygenase enzymes, such as the ten-eleven translocation (TET) family of DNA hydroxylases. [bib_ref] Succinate dehydrogenase deficiency is associated with decreased 5-hydroxymethylcytosine production in gastrointestinal stromal..., Mason [/bib_ref] TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, which is essential for subsequent DNA demethylation. Therefore, succinate accumulation due to SDH deficiency could potentially drive tumorigenesis via the inhibition of TET family proteins and subsequently alter global DNA methylation; this might thereby influence gene expression.
KIT/PDGFRA-mutated GISTs can occur anywhere in the GI tract, have an equal sex distribution, usually show a spindled morphology, rarely metastasize to lymph nodes or distant organs and frequently respond to imatinib. By contrast, a small number of reports have determined that SDH-deficient GISTs are located exclusively in the stomach, show a predilection for children and young adults, have a female preponderance, and are characterized by a distinctive multinodular/plexiform architecture and an epithelioid or mixed histologic subtype. [bib_ref] Succinate dehydrogenase deficiency in pediatric and adult gastrointestinal stromal tumors, Belinsky [/bib_ref] Occasional cases of SDH-deficient GISTs show symptoms related to those of metastatic tumors in the liver or abdomen. Furthermore, SDHdeficient GISTs run a relatively indolent course despite their frequent lymph node or distant metastasis and exhibit consistent primary resistance to imatinib therapy.
Due to the rarity of SDH-deficient GISTs and the mostly recent interest in these tumors, data regarding SDH-deficient GISTs, particularly within Asian populations, are relatively limited. In this study, we performed SDHB IHC on 335 GISTs diagnosed at our institution from October 31, 2013 to October 31, 2016, ultimately selecting 12 cases of SDH-deficient GISTs, aiming to characterize the clinicopathologic characteristics, treatment, and prognosis of these uncommon GISTs.
# Materials and methods
## Study design, patients, and setting
Of 842 patients with GISTs who were evaluated since 2005 at our clinic, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology in China, the data of 335 patients who were diagnosed between October 31, 2013 and October 31, 2016 were retrospectively evaluated and included in the study. This retrospective study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology according to the 1964 Helsinki Declaration and its later amendments or comparable ethical standards, and written informed consent was obtained from all 335 study participants.
## Tumor samples and clinicopathologic data
For each case, hematoxylin and eosin and IHC slides and pathology reports were reviewed by 2 experienced GI pathologists. Data concerning tumor location, maximal tumor diameter, multifocality, multinodular (plexiform) architecture, morphologic subtype, necrosis, lymphovascular invasion, lymph node metastasis, and mitotic figures per 50 high-power fields (HPFs) were recorded. Detailed clinical history, including history of genetic testing and clinical follow-up, were collected from medical records, outpatient recheck, and telephone interviews with physicians. The tumors were classified into prognostic risk groups according to the modified NIH stratification criteria. [bib_ref] Risk stratification of patients diagnosed with gastrointestinal stromal tumor, Joensuu [/bib_ref] 2.3. Succinate dehydrogenase-B immunohistochemistry IHC for SDHB was performed on 4-mm-thick formalin-fixed paraffin-embedded whole tissue sections using commercially available mouse monoclonal antibodies directed against SDHB (1:100 dilution; 40 minutes incubation; clone 21A11AE7; Abcam, Cambridge, MA) following pressure cooker antigen retrieval (Target Retrieval Solution, pH 6.1; Dako, Carpinteria, CA), as previously described. [bib_ref] Gastrointestinal stromal tumours: from KIT to succinate dehydrogenase, Doyle [/bib_ref] [bib_ref] Succinate dehydrogenase-deficient gastrointestinal stromal tumors, Wang [/bib_ref] Cases showing definite granular cytoplasmic staining were classified as positive. Cases not showing cytoplasmic staining in the presence of an internal positive control of nonneoplastic cells (e.g., vascular, smooth muscle, and epithelial elements) were classified as negative. If the findings of 2 pathologists were different or uncertain (e.g., due to weak or absent internal positive controls), IHC was repeated on a whole section and a third pathologist was consulted.
# Results
## Baseline features
Of the 335 total GISTs cases, 162 arose primarily from the stomach, among which 12 cases (7.4%) of SDH-deficient GISTs were identified. The clinical and pathologic characteristics of the patients are summarized in [fig_ref] Table 1: Clinicopathological characteristics of 12 patients with SDHdeficient GISTs [/fig_ref]. There were 8 male and 4 female patients in our study (male/female ratio of 2:1), with a median age of 57 years (range, 21-73 years). Of note, 10 patients (83.3%) were diagnosed at or after the age of 40 years, and the remaining 2 patients were diagnosed at 21 and 24 years, respectively; the 10 patients represented 7.2% (10/138) of the entire population of elderly patients with gastric GISTs. All 12 cases of SDH-deficient GISTs were located in the stomach, including 5 (41.7%) in the corpus, 5 (41.7%) in the antrum, and 2 (16.6%) in the fundus. One patient (8.3%) was presumed to have a Carney triad (CTr) due to the asynchronous occurrence of a pulmonary chondroma. Of the 12 SDH-deficient patients, 4 (33.3%) had metastasis at the time of the initial diagnosis: 3 (75%) involved metastasis to the liver [fig_ref] Figure 1: SDH-deficient GISTs [/fig_ref] and B) and 1 (25%) to the peritoneum. Three of the 4 metastatic masses were removed along with the primary tumor, and the remaining case of metastasis was not removed because of its deep location in liver segment I.
## Pathologic characteristics
The tumor size varied from 3 to 19 cm (median, 7 cm). The mitotic rate varied from 2 to 18 per 50 HPFs (median mitotic count, 6/50 HPFs). Ten cases (83.3%) showed pure epithelioid morphology [fig_ref] Figure 2: Representative histological and IHC features of SDH-deficient GISTs [/fig_ref] , with the remaining 2 cases (16.7%) showing a mixed histologic subtype [fig_ref] Figure 2: Representative histological and IHC features of SDH-deficient GISTs [/fig_ref]. Six cases of primary tumors (50%) were multifocal [fig_ref] Figure 1: SDH-deficient GISTs [/fig_ref] and D), with 10 cases (83.3%) showing a multinodular or plexiform architecture [fig_ref] Figure 2: Representative histological and IHC features of SDH-deficient GISTs [/fig_ref]. Nine cases (75%) had tumor necrosis. Lymphovascular invasion was observed in 3 (25%) cases [fig_ref] Figure 2: Representative histological and IHC features of SDH-deficient GISTs [/fig_ref] , and lymph node metastasis were encountered in 3 of 6 cases [fig_ref] Figure 2: Representative histological and IHC features of SDH-deficient GISTs [/fig_ref]. Of the total 162 cases of GIST located in the stomach, most (150, 92.6%) were immunohistochemically positive for SDHB [fig_ref] Figure 2: Representative histological and IHC features of SDH-deficient GISTs [/fig_ref] , with granular cytoplasmic staining of various intensities. IHC for SDHB staining of the 12 SDH-deficient GISTs showed loss of expression [fig_ref] Figure 2: Representative histological and IHC features of SDH-deficient GISTs [/fig_ref]. Furthermore, all cases had positive CD117, CD34, and DOG-1 expression, with negative SMA and S-100 expression. In addition, genetic analysis was performed in 7 patients and that all had WT GISTs.
## Treatment and follow-up
All 12 cases underwent surgical resection, and the detailed interventions are shown in . Follow-up data for all patients were available, ranging from 9 to 34 months (mean 21 months), with no deaths. According to modified NIH stratification criteria, 9 patients were at high-risk, 1 at moderate-risk, and 2 at low-risk. Of the 10 patients at high-or moderate-risk, 9 were administered targeted therapy (imatinib or sunitinib). Four patients experienced disease progression during imatinib treatment after the initial resection, but all of them showed progression-free survival when the treatment was changed to sunitinib targeted therapy. The remaining patients who were administered targeted therapy had a favorable prognosis.
# Discussion
SDH-deficient GISTs represent the largest proportion of the 10% to 15% of GISTs in adults and the 90% in children that lack KIT or PDGFRA mutations, and they are often grouped together and considered as WT GISTs. [bib_ref] Gastrointestinal stromal tumors of the stomach in children and young adults. A..., Miettinen [/bib_ref] Although several studies in America and Europe have revealed the clinicopathologic and IHC features and prognosis of SDH-deficient GISTs, the data in Asian populations remains relatively limited. Herein, we presented the first study of SDH-deficient GISTs described in China. In this study, we examined SDHB levels by IHC in 335 primary GISTs treated at our institution within the recent 3 years and demonstrated that SDHB staining was absent in 7.4% (12/162) of gastric GISTs. Similarly, the rate that Miettinen et al [bib_ref] Succinate dehydrogenase-deficient GISTs: a clinicopathological, immunohistochemical, and molecular genetic study of 66..., Miettinen [/bib_ref] predicted based on their cohort of 66 SDH-deficient GISTs was 7.5%. In addition, the largest study (76 cases) of SDH-deficient GISTs conducted by Mason and Hornick [bib_ref] Conventional risk stratification fails to predict progression of succinate dehydrogenase-deficient gastrointestinal stromal..., Mason [/bib_ref] showed a frequency of approximately 7.7%. Previous studies have demonstrated that SDH-deficient GISTs were more likely to occur in younger, female patients. Mason and Hornick [bib_ref] Conventional risk stratification fails to predict progression of succinate dehydrogenase-deficient gastrointestinal stromal..., Mason [/bib_ref] reported a median age of 32 and a male/female ratio of 1:1.5. This finding conflicts with the present study, where an overwhelming majority of patients (10/12, 83.3%) were diagnosed at or after the age of 40 years, representing 7.2% (10/ 138) of the study population of elderly patients with gastric GISTs. Furthermore, there were 8 male and 4 female patients, resulting in a male/female ratio of 2:1. The apparent bias in this ratio might be in part due to the small number of cases. However, the large proportion of elderly patients may suggest that SDHdeficient GISTs affecting elderly people are not uncommon.
In addition to sporadic SDH-deficient GISTs, a small subset of patients with SDH-deficient GISTs fulfills criteria for 1 of 2 tumor syndromes, Carney-Stratakis syndrome (CSS) or CTr. [bib_ref] The triad of paragangliomas, gastric stromal tumours and pulmonary chondromas (Carney triad),..., Stratakis [/bib_ref] CTr is a nonhereditary syndrome involving gastric GISTs along with pulmonary chondroma and extraadrenal paraganglioma, with a predominance in young female. [bib_ref] SDHB immunohistochemistry: a useful tool in the diagnosis of Carney-Stratakis and Carney..., Gaal [/bib_ref] CSS is a hereditary syndrome characterized by the occurrence of GISTs and paragangliomas. Unlike CTr, CSS has an equal sex distribution and more Particularly in CTr, paragangliomas often occur in a long span time apart from GISTs and only 25% of the patients have all 3 tumors; therefore, the presence of any 2 of the components is sufficient for the diagnosis. [bib_ref] A syndrome featuring paraganglionic, adrenocortical, and possibly other endocrine tumors, Carney [/bib_ref] In our review, 1 female patient had CTr with pulmonary chondroma diagnosed at 21 years, which was the youngest observed at our institution. Recently, Boikos et al [bib_ref] Molecular subtypes of KIT/ PDGFRA wild-type gastrointestinal stromal tumors: a report from..., Boikos [/bib_ref] reported that of the 95 total patients in their study, 9 (9.5%) had incomplete CTr, 7 (77.8%) of whom were female, with a median age of 21 years (range, 13-37 years).
Although we were only able to analyze 12 cases, our findings suggested that SDH-deficient GISTs arose exclusively in the stomach, showing epithelioid (10/12) or mixed (2/12) morphology, multifocal disease (6/12), and common lymph node metastasis (3/6), whereas these features were extraordinarily rare in conventional KIT/PAGFRA-mutant GISTs. Similarly, the rate of lymph node metastasis reported by Miettinen et al [bib_ref] Succinate dehydrogenase-deficient GISTs: a clinicopathological, immunohistochemical, and molecular genetic study of 66..., Miettinen [/bib_ref] was 41.7% (5/12) and that reported by Boikos et al [bib_ref] Molecular subtypes of KIT/ PDGFRA wild-type gastrointestinal stromal tumors: a report from..., Boikos [/bib_ref] was 51% (18/ 35). Moreover, blood vessel invasion (BVI) was detected in 3 cases (25%) in the present study. Yamamoto et al [bib_ref] Prognostic impact of blood vessel invasion in gastrointestinal stromal tumor of the..., Yamamoto [/bib_ref] found that BVI was a strong indicator of liver metastasis in GIST and they noted that when BVI was present in the primary localized GIST, approximately 80% of patients subsequently developed liver metastasis. Interestingly, the 1 patient who developed liver metastasis at 8 months after the initial resection in our observations was found to have BVI in the tumor sample. Not surprisingly, IHC examinations for all cases showed positivity for CD117, CD34, and DOG-1 and negativity for SMA and S-100. All cases with an available genetic analysis in our study exhibited WT, which is consistent with the results of previous investigations. [bib_ref] The triad of paragangliomas, gastric stromal tumours and pulmonary chondromas (Carney triad),..., Stratakis [/bib_ref] [bib_ref] SDHB immunohistochemistry: a useful tool in the diagnosis of Carney-Stratakis and Carney..., Gaal [/bib_ref] Loss of SDHB expression is a consistent feature of SDHdeficient GISTs, whereas SDHB expression is intact in conven-tional GISTs. [bib_ref] Gastrointestinal stromal tumors: what do we know now?, Corless [/bib_ref] Approximately half of SDH-deficient GISTs have SDH subunit gene mutations, often germline and most commonly A (30%), and B, C, or D (together 20%), with both alleles inactivated in the tumor cells according to the classic tumor suppressor gene model. [bib_ref] The genetic landscape of gastrointestinal stromal tumor lacking KIT and PDGFRA mutations, Boikos [/bib_ref] However, the remaining half of SDH-deficient GISTs lack identifiable SDHx mutations; therefore, the mechanism of inactivation of SDH in those cases remains unclear. In 2014, Killian et al [bib_ref] Recurrent epimutation of SDHC in gastrointestinal stromal tumors, Killian [/bib_ref] uncovered a recurrent gene silencing epimutation of SDHC highly specific to SDHx-WT SDH-deficient GISTs by examining the genomes, methylomes, gene expression profiles, and SDHx mutation status of a cohort of 59 SDH-deficient GISTs patients.
Perhaps the most significant characteristic of SDH-deficient GISTs is that those tumors generally pursue an indolent course despite the high rate of local recurrence and distant metastasis. [bib_ref] Good survival outcome of metastatic SDH-deficient gastrointestinal stromal tumors harboring SDHA mutations, Pantaleo [/bib_ref] In our study, at the longest follow-up of 34 months, none of the patients died of disease even though five cases (41.7%) presented distant metastasis at or after diagnosis and those underwent disease progression obtained disease control again when the targeted therapy was changed. Similarly, Miettinen et al [bib_ref] Immunohistochemical loss of succinate dehydrogenase subunit A (SDHA) in gastrointestinal stromal tumors..., Miettinen [/bib_ref] reported that 37 patients in their study had distant metastasis. However, of these patients, only 13 patients (35.1%) died of disease, and there was a favorable median survival of 8.8 years. The remaining 24 patients (64.8%) were alive with metastasis 2 to 43 years after surgery. More strikingly, Mason and Hornick [bib_ref] Conventional risk stratification fails to predict progression of succinate dehydrogenase-deficient gastrointestinal stromal..., Mason [/bib_ref] reported in their observations that even patients who were classified by conventional criteria as having very low-or low-risk features will often eventually develop metastatic disease. Sixty-seven percent of patients (8/12) with very low-risk and 60% of patients (6/10) with low risk classifications in their study had distant metastasis at a mean follow-up of 8.2 years. Furthermore, in a study of 76 patients regarding the surgical management of WT-GISTs, Weldon et al [bib_ref] Surgical management of wildtype gastrointestinal stromal tumors: a report from the National..., Weldon [/bib_ref] revealed that metastatic disease at diagnosis and mitotic count >5/50 HPFs were prognostic risk factors of event-free survival by multivariate analysis. For those Manifestation, treatment, and follow-up of 12 patients with SDH-deficient GISTs. reasons, the conventional risk stratification based on the modified NIH criteria to predict disease progression might not suitable for patients with SDH-deficient GISTs. Due to the rarity of SDH-deficient GISTs, the treatment experience of these tumors is relatively limited. Complete surgical removal of the primary tumor and locoregional metastasis is recommended whenever possible, whereas repeated resection after the initial resection significantly decreases postoperative event-free survival. [bib_ref] Surgical management of wildtype gastrointestinal stromal tumors: a report from the National..., Weldon [/bib_ref] In addition, SDH-deficient GISTs generally respond poorly to imatinib due to a lack of activating tyrosine kinase mutations. [bib_ref] Pediatric type" gastrointestinal stromal tumors in adults: distinctive histology predicts genotype and..., Rege [/bib_ref] A recent study noted that imatinib directly inhibits metabolic pathways in the submolecular arena of ATP yielding energy producing mitochondrial protein nanomotor functions, where the lack of a cellular ketogenic substrate reserve may explain its failure in SDH-deficient GISTs as well. [bib_ref] 13 C and 15 N natural isotope abundance reflects breast cancer cell..., Tea [/bib_ref] [bib_ref] Structural homologies between phenformin, lipitor and gleevec aim the same metabolic oncotarget..., Somlyai [/bib_ref] As mentioned above, in SDH-deficient GISTs, SDH inactivation leads to the accumulation of HIF-1a. Meanwhile, HIF, acting as an active transcription factor, induces the expression of downstream genes, including the insulin-like growth factor gene (IGF) and vascular endothelial growth factor (VEGF). This may explain how sunitinib, a small-molecule that possesses direct cytotoxicity and is an inhibitor of multiple receptor tyrosine kinases, including the VEGFR receptor in addition to PDGFRA and KIT receptors, works well in SDH-deficient GISTs. Interestingly, 4 patients in our study who suffered disease progression during imatinib treatment obtained disease control when the targeted therapy was changed to sunitinib. Furthermore, there is an apparent lack of fumaric acid formation due to SDH deficiency that decreases mitochondrial metabolic matrix water recycling, whereby the natural deuterium depleting capacity of tumor cells from fatty acids upon complete oxidation of the beta carbon are diminished. This is important as the deuterium depleted matrix water yield of mitochondrial ketogenic substrates is strictly dependent on Krebs-Szent--Györgyi cycle functions, where SDH plays a major role. Therefore, it may be clinically important to provide deuterium depleted water for cytoplasmic hydrogen exchange reactions that needs to be incorporated in dieting protocols to achieve more favorable clinical outcomes. [bib_ref] Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells, Lewis [/bib_ref] [bib_ref] Naturally occurring deuterium is essential for the normal growth rate of cells, Somlyai [/bib_ref] [bib_ref] Natural deuterium distribution in fatty acids isolated from peanut seed oil: a..., Duan [/bib_ref] [bib_ref] Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in..., Boros [/bib_ref] Moreover, IGF and its receptor show potential as therapeutic targets. [bib_ref] Succinate dehydrogenase-deficient GISTs are characterized by IGF1R overexpression, Chou [/bib_ref] In conclusion, although we are only able to study 12 cases in our institution, we found that SDH-deficient GISTs comprise a subgroup of a relatively rare tumor type and show a number of clinicopathologic and IHC unique features. The use of IHC to analyze loss of SDHB is reliable for detecting these tumors. SDHdeficient GISTs are restricted to stomach and do not occur infrequently in people older than 40 years. Lymph node metastasis, BVI, local recurrence, and distant metastasis are much more commonly in SDH-deficient GISTs than KIT/ PDGFRA-mutant GISTs. Nevertheless, they often follow an indolent course despite the high rate of metastasis. Moreover, SDH-deficient GISTs respond poorly to imatinib due to their lack of KIT/PDGFRA mutation, whereas sunitinib may work well on account of its ability to inhibit multiply receptor tyrosine kinases.
[fig] Figure 1: SDH-deficient GISTs (A and B, white arrows) are common with live metastasis (A and B, red arrows) at the time of initial diagnosis and often show multifocal gastric masses (C and D). [/fig]
[fig] Figure 2: Representative histological and IHC features of SDH-deficient GISTs. Tumors present epithelioid (A, H&E, Â100) or mixed epithelioid and spindle cell (B, H&E, Â100) morphology and characteristically show a multinodular or plexiform architecture (C, H&E, Â20). Lymphovascular invasion in the primary resection (D, H&E, Â400) and lymph node metastasis (E, H&E, Â100) are common findings. Traditional GISTs with SDHB expression (F, IHC staining, Â100). SDH-deficient GISTs present completely negative for SDHB immunostaining in tumor cells (G, IHC staining, Â100) but positive in mucosal cells (H, IHC staining, Â100, positive internal control). Liu et al. Medicine (2017) 96:32 Medicine commonly occurs in elderly patients. Both of these syndromes are underrecognized because of the rare asynchronous occurrence of the different tumor components in the same individual. [/fig]
[table] Table 1: Clinicopathological characteristics of 12 patients with SDHdeficient GISTs. [/table]
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Update on newer approaches to prevent or treat COVID-19 infection: What we all need the most right now!!
# Introduction
The COVID-19 pandemic is a new critical emerging human disease, where no vaccines, or monoclonal antibodies (mAbs), or drugs are currently available for preventative measures or therapy. Active vaccination, as the first choice, requires the induction of an immune response against a given agent by a susceptible individual for the purpose of preventing or treating the disease. Development of an effective vaccine can take time to be fully validated for use. The duration of effectiveness of a vaccine must be established, so is its capability and effectiveness as a public health tool can be established. Moreover, to develop an effective and appropriate of vaccine to the SARS-CoV-2 virus, it is advisable to target the spiky crown of the virus to prevent viral propagation via entry of the virus into the cell and to target the viral development machinery. Trials underway in the UK indicate that there is promising progress with the development of an effective vaccine. In addition, the use of available drug therapies, such as dexamethasone to treat lung inflammation or the antiviral Remdesivir, can reduce mortality. More data are expected in the future with the use of other relevant drugs.
COVID-19 convalescent plasma (CCP) therapy involves the use of circulating antibodies (Abs) administration from recovered COVID 19 patients, in particular after 2− 4weeks recovery, as a practical strategy to provide immediate passive immunity in susceptible recipients in need. The success of therapeutic use of convalescent plasma in Ebola cases is well established [bib_ref] Ebola virus convalescent blood products: where we are now and where we..., Burnouf [/bib_ref] and this has led many investigators worldwide to pursue developing COVID-19 convalescent plasma programs for compassionate and emergency use [bib_ref] Treatment for emerging viruses: convalescents plasma and COVID-19, Brown [/bib_ref] [bib_ref] Covid-19, induced activation of hemostasis, and immune reactions: Can an auto-immune reaction..., Amiral [/bib_ref] [bib_ref] Convalescent plasma, an apheresis research project targeting and motivating the fully recovered..., Seghatchian [/bib_ref] [bib_ref] How do we plan hematopoietic cell transplant and cellular therapy with the..., Dholaria [/bib_ref] [bib_ref] Treatment of 5 critically ill patients with COVID-19 with convalescent plasma, Shen [/bib_ref] [bib_ref] Reflection on passive immunotherapy in those who need most: some novel strategic..., Lanza [/bib_ref] [bib_ref] Coronavirus infections and immune responses, Li [/bib_ref] [bib_ref] Research and development of therapeutic agents and vaccines for COVID-19 and related..., Liu [/bib_ref].
Currently, to respond to the COVID-19 pandemic crisis many countries have adopted widespread public health initiatives for the testing, tracking, tracing, and isolation of those that are at risk of transmitting the SARS-CoV-2 virus. Through these initiatives, it has become possible to identify recovered COVID-19 individuals who can participate in large CCP programs. Currently in the USA and other countries, multiple randomized control trials are underway to evaluate the efficacy of CCP therapy. Such programs are highly complex undertakings and have required rigorous risk assessments, and detailed planning in order to ensure that there are the required resources and staff available to perform the targeted donor recruitment, plasma collection, testing, and product inventory management required [bib_ref] Treatment for emerging viruses: convalescents plasma and COVID-19, Brown [/bib_ref]. Establishing appropriate SARS-CoV-2 antibody titres for donor selection, evaluating concerns over residual levels of virus antigen in asymptomatic individuals and the need to adopt pathogen inactivation processes as part of standardizing apheresis collections are some of the additional challenges facing the rapid establishment of CCP programs.
At the time of preparing this article, more than 53,000 people in the United States have received COVID-19 convalescent plasma (www. uscovidplasma.org) and controlled clinical trials of CCP are underway around the world. Given the lack of other therapies available, many countries are making available CCP under "Expanded Access" or compassionate use programs, while at the same time conducting the clinical trials that will establish if CCP has any clinical benefit in patients affected by the disease. While limited studies have reported on the effectiveness of CCP in treating patients with severe disease [bib_ref] Treatment of 5 critically ill patients with COVID-19 with convalescent plasma, Shen [/bib_ref] [bib_ref] Effectiveness of convalescent plasma therapy in severe COVID-19 patients, Duan [/bib_ref] , these studies have served in guiding important decisions around donor selection and appropriate source and dose of neutralizing antibodies in CCP products. Importantly, as evidence emerges from large multi-site clinical trials, new indications for the collection, production and use of CCP will emerge which will allow for more targeted use of this product.
Global concern over the potential for "second" or "third" waves of infection to occur before effective vaccines or drug therapies are available has many looking at other biological sources for large-scale production of neutralizing SARS-CoV-2 antibodies. This report summarizes some of the novel strategies for developing alternative safe sources of therapeutic autologous antibodies from COVID -19 infected patients, and provides some original thoughts on how to rapidly implement a safe passive immunity in those COVID-19 patients who are most in need of intervention.
## Alternative sources of covid-19 antibodies for clinical use
To support the development of guidelines for COVID-19 management, systematic reviews and meta-analyses on the use of convalescent plasma in COVID-19 and other severe respiratory viral infections, including influenza and Ebola viral infections, have been performed [bib_ref] How do we plan hematopoietic cell transplant and cellular therapy with the..., Dholaria [/bib_ref] [bib_ref] Treatment of 5 critically ill patients with COVID-19 with convalescent plasma, Shen [/bib_ref] [bib_ref] Reflection on passive immunotherapy in those who need most: some novel strategic..., Lanza [/bib_ref] [bib_ref] Coronavirus infections and immune responses, Li [/bib_ref]. Intriguingly, in one meta-analysis the quality of evidence was very low for all efficacy outcome and raises the possibility that convalescence plasma has minimal or no benefit in the treatment of COVID-19. This highlights that there is an urgent need for optimizing the products to be used, more carefully establishing antibody titers, dose, and at what stage of disease such preventative or therapeutic modality can provide the most favorable clinical outcome to recipients.
In general, convalescent plasma is collected from eligible donors who have previously been confirmed positive for COVID-19 by a laboratory test but have recovered and are symptom-free for 14-28 d. Leukodepleted CCP from whole blood donation can be stored fresh as liquid plasma for up to 40 d or frozen for up to 12 months prior to use. However, COVID-19 antibodies can be isolated or delivered using a number of other techniques including: i) Plasmapheresis: Standardisation of plasma collection using modern mobile apheresis procedures can be used to collect single or double doses of CCP from an individual donor. As plasmapheresis infrastructure is well established and readily accessible to a large percentage of the population, this method of collecting large numbers of CCP has been adopted by many of the regional and national programs that have been launched to support ongoing clinical trials and compassionate / emergency access programs.
ii) Plasma Cryoprecipitate Reduced (Cryosupernatant) -Recently we have proposed the use of Plasma Cryoprecipitate Reduced (US) or Plasma, Fresh Frozen Cryoprecipitate Depleted (Europe) instead of apheresis plasma from recovered COVID-19 patients as a source of neutralizing SARS-CoV-2 antibodies [bib_ref] Convalescent plasma, an apheresis research project targeting and motivating the fully recovered..., Seghatchian [/bib_ref]. Since originally proposed by Judith Pool in 1964 [bib_ref] High-potency antihaemophilic factor concentrate prepared from cryoglobulin precipitate, Pool [/bib_ref] , most blood manufacturers are able to produce cryoprecipitate supernatant from rapidly frozen plasma. The plasma containing the neutralizing SARS-CoV-2 antibodies is rapidly frozen to less than -70 - C, in liquid nitrogen or a deep freezer and the cryoprecipitate, containing FVIII, fibrinogen and fibronectin is isolated after thawing overnight at 4 - C using a cold centrifuge and the cryosupernatant containing all immunoglobulins is transferred to a new bag and store in the refrigerator for up to 5 days if required before freezing. This cryosupernatant has found a unique place in therapeutic exchanges of thrombotic thrombocytopenic purpura (TTP) patients with enormous success, as it lacks the high molecular substances in plasma. Our suggestion is that leukocyte-depleted and virally inactivated cryosupernatant would remove the residual risk of SARS-CoV-2 infection and provide a safer product due to the depletion of large molecular weight plasma proteins than whole blood-derived and apheresis CCP. iii) Affinity Column Preparation of Antibody Hyperconcentrate -Affinity columns can be used to remove either the COVID-19 viral antigen or its antibodies from COVID-19 patients, blood products or the circulation of recovered COVID-19 donors to produce a hyperconcentrate for therapeutic use. Such procedures, purposely designed by some manufacturers of affinity columns to removal FIX antibodies, have been already developed and used with success in Malmo, Sweden and can be easily adopted to COVID-19 antibody removal. In fact, a capture ELISA, with a plate coated with recombinant ACE-2, or its complexes with S-Protein or its S1 subunit, as a specific receptor, have been designed for capturing allo-or autoantibodies present in COVID 19 patients; especially useful for those with delayed severe complications. This would allow measurement of the kinetics of these antibodies during disease evolution. If present, these antibodies are expected to be alloantibodies, induced by the association of the viral protein. A similar design could be constructed on an affinity column matrix to capture antibodies or antigens as required either in the circulation or from FFP from COVID-19 plasma. To enhance the safety of the antibody hyperconcentrate, the final products should be pathogen inactivated or UVC sterilized. iv) Other Therapeutic Alternatives -Several other protocols might be helpful to consider including platelet exchange instead of plasma exchange from COVID-19 patients to combat severe viral infection of the lungs. Platelet therapy or viral-inactivated platelet lysates which contain all of the active components in a platelet product are a routine procedure in many establishments and may be a new mode of therapy for COVID-19 patients. v) Advanced Cell-Based Delivery Systems -Instead of passive immune therapy through plasma exchange, local delivery of hyperconcentrated COVID-19 antibodies using engineered RBCs or other carriers may be a future consideration. RBC are considered to be the best natural drug delivery system due to their abundance, long circulation half-life and their well-established immune-biocompatibility and biodegradability. With wellestablished protocols for their safe collection, processing and storage this makes RBCs readily available to be used as delivery systems. The emergence of stem-cell derived RBCs provides further opportunities for developing customized antibody delivery systems for immune therapy [bib_ref] Drug delivery by erythrocytes, Villa [/bib_ref]. Currently, several drug-loading techniques such as encapsulation and surface conjugation have been used for the effective treatment of infections, cancer, and chronic autoimmune diseases [bib_ref] Drug delivery by erythrocytes, Villa [/bib_ref]. More recently, there is progressive evolution in the use of inhalable drug delivery systems for lung cancer therapy. Inhalation offers many advantages as it is a non-invasive method of drug administration as well as provides for localized delivery of anti-cancer drugs to lung tumors. Currently various inhalable colloidal systems exist for tumor-targeted drug delivery including polymeric, lipid, hybrid and inorganic nanocarriers. These approaches for enhanced delivery of nanocarriers to lung cancer cells were illustrated by the recent advances of inhalable microparticles-based drug delivery systems for lung cancer therapy including: large porous, solid lipid and drug-complex microparticles [bib_ref] Red cell membrane processing for biomedical applications, Rossi [/bib_ref]. Despite enormous progress in this field, nevertheless,s little is done in the direction of the potential application of RBC-based DDS in transfusion therapy in particular hypoxia that appears to be a common feature of COVID-19 patients even in the preclinical stages. Hence, deserved to be explored further by investigators with appropriate know how as a research protocol.
# Conclusions
In countries without access to advanced blood-processing technologies, choices may initially be restricted to convalescent plasmapheresis using modern apheresis tools that provide leukoreduced plasma for the treatment of COVID-19 patients. As ongoing clinical trials establish the optimal conditions for CCP therapy, data on effective neutralizing antibody concentrations and dose will help inform the development of more efficient plasmapheresis protocols. The use of two units of plasma, or plasma from two different individuals and /or even preparation of pools of immunoglobulins containing antibodies from various individuals may emerge as the most effective therapy. Cryosupernatant plasma or pools of cryosupernatant plasma may also be considered as evidence on the effectiveness of these protocols is collected. In technologically more advanced countries, additional options for pathogen inactivated and sterilized convalescent plasma or blood products such as immunoglobulins to SARS-CoV-2, including virally inactivated minipool plasma may be considered. This commentary has introduced various alternatives to the current use of COVID-19 convalescent plasma to treat COVID-19 patients. While the proposed technological options may, in some cases, be theoretical, the growing concern over the rapid spread of the SARS-CoV-2 virus has prompted many to pursue innovative and creative solutions to reduce the mortality and morbidity resulting from the current global pandemic. |
Application of the Biospeckle Method for Monitoring Bull’s Eye Rot Development and Quality Changes of Apples Subjected to Various Storage Methods—Preliminary Studies
# Introduction
Bull's eye rot, caused by species of Neofabraea, better known as Pezicula, is one of the most frequent and damaging fungal diseases affecting stored apples. It starts in the orchard, but its symptoms appear only several months after harvest (generally after 3-4 months in cold storage). Late maturing apple cultivars like 'Pinova' and 'Topaz' are particularly susceptible to the disease, with an incidence that can exceed 15-30% after 120 days of cold storage [bib_ref] Phylogenetic relationships among Neofabraea species causing tree cankers and bull's-eye rot of..., Jong [/bib_ref] [bib_ref] Effects of environmental factors and cultural practices on bull's eye rot of..., Henriquez [/bib_ref]. Early detection of infection is important for ensuring microbiological quality and safety of food commodities. Actually, non-destructive techniques has been applied in the evaluation and monitoring of biological properties [bib_ref] Actual pathogen detection: Sensors and algorithms-A review, Hahn [/bib_ref].
The biospeckle technique is a relatively new, non-invasive method to analyze the vitality of biomaterials. It is based on the optical phenomenon occurring during illumination of samples by coherent light. The scattered rays interfere with each other and form random, granular patterns consisting of dark and bright spots. If the illuminated sample does not show any biological activity, the images obtained are invariant. In the case of biological samples, however, the intensity distribution evolves and fluctuates in time [bib_ref] Temporal and spatial properties of the time-varying speckles of botanical specimens, Xu [/bib_ref] [bib_ref] Point-wise and whole-field laser speckle intensity fluctuation measurements applied to botanical specimens, Zhao [/bib_ref]. This was first observed by Abramson at the Stockholm Royal Institute of Technology [bib_ref] Wavelength dependence of intensity fluctuations in speckle patterns from biological specimens, Briers [/bib_ref] , who noticed that 'when an apple is illuminated with laser light the speckles move!'. According to Braga et al. [bib_ref] Live biospeckle laser imaging of root tissues, Braga [/bib_ref] processes such as cytoplasmic streaming, organelle movement, cell growth and division during fruit maturation and biochemical reactions are responsible for certain biospeckle activity. Brownian motion should also be taken into account [bib_ref] Point-wise and whole-field laser speckle intensity fluctuation measurements applied to botanical specimens, Zhao [/bib_ref]. Nevertheless knowledge about the biospeckle phenomenon in relation to fruits and vegetables is still limited, and there is a lack of consistent biological interpretations of the phenomenon.
Applications of the biospeckle technique in the agricultural area include monitoring of quality and ripening of fruits and vegetables, analysis of seeds or assessment of motility parameters [bib_ref] Temporal and spatial properties of the time-varying speckles of botanical specimens, Xu [/bib_ref] [bib_ref] Point-wise and whole-field laser speckle intensity fluctuation measurements applied to botanical specimens, Zhao [/bib_ref] [bib_ref] Biological activity measurement on botanical specimen surfaces using a temporal decorrelation effect..., Oulamara [/bib_ref] [bib_ref] Bio-speckle assessment of bruising in fruits, Pajuelo [/bib_ref] [bib_ref] Assessment of seed viability by laser speckle techniques, Braga [/bib_ref] [bib_ref] Laser speckle techniques in quality evaluation of orange fruits, Rabelo [/bib_ref] [bib_ref] Bio-speckle activity applied to the assessment of tomato fruit ripening, Romero [/bib_ref] [bib_ref] New nondestructive method based on spatial-temporal speckle correlation technique for evaluation of..., Zdunek [/bib_ref] [bib_ref] Comparison of puncture test, acoustic emission and spatial-temporal speckle correlation technique as..., Zdunek [/bib_ref] [bib_ref] Relation of biospeckle activity with quality attributes of apples, Zdunek [/bib_ref] [bib_ref] Relation of biospeckle activity with chlorophyll content in apples, Zdunek [/bib_ref] [bib_ref] Dynamic laser speckle to detect motile bacterial response of Pseudomonas aeruginosa, Sendra [/bib_ref] [bib_ref] Motility parameters assessment of bovine frozen semen by biospeckle laser (BSL) system, Carvalho [/bib_ref]. In all cases the biospeckle activity changed with the state of investigated sample. Analysis of temporal variation of the speckles was used to evaluate the presence of fungi in beans [bib_ref] Detection of fungi in beans by the laser biospeckle technique, Braga [/bib_ref]. Due to the fact that the laser light can penetrate apple tissue to a depth of 7-10 mm [bib_ref] Temporal and spatial properties of the time-varying speckles of botanical specimens, Xu [/bib_ref] it is possible to obtain information about biospeckle activity from tissue localized under the skin. This means that, there would be a chance to detect and monitor the development of different defects before their symptoms become visible on the fruit surface.
The aim of this work was to monitor bull's eye rot development by means of the biospeckle technique. Up to now, examples of biospeckle application for estimation of fruits quality have been shown [bib_ref] Bio-speckle assessment of bruising in fruits, Pajuelo [/bib_ref] [bib_ref] Laser speckle techniques in quality evaluation of orange fruits, Rabelo [/bib_ref] [bib_ref] New nondestructive method based on spatial-temporal speckle correlation technique for evaluation of..., Zdunek [/bib_ref] [bib_ref] Comparison of puncture test, acoustic emission and spatial-temporal speckle correlation technique as..., Zdunek [/bib_ref] [bib_ref] Relation of biospeckle activity with quality attributes of apples, Zdunek [/bib_ref] [bib_ref] Relation of biospeckle activity with chlorophyll content in apples, Zdunek [/bib_ref] , however the influence of storage methods on biospeckle dynamics in post-storage shelf life was not tested yet. The second aim was to analyze changes of biospeckle activity and quality attributes of apples during shelf life after storage under normal and controlled atmosphere conditions, including 1-metylocyclopropene treatment.
# Materials and methods
Apples (Malus domestica Borkh.) of the cultivars 'Pinova' and 'Topaz' were obtained from the Research Institute of Horticulture in Skierniewice, Poland. These cultivars were chosen due to their known susceptibility to bull's eye root. About 480 apples of each cultivar were harvested in October 2010 at their optimal harvest windows. For two experiments, the fruit were divided: (1) into a batch of 100 apples for non-destructive monitoring of bull's eye rot development, and (2) into a batch of about 380 apples for destructive measurements during shelf life after storage under various conditions.
In the first experiment, fruits were stored under a normal atmosphere (NA) at 2 °C for about 140 d. One hundred apples were tested non-destructively at eight dates with 20 d intervals. About eight apples were removed from NA, immediately tested at room conditions and then placed back to NA. In total, about 15 min were required for all measurements.
In the second experiment apples were stored under normal atmosphere at 2 °C (1NA, 2NA, 3NA, which mean respectively 1, 2 and 3 months of storage), and under controlled conditions (1 °C, 2% CO 2 , 2% O 2 ) for eight months (4CA). Additionally, one batch (5MCP) was treated by 1-metylocyclopropene (625 ppb, SmartFresh 03 VP, Rohm and Haas, Philadelphia, PA, USA) and then stored for eight months under CA atmosphere. In each of the five storage variants 75 apples were tested in shelf life simulation. Biospeckle activity and quality attributes (firmness, soluble solids content, dry matter content) were evaluated at 1, 3, 5, 7 and 10th day of shelf life.
## Biospeckle measurement
The device for biospeckle measurement was similar to that previously used by Zdunek et al. [bib_ref] New nondestructive method based on spatial-temporal speckle correlation technique for evaluation of..., Zdunek [/bib_ref] [bib_ref] Comparison of puncture test, acoustic emission and spatial-temporal speckle correlation technique as..., Zdunek [/bib_ref] [bib_ref] Relation of biospeckle activity with quality attributes of apples, Zdunek [/bib_ref] [bib_ref] Relation of biospeckle activity with chlorophyll content in apples, Zdunek [/bib_ref]. Its schematic illustration is presented in [fig_ref] Figure 1: Scheme of the biospeckle setup [/fig_ref]. In this study, two similar systems, equipped with differed laser for illuminating the sample were used. In the first experiment, and in the case of 1NA, 2NA and 3NA series from the second experiment, a He-Ne laser (1 mW, λ = 632.8 nm, LLR 811, Optel, Opole, Poland) was used. The laser beam was expanded by a microscope objective (10/0.24, 160/-, PZO, Poland).
For the 4CA and 5MCP experiments, a diode laser (8 mW, λ = 635 nm, LQC635-08C, supplied with Laser Diode Control Unit, Newport, Irvine, CA, USA) was used. The laser beam was expanded by a beam expander 20× (Edmund Optics GmbH, Karlsruhe, Germany).
In both systems, a CCD camera (Monochrome FireWire Astronomy Camera DMK 21AF04.AS, The Imaging Source Europe GmbH, Bremen, Germany) with a 25 mm TV lens 1:14 and 20 mm extension ring (Pentax Corporation, Tokyo, Japan) was used as detector of scattered light. The distance between camera and apple was about 100 mm, and that between laser and apple 180 mm. The illumination angle was Θ ≈ 30°. Biospeckle movies lasting 4 s were recorded in uncompressed AVI film form (8 bits, RGB24 codec) at a 15 fps rate. The image exposure time of the CCD camera was 1/250, gain and brightness were set experimentally to avoid pixel overexposure on the image histogram. The image resolution was 320 × 240 pixels which corresponded to observation area of about 3 × 2 mm. These settings ensured avoidance of apple curvature. The parameters of image acquisition were kept unchanged during the experiment.
Biospeckle activity was evaluated using the correlation coefficient C kτ , where k is a frame number and τ is a lag time (1/15 s) [bib_ref] Relation of biospeckle activity with quality attributes of apples, Zdunek [/bib_ref] [bib_ref] Relation of biospeckle activity with chlorophyll content in apples, Zdunek [/bib_ref]. C kτ was calculated as the correlation coefficient of data matrix, consisting of intensities of pixels, of the first frame with the data matrixes of the following frames from the analyzed biospeckle movies. In this study, C 4 was analyzed only as the correlation coefficient between the first frame and the frame at kτ = 4 s. The time of 4 s was chosen to obtain a reasonable decrease of C kτ which was down to 0.2 for some apples and in the case of lasers and optics used. Then, a BA = 1-C 4 value was determined as the biospeckle activity parameter for a certain sample. Higher biospeckle activity corresponds to higher 1-C 4 value. Correlation coefficient C was calculated using "corrcoef" function in Matlab® R2010a software (MathWorks, Natick, MA, USA). Each apple was tested at six points localized on the fruit equator in the experiment 1, or at two opposite sites in experiment 2. As a result, mean values of BA were calculated.
## Firmness measurements
Firmness of the apples was measured in a non-destructive way with an acoustic impulse response technique (AFS impact, AWETA, G&P, Nootdorp, The Netherlands). In this method only two parameters are needed to obtain the firmness index (FI): the resonant frequency of the first elliptical mode and the mass of the fruit [bib_ref] An interpretation of the resonant behaviour of intact fruits and vegetables, Cooke [/bib_ref] [bib_ref] Postharvest firmness changes as measured by acoustic and low-mass impact devices: A..., De Ketelaere [/bib_ref]. In experiment 2 each sample was measured at two opposite sites and as a result the mean value of FI for each fruit was obtained.
Moreover, in experiment 2, apple firmness was also measured destructively using a universal testing machine Lloyd LRX (Lloyd Instruments Ltd, Hampshire, UK) with a 500 N load cell. Ten apples were punctured by a cylindrical Magness-Taylor probe (11.1 mm diameter) at a speed of 20 mm min −1 . Before the test, the apple skin was removed. Maximum force, needed to penetrate the flesh over a distance of 8 mm was read as the apple firmness (F) and expressed in N.
## Soluble solids content
Soluble solids content (SSC) was determined using a digital refractometer (PAL-BX/RI, Atago Co. Ltd., Tokyo, Japan). Freshly squeezed juice was poured onto the prism. The results were obtained in Brix degrees. Measurements were carried out for five apples in four replicates.
## Dry matter content
Samples of five apples (approximately 20 g) were cut into small pieces and dried (SUP-30W, Wamed, Poland) at 105 °C to constant mass. Dry matter content (DM) was calculated as DM = (m 2 /m 1 ) × 100, where m 1 was the mass of the fresh sample and m 2 -mass of the dried sample.
# Statistical analysis
Statistical analysis was performed using Statistica 8.0 (StatSoft, Inc., Tulsa, OK, USA). In the experiment 1 storage effect was tested by one-way ANOVA to show differences between measured biospeckle activity during apple fungal disease development. Post-hoc analysis were performed using the HSD Tukey test. In the experiment 2 basic statistics (mean values and standard deviations) were calculated for quality attributes and biospeckle activity measured in the shelf life simulations. Moreover two-way ANOVA was used for testing series*day effect. Pearson's correlation coefficients between mean BA values and other mean quality attributes were also determined. The effects were tested using F-value and the significance level was evaluated at p < 0.05.
# Results
## Infection development in experiment 1
After about two months of storage, the first hardly visible spots appeared on the skin of two apples of both cultivars [fig_ref] Figure 2: Biospeckle activity BA [/fig_ref]. The circular and light-brown areas constantly increased in size and darkened with time. As a result, 34% of 'Pinova' and 21% of 'Topaz' apples were severely diseased after ~140 d. Symptoms of infections were always more pronounced in 'Pinova' apples [fig_ref] Figure 2: Biospeckle activity BA [/fig_ref]. Bull's eye rots in this case were mostly observed close to the equator of apples, whereas in 'Topaz', the spots usually appeared close to the stem or calyx end.
Each mean BA value, presented in [fig_ref] Figure 2: Biospeckle activity BA [/fig_ref] , was calculated from 600 measurements (100 apples × 6 places on the equator). Standard deviations of BA are relatively large however due to number of repetitions observed changes were often significant and allowed making conclusions about trends. Overall effect of storage showed significant change of biospeckle activity (F-value > 38, p < 0.05). With one exception, BA change trends were similar for apples of both cultivars. On day 26 of storage, however, BA of 'Topaz' apples occasionally increased [fig_ref] Figure 3: Biospeckle activity BA [/fig_ref]. Despite this, BA of healthy apples seemed to decrease ('Pinova') or, at least, remained constant ('Topaz') during the initial 40 d of storage. In the next stage, (~40-100 storage days) BA significantly increased as the post-hoc analysis showed. In this period small, light-brown, circular spots appeared. Prolonged storage (~100-140 d) caused pronounced visible symptoms and death of the tissue. This is accompanied by significant decrease of biospeckle activity .
Due to the experimental set up, the locations of BA measurements were preliminary fixed and were monitored in the same places. Since bull's eye rot development was not controlled, as a result, direct BA measurements of bull's eye rot spots exactly in the same place were very rare. Comparison of biospeckle activity changes for infected and health place on the same apple in a specific case where rotting appeared exactly in monitoring area confirmed results in [fig_ref] Figure 2: Biospeckle activity BA [/fig_ref]. BA decreased during the 40 d of cold storage. Then in both healthy and infected places biospeckle activity increased but for the decaying place it happened faster. The first bull's eye rot hardly visible symptoms occurred at 85th day of NA storage in this case. It suggests that maybe the increase of BA at 60th day, when the change 40-60 d was already significant, would be used for prediction of infection. Finally, BA decreased and especially infected part showed very low activity.
## Figure 4.
Biospeckle activity BA of one selected 'Pinova' apples for healthy and decaying tissue during storage. Photographic documentation of apple bull's eye rot development is also given. Lesion area is marked by black arrow and red circle presents laser illumination point.
## Biospeckle activity after various storage methods (experiment 2)
Decrease in BA during shelf life was observed for 1NA series of both tested cultivars. In 'Pinova' apples, biospeckle activity changed slightly during 2NA shelf life simulation and increased during 3NA . The highest value of biospeckle activity was obtained in general for 4CA series but also a decrease of BA was noted. After 1-MCP treatment (5MCP) biospeckle activity also decreased. BA of 'Topaz' decreased in the case of 1NA and 2NA shelf-life experiments . For 3NA biospeckle activity increased from 0.48 (1st day) to 0.59 (10th day). During storage under 4CA and 5MCP conditions, no clear trend in changes of BA could be detected. Firmness gradually decreased in the case of 1NA, 2NA, 4CA and 5MCP, both in 'Pinova' and in 'Topaz' apples . The lowest values of IF and F and the least pronounced shelf life effect was obtained in the 3NA experimental stage. Dry matter content DMC and total soluble solids content SSC generally increased during apple storage. The highest values of DMC were obtained at day 10 in the 5MCP series (18.60% for 'Pinova' and 18.03% for 'Topaz'). 2NA program was characterized by the highest mean SSC, and it fluctuated from 14.2 to 15.7° Brix for cv. 'Pinova' and 12.7-14.1° Brix for cv. 'Topaz'. In turn the lowest mean values of SSC was obtained for 4CA series in both cases. . Mean values of biospeckle activity (BA) and quality attributes for 'Pinova' (F-firmness, FI-firmness index, SSC-soluble solids content, DMC-dry matter content) of apple during shelf-life. SD-standard deviation, the same letters a-e mean no significant difference at the level of α = 0.05 between superscripted values.
## Series
Shelf life (days) BA ± SD F (N) ± SD FI ± SD SSC (°Brix) ± SD DMC (%) ± SD . Mean values of biospeckle activity (BA) and quality attributes for 'Topaz' (F-firmness, FI-firmness index, SSC-soluble solids content, DMC-dry matter content) of apple during shelf-life. SD-standard deviation, the same letters a-e mean no significant difference at the level of α = 0.05 between superscripted values.
## Series
Shelf life (days) BA ± SD F (N) ± SD FI ± SD SSC (°Brix) ± SD DMC (%) ± SD Two-way ANOVA [fig_ref] Table 3: F-value and p-values of two-way ANOVA for combined series*day effect for individual... [/fig_ref] showed that combined effects of storage method and shelf life were significant (p < 0.05) for measured parameters with the exception of DMC for 'Pinova' (p = 0.19). presents summarized Pearsons' correlation coefficients (R) calculated among mean values of variables analyzed in this experiment. Strong correlation between BA and FI was observed for 'Topaz' (R = 0.84, p < 0.05), whereas in other cases R-values were weak but still significant at the level of p < 0.05. For 'Pinova' apples, correlations between BA and quality attributes were not significant (p > 0.05) with the exception of SSC, for which R = −0.40 (p < 0.05).
# Discussion
It is believed that biospeckle fluctuations result from the elastic light scattering on moving particles such as cellular organelles. Any disturbance of this movement would be detected as a BA change. Thus, biospeckle activity fluctuation, presented in [fig_ref] Figure 2: Biospeckle activity BA [/fig_ref] , may be related to pathological changes occurring in apple tissues during the development of fungal infection. In the first period, before visible symptoms appeared, BA decreased, probably due to starch granule degradation [bib_ref] Relation of biospeckle activity with quality attributes of apples, Zdunek [/bib_ref]. The decrease of biospeckle activity during storage was previously reported by other authors [bib_ref] Temporal and spatial properties of the time-varying speckles of botanical specimens, Xu [/bib_ref] [bib_ref] Point-wise and whole-field laser speckle intensity fluctuation measurements applied to botanical specimens, Zhao [/bib_ref] [bib_ref] Biological activity measurement on botanical specimen surfaces using a temporal decorrelation effect..., Oulamara [/bib_ref] [bib_ref] Laser speckle techniques in quality evaluation of orange fruits, Rabelo [/bib_ref] [bib_ref] New nondestructive method based on spatial-temporal speckle correlation technique for evaluation of..., Zdunek [/bib_ref] [bib_ref] Comparison of puncture test, acoustic emission and spatial-temporal speckle correlation technique as..., Zdunek [/bib_ref].
According to the literature data [bib_ref] Plant physiology meets phytopathology: Plant primary metabolism and plant-pathogen interactions, Berger [/bib_ref] fungal attack results in elevated level of ethylene concentration and increase in transpiration and this could be a reason for observed increase in biospeckle activity between the ~40th and ~100th day.
During infection, a number of biologically active substances, like enzymes, toxins and growth regulators, are released by the fungal pathogens, which may affect the structural integrity of the host cells or their physiological process. Production of pectolytic enzymes causes pectin degradation and leads to plant tissue maceration, i.e., softening, loss of coherence and separation of individual cells, which eventually die [bib_ref] The importance of fungal pectinolytic enzymes in plant invasion, host adaptability and..., Reignault [/bib_ref] [bib_ref] Pectin-degrading enzymes and plant-parasite interactions, Alghisi [/bib_ref]. Death of the apple tissue was responsible for decrease in biospeckle activity during prolonged storage (~100-140 d).
During apple ripening many biochemical reactions take place that could be responsible for variable biospeckle. Chlorophyll degradation causes a BA increase due to a decrease of light absorption and deeper light penetration [bib_ref] Relation of biospeckle activity with chlorophyll content in apples, Zdunek [/bib_ref] , whereas starch degradation has an opposite effect: BA increases due to decreases in the number of particles acting as light scattering centers [bib_ref] Relation of biospeckle activity with quality attributes of apples, Zdunek [/bib_ref]. These two processes usually occur during fruit ripening. In previous studies apparent biospeckle activity decreased just after harvest in shelf life experiments [bib_ref] Comparison of puncture test, acoustic emission and spatial-temporal speckle correlation technique as..., Zdunek [/bib_ref]. Similar results were obtained in present experiment, suggesting that starch hydrolysis plays a more important role than chlorophyll degradation for biospeckle activity. NA, CA and 1-MCP treatments are followed by a decrease in BA during shelf life. showed that soluble solids content also affects biospeckle activity: higher SSC was reflected as lower BA. However, at present it is difficult to interpret the reasons behind this relationship.
# Conclusions
This work revealed that biospeckle reflects biological activity occurring inside apples and on apple skins during bull's eye rot development. An increase of biospeckle activity was observed when disease symptoms were hardly visible. Senescence resulted in a decline of biospeckle activity due to reduction of life processes within tissue. This showed that this method would be used in a future for nondestructive monitoring of pathogen infection development. This experiment showed that a limit of detection would be at least comparable to that of visual inspection. However to estimate this limit precisely further study is needed in model experiments, with artificial inoculation, where biospeckle activity will be evaluated exactly in the place of infection.
This study also showed that, irrespective of storage conditions, biospeckle activity decreased during shelf life. In the case of 'Topaz' BA correlated significantly (p < 0.05) with quality attributes (firmness, firmness index, soluble solids content, dry matter content), whereas for 'Pinova' a significant correlation was obtained only between BA and SSC. It can be concluded that there is a chance to monitor postharvest quality of apples, in relation to biochemical changes, by this non-destructive method.
[fig] Figure 1: Scheme of the biospeckle setup. [/fig]
[fig] Figure 2: Biospeckle activity BA (black line) and percentage of infected fruit (red line) during development of bull's eye rot of 'Pinova' apples. Bars indicate standard deviation; different letters denote significant differences (at α = 0.05) between means. [/fig]
[fig] Figure 3: Biospeckle activity BA (black line) and percentage of infected fruit (red line) during development of bull's eye rot of 'Topaz' apples. Bars indicate standard deviation; different letters denote significant differences (at α = 0.05) between means. [/fig]
[table] Table 3: F-value and p-values of two-way ANOVA for combined series*day effect for individual cultivars. BA means biospeckle activity, F-firmness, FI-firmness index, SSC-soluble solids content, DMC-dry matter content.Table 4. Pearson's correlation coefficients (R) for the correlation between biospeckle activity (BA) and other quality attributes (F-firmness, FI-firmness index, SSC-soluble solids content, DMC-dry matter content), measured for 'Pinova' and 'Topaz' apples. Asterisks denote significance at p < 0.05. [/table]
|
Long Noncoding RNA Serve as a Potential Predictive Biomarker for Breast Cancer: A Meta-Analysis
Purpose. The detection of long noncoding RNA (lncRNA) is a novel method for breast cancer diagnosis. The purpose of this metaanalysis was to evaluate the clinical significance of lncRNAs in identification of human breast cancer. Methods. Electronic databases, including PubMed (176), EMBASE (167), Cochrane Library (4), Web of Science (273), CNKI (41), VIP (18), and wanfang (21), were searched for relevant original articles. Diagnostic capacity of lncRNAs was assessed by pooled sensitivity and specificity, area under the summary receiver operating characteristic curve (AUC), diagnostic odds ratio (DOR), and subgroup and metaregression analysis. Stata and Meta-Disc software were used to conduct the meta-analysis. Results. 33 articles including 4500 cases were identified in our meta-analysis. lncRNAs sustained a high diagnostic efficacy; the pooled sensitivity, specificity, AUC, and DOR of lncRNAs in differentiating BC from controls were 0.74 (95% CI: 0.69-0.78), 0.78 (95% CI: 0.72-0.83), 0.82 (95% CI: 0.79-0.85), and 10.01 (95% CI: 7.13-14.06), respectively. The subgroup analysis showed that the diagnostic efficacy of lncRNAs in Asian populations was higher than that in Caucasians; lncRNAs in BC were lower than those in TNBC and were higher in plasma and serum specimens than in tissues. In addition, heterogeneity was clearly apparent but was not caused by the threshold effect. Conclusion. This meta-analysis suggested that lncRNAs might be promising biomarkers for identifying breast cancer, and its clinical application warrants further investigation.
# Introduction
Breast cancer (BC) is the most common type of cancer in women worldwide. The proportion of BC in the incidence and mortality of female malignant tumors is increasing year by year. The incidence of BC ranks first among female malignant tumors in 161 countries, and the mortality of BC ranks first among 98 countries. Meanwhile, according to the 2013 Global Burden of Disease study, BC represented 24.2% of all cancer cases and 15% of all cancer-related deaths among females. In 2018, the American cancer center estimated that new cases of ductal and lobular carcinoma in situ (CIS) breast cancer in the United States would be about 266,120 and 63,960, respectively. And it alone was anticipated to account for 30% of all new cancer diagnoses in women. According to an annual report on status of cancer in China, 2017, about 279,000 new BC cases are reported in China each year, up more than 2% each year.
Histological evaluation of biopsy is the gold diagnosis standard for BC, which is invasive and fails to diagnose cancer at an early stage. Mammography X-ray imaging is widely used in BC screening and detection, but its application in radiation is limited. Currently, several specific biomarkers, such as the carcinoembryonic antigen (CEA), carbohydrate antigen , progesterone receptor (PR), and estrogen receptor (ER), have been employed extensively to diagnose BC in clinic. However, they are not sensitive and accurate enough to predict the prognosis of BC. Therefore, highly effective diagnostic and biomarkers are needed for early detection of BC to provide precise and personalized treatment for patients.
Recently, genome-wide transcriptome studies have confirmed the existence of a large number of long noncoding RNAs (lncRNAs) in the organism, which in the past were dismissed as simply transcriptional "noise". lncRNAs are non-protein-coding RNA molecules with a sequence longer than 200 nucleotides. Mounting evidence indicated that lncRNAs are an important layer of the genome regulatory network and work via diverse mechanisms in a series of biological processes, including chromatin modification, transcriptional regulation, and posttranscriptional regulation. lncRNAs play important roles in BC biological processes, such as increasing cell proliferation, migration, and invasion abilities, as well as epithelial-to-mesenchymal transition. One of the most studied lncRNAs, MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) promotes tumor growth by regulating cell cycle. Downregulation of MALAT1 in vitro in cell lines of different cancer types leads to reduced cell proliferation by cell cycle arrest in G2/M phase and to cell apoptosis. This ultimately leads to a decrease in the ability of cells to invade and migrate. The lncRNA-HOTAIR (Hox transcript antisense intergenic RNA) is upregulated in primary breast tumors and metastases, and its overexpression is associated with enhanced BC metastasis. Multiple tumor-related lncRNAs have been found in cell lines, tissues, and body fluid of cancer patients. Therefore, these molecules are considered as potential molecular biomarker for cancer diagnosis, prognosis prediction, and therapeutic targets.
The diagnostic efficacy of lncRNAs in BC has been proved by many recent studies. However, which kind of lncRNA has higher diagnostic efficacy or lncRNA in which kind of sample might be more appropriate for BC diagnosis is inconsistent in different studies. For instance, the sensitivity and specificity of RP11-445H22.4 for BC were 92 and 74%, respectively, while those of MALAT1 were 71% and 75%, respectively. Thus, we conducted a metaanalysis to evaluate the diagnostic efficacy of lncRNAs in identification of BC, which was intended to provide valid evidence for further studies about lncRNA.
# Materials and methods
2.1. Literature Search Strategy. This review was registered in the PROSPERO International Prospective Register of Systematic Reviews and the registration number was CRD42019139914. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) was followed. Two researchers searched the electronic databases (PubMed, EMBASE, Cochrane Library, Web of Science, Chinese National Knowledge Infrastructure Database (CNKI), VIP, and wanfang) from the start of each database up to June 1, 2019. The search strategy used both MeSH terms and free textwords to increase the sensitivity of the search. The following search terms were included: "breast cancer", "lncRNA", and "diagnosis". We searched PubMed using the following strategy: ("Breast Neoplasms" OR "Breast Neoplasm" OR "Neoplasm, Breast" OR "Breast Tumors" OR "Breast Tumor" OR "Tumor, Breast" OR "Tumors, Breast" OR "Neoplasms, Breast" OR "Breast Cancer" OR "Cancer, Breast" OR "Mammary Cancer" OR "Cancer, Mammary" OR "Cancers, Mammary" OR "Mammary Cancers" OR "Malignant Neoplasm of Breast" OR "Breast Malignant Neoplasm" OR "Breast Malignant Neoplasms" OR "Malignant Tumor of Breast" OR "Breast Malignant Tumor" OR "Breast Malignant Tumors" OR "Cancer of Breast" OR "Cancer of the Breast" OR "Mammary Carcinoma, Human" OR "Carcinoma, Human Mammary" OR "Carcinomas, Human Mammary" OR "Human Mammary Carcinomas" OR "Mammary Carcinomas, Human" OR "Human Mammary Carcinoma" OR "Mammary Neoplasms, Human" OR "Human Mammary Neoplasm" OR "Human Mammary Neoplasms" OR "Neoplasm, Human Mammary" OR "Neoplasms, Human Mammary" OR "Mammary Neoplasm, Human" OR "Breast Carcinoma" OR "Breast Carcinomas" OR "Carcinoma, Breast" OR "Carcinomas, Breast") AND ("RNA, Long Noncoding" OR "Noncoding RNA, Long" OR "lncRNA" OR "Long ncRNA" OR "ncRNA, Long" OR "RNA, Long Non-Translated" OR "Long Non-Translated RNA" OR "Non-Translated RNA, Long" OR "RNA, Long Non Translated" OR "Long Non-Coding RNA" OR "Long Non Coding RNA" OR "Non-Coding RNA, Long" OR "RNA, Long Non-Coding" OR "Long Non-Protein-Coding RNA" OR "Long Non Protein Coding RNA" OR "Non-Protein-Coding RNA, Long" OR "RNA, Long Non-Protein-Coding" OR "Long Noncoding RNA" OR "RNA, Long Untranslated" OR "Long Untranslated RNA" OR "Untranslated RNA, Long" OR "Long ncRNAs" OR "ncRNAs, Long" OR "Long Intergenic Non-Protein Coding RNA" OR "Long Intergenic Non Protein Coding RNA" OR "LincRNAs" OR "LINC RNA") AND ("Sensitivity and Specificity" OR "Specificity and Sensitivity" OR "Sensitivity" OR "Specificity" OR "Diagnose" OR "Diagnosis" OR "Diagnostic" OR "Predictive Value of Tests" . Additionally, the reference lists of all included articles were also consulted to locate additional references of interest.
## Inclusion and exclusion
Criteria. The inclusion criteria were as follows: (1) patients were diagnosed with BC by histopathological examination; (2) studies evaluated the diagnosis capacity of lncRNA for BC; (3) trials were described as case-control study; (4) studies must provide enough data to evaluate diagnosis value of lncRNA in BC; and (5) all the publication languages were restricted to Chinese and English.
Trials were excluded if they did not meet the criteria above and included the following: (1) reviews, abstracts, letters, meeting, and case reports;animal studies or in vitro studies; (3) studies in respect of survival or prognosis of BC; (4) the research could not find the outcome measurements; and (5) duplicate publications. Two independent researchers assessed and selected related studies to exclude the references which did not meet the inclusion criteria. If the content of a study arouse controversy, it would be discussed until agreement was reached.
## 2
BioMed Research International 2.3. Data Extraction and Quality Assessment. Two independent investigators performed the data extraction using a standardized data collection form. The following information was collected from each study: (1) basic information including first author, publication year, and country;the information about patients: ethnicity, sample type, sample size, and specimen source; (3) the information of methods: detection methods, lncRNA type, expression status, and reference gene; and (4) the outcomes: cutoff value and diagnostic 4-fold contingency table (TP, FP, FN, and TN). The Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) was applied to assess the quality of eligible studies and the risk of bias. In QUADAS-2 tool, patients through the patient selection, index test, and reference standard as well as flow and timing were discussed by 4 key domains and each of which contained applicability concerns and risk of bias. A score of 1 was given to low risk of bias or high concern about applicability, 0 to high or unclear risk or low or unclear concern. And any discrepancies were resolved through discussion.
## Statistical
Analysis. The application value of lncRNA for diagnosing BC was assessed through calculating the corresponding 95% CI, sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic OR (DOR). The property of diagnostic tests was evaluated by DOR and summary receiver operator characteristics (SROC), and the pooled AUC value was calculated. Heterogeneity among studies was estimated using Cochran's Q statistic and I 2 tests. P < 0:05 or I 2 > 50% was defined to have heterogeneity. We calculated sensitivity and specificity with their corresponding 95% CI using a bivariate model. The random effects model was used when there was significant statistical heterogeneity; otherwise, the fixed effects model was used. It was necessary to make a subgroup analysis and sensitivity analysis to seek the source of the heterogeneity. Visual funnel plot and quantifiable Deeks' funnel plot were used to identify the potential publication bias. P < 0:05 was considered significant. All these analyses were conducted using Meta-Disc 1.4 (XI Cochrane Colloquium, Barcelona, Spain) and Stata 15.0 (Stata Corporation, College Station, TX, USA).
# Results
## Study
Characteristics and Quality Assessment. The detailed screening process was shown in. A total of 700 articles were identified from the online database, and 480 articles were left after removing duplication. After screening the titles and abstracts and assessing the full articles, 33 eligible studieswere identified in the meta-analysis. These diagnostic studies involved 2425 patients and 2075 paired controls, and all participants were Asian and Caucasian. Most of the selected articles were published within the last 5 years. Regarding the specimen types, 9 studies examined tissue samples, 10 studies examined serum samples, 11 studies examined plasma samples, 2 studies examined plasma exosomal samples, and 1 study examined urine samples. Method of testing in all studies was real-time PCR, and lncRNAs were normalized by GAPDH, β-actin, and U6. These 33 selected cohorts were divided into 46 individual studies according to 30 lncRNAs (such as MALAT1, HOTAIR, and H19). The characteristics of the included studies were shown in. The Quality Assessment of Diagnostic Accuracy Studies 2 confirmed that all enrolled articles achieved a relatively high score equal or larger than 4, suggesting that these studies were reliable to be synthesized in the meta-analysis. The quality assessment was shown in .
3.2. Diagnostic Efficacy of lncRNAs. Significant heterogeneity was observed between studies for the high I 2 values in sensitivity (88.90%, P < 0:001), specificity (87.50%, P < 0:001), PLR (83.14%, P < 0:001), and NLR (90.56%, P < 0:001). Therefore, the random effects model was adopted for further analysis. Forest plots of the results for the enrolled studies on the pooled sensitivity, specificity, and DOR were shown in Figures 3 and 4. The pooled sensitivity, specificity, and DOR of lncRNAs in differentiating BC from controls were 0.74 (95% CI: 0.69-0.78), 0.78 (95% CI: 0.72-0.83), and 10.01 (95% CI: 7.13-14.06), respectively. The summary receiver operator characteristic (SROC) curve was plotted and the AUC was calculated to be 0.82 (95% CI: 0.79-0.85) , indicating an overall high diagnostic accuracy as a diagnostic test. Moreover, the pooled positive likelihood ratio (PLR) and negative likelihood ratio (NLR) also were calculated 3.35 (95% CI: 2.67-4.21) and 0.33 (95% CI: 0.28-0.40), respectively.
# Analysis diagnostic threshold
Effect. Threshold effect and nonthreshold effect are two important reasons for heterogeneity of diagnostic tests. In this study, Spearman's correlation coefficient of sensitivity and specificity was selected as the representative way of exploring the threshold effect. Since the corresponding Spearman correlation coefficient was 0.178 with a P value of 0.236 (P > 0:05), there was no heterogeneity from the threshold effect. In addition, the value of I 2 > 50% indicated that there was heterogeneity of nonthreshold effect among these studies.
## Subgroup and meta-regression analysis.
In order to illustrate the source of heterogeneity, we performed subgroup analyses based on ethnicity, pathologic type, specimen, dysregulated state, and type of lncRNA. The details were shown in. We found that the pooled sensitivity, specificity, and AUC of the studies were 0.76 (95% CI: 0.71-0.80), 0.80 (95% CI: 0.75-0.84), and 0.84 (95% CI: 0.81-0.87) for Asian versus 0.75 (95% CI: 0.71-0.78), 0.67 (95% CI: 0.49-0.80), and 0.72 (95% CI: 0.52-0.86) for Caucasian. When stratified by pathologic types, the diagnostic effect of lncRNAs in BC was lower than that in TNBC (AUC: 0.81 (95% CI: 0.77-0.84) versus 0.87 (95% CI: 0.84-0.90)). Subgroup analysis of specimens showed no significant difference in diagnostic accuracy between plasma and serum, with AUC of 0.86 (95% CI: 0.82-0.89) versus 0.86 (95% CI: 0.83-0.89), sensitivity of 0.76 (95% CI: 0.70-0.81) versus 0.78 (95% CI: 0.70-3 BioMed Research International 0.85), and specificity of 0.83 (95% CI: 0.76-0.88) versus 0.80 (95% CI: 0.70-0.87). However, both of these exhibited higher diagnostic accuracy than tissues, for which the pooled sensitivity, specificity, and AUC were 0.65 (95% CI: 0.52-0.76), 0.71 (95% CI: 0.57-0.83), and 0.73 (95% CI: 0.69-0.77), respectively. The diagnostic performance of lncRNA expression status suggested that upregulated lncRNAs (AUC: 0.84 (95% CI: 0.81-0.87)) were higher than downregulated lncRNAs (AUC: 0.70 (95% CI: 0.65-0.73)). In the metaanalyzed data based on types of lncRNA, lncRNA-MALAT1 sensitivity was 0.81 (95% CI: 0.71-0.88), specificity was 0.77 (95% CI: 0.68-0.84), and AUC was 0.85 (95% CI: 0.82-0.88), which were superior to lncRNA-HOTAIR and lncRNA-H19.
Meta-regression analysis indicated that ethnicity (P = 0:186), pathologic type (P = 0:428), specimen (P = 0:157), and type of lncRNA (P = 0:296) did not significantly affect the pooled results. However, lncRNA expression status was associated with study heterogeneity (P = 0:070).
## Sensitivity analysis and publication
Bias. Sensitivity analysis was used to test the stability of the overall effects and the change of heterogeneity by excluding a single study. None of the individual studies was out of the upper or lower CI limits, indicating that the selected studies were homogeneously distributed .
The publication bias among studies was assessed by Deeks' funnel plot asymmetry test. The symmetry of the funnel plot and a P value of 0.35 indicated that there was no significant publication bias in the diagnostic meta-analysis for lncRNAs .
3.6. Clinical Utility of lncRNAs in the Diagnosis of BC. Fagan's nomogram was used to describe the diagnosis value of lncRNAs for BC . When 50% was selected as the pretest probability, the data indicated that, if the result of a diagnostic test was positive, the posttest probability that the individual suffered BC would be approximately 77% (red line). If the result was negative, the posttest probability that the participant was affected with BC would be approximately (108) Not diagnosis (133) Not breast cancer (68) Not lncRNA (38) Studies included in quantitative synthesis (meta-analysis) (N = 33)
Records were excluded by details (N = 40): Reviews and case reports (15) Survival or prognosis studies (7) Without complete data (5) Data duplication
# Discussion
BC is one of the top killers in women health. Given that the disease in early stages often appears with nonspecific symptoms, advanced or terminal diagnosis may occur by the time symptoms develop, with poor prognosis and poor treatment effect. Mammography, ultrasonography, and magnetic resonance imaging (MRI) are routinely used for the detection of breast abnormalities. However, the sensitivity of mammography is moderate and can be affected by age and the density of the breast tissue. Breast ultrasonography has a high rate of false-negative results when used in BC screening, particularly in women with dense breast tissue. The high cost of breast MRI makes it inappropriate for use in screening for early BC. Thus, there remains a considerable need for identification of novel biomarkers and better understanding of the molecular mechanisms underlying BC. Increasing evidence suggested that lncRNAs are dysregulated in many different cancers, including BC. And the expression patterns of lncRNAs exhibit greater tissue specificity compared with protein-coding genes. The functions of lncRNAs are complicated because they can function as oncogenic or tumor suppressor genes and. Recent studies have shown that lncRNAs are tumor-derived nucleic acids that can be released into the peripheral circulation and detected in the plasma and serum. They can function as a competing endogenous RNA (ceRNA) that regulates other RNA transcripts by regulating specific miRNAs and the corresponding target genes. For instance, lncRNA-HOTAIR is overexpressed in patients with BC, and its deregulation is correlated with enhanced BC metastasis. lncRNA GAS5 inhibits the pathogenesis and progression and regulates the expression of PDCD4 of BC by acting as miR-21 "sponge". However, the function and overall clinical significance of the vast majority of lncRNAs in BC remain largely undetermined. Moreover, recent studies have demonstrated that lncRNAs exhibit greater expression restriction, and several lncRNAs have shown the potential as biomarkers for cancer diagnosis and prognosis. Thus, it is definitely worthwhile to further investigate their potential roles and clinical utility in cancer progression.
lncRNAs have been proved to be a potential diagnostic biomarker for BC, although a meta-analysis about application value of lncRNAs on diagnosis of breast carcinoma had been published. Yu et al. evaluated 10 studies and showed that lncRNAs were highly sensitive (0.79 (95% CI: 0.70-0.85)) and specific (0.80 (95% CI: 0.73-0.85)) to diagnosis of breast carcinoma. However, they included a limited number of studies and only assessed the pooled diagnostic efficacy of upregulated lncRNAs, but not the diagnostic performance of downregulated ones. According to the full text, Zhang et al. carried out a quantitative real-time PCR method to examine the expression levels of plasma H19 in 102 BC patients and 96 healthy controls, but only evaluated the diagnostic values of plasma H19 between 30 early-stage BC patients and 30 healthy controls. Therefore, the number of samples included in Yu's meta-analysis may be biased. Furthermore, several studies about the diagnostic efficacy of lncRNAs have been published recently. So we conducted this meta-analysis to clarify the diagnostic effect of lncRNAs in BC.
The present meta-analysis for lncRNA expression profile in BC revealed that the pooled specificity and sensitivity were 0.74 (95% CI: 0.69-0.78) and 0.78 (95% CI: 0.72-0.83), which indicated its potential diagnostic capability. Diagnostic test performance was represented by DOR and area under SROC (AUC). The ideal SROC curve position for a perfect test is near the upper-left corner. The DOR and AUC of lncRNAs were 10.01 (95% CI: 7.13-14.06) and 0.82 (95% CI: 0.79-0.85), respectively. Meanwhile, we also conducted a meta-analysis on the diagnostic efficacy of other serum biomarkers commonly used in clinic in the included literatures 8.50 35.79 8.61 4.44 47.67 6.68 11.14 126.58 6.10 6.82 6.00 3.50 5.69 11.54 13.34 9.47 For tumor types, the diagnostic effect of lncRNAs in BC was lower than that in TNBC (AUC: 0.81 (95% CI: 0.77-0.84) versus 0.87 (95% CI: 0.84-0.90)). The plasma and serum specimen sensitivity (0.76 (95% CI: 0.70-0.81) and 0.78 (95% CI: 0.70-0.85)), specificity (0.83 (95% CI: 0.76-0.88) and 0.80 (95% CI: 0.70-0.87)), and AUC (0.86 (95% CI: 0.82-0.89) and 0.86 (95% CI: 0.83-0.89)) were higher than tissue sensitivity (0.65 (95% CI: 0.52-0.76)), specificity (0.71 (95% CI: 0.57-0.83)), and AUC (0.73 (95% CI: 0.69-0.77)). The diagnostic performance of lncRNA expression status suggested that upregulated lncRNAs (AUC: 0.84 (95% CI: 0.81-0.87)) were better than downregulated lncRNAs (AUC: 0.70 (95% CI: 0.65-0.73)). lncRNA-MALAT1 sensitivity was 0.81 (95% CI: 0.71-0.88), specificity was 0.77 (95% CI: 0.68-0.84), and AUC was 0.85 (95% CI: 0.82-0.88), which were superior to lncRNA-HOTAIR and lncRNA-H19. Our findings may further help guide therapeutic strategy in the clinic.
Exploring the sources of heterogeneity is critical to a meta-analysis. Since heterogeneity was obviously revealed by test results, we attempted to explain its sources. Threshold effect is a primary cause of heterogeneity in test accuracy studies. The Spearman correlation coefficient was 0.178 (P = 0:236), which suggested that threshold effect was excluded from the factors causing heterogeneity in the current study. Sensitivity analysis was next used to test if the heterogeneity came from any individual study. Our results indicated that the selected studies were homogeneously distributed. Subgroup and meta-regression analyses were utilized for other factors causing heterogeneity. We validated five covariates, including ethnicity (P = 0:186), pathologic type (P = 0:428), specimen (P = 0:157), type of lncRNA (P = 0:296), and expression status (P = 0:070). The data suggested that lncRNA expression status was associated with study heterogeneity. Finally, Deeks' funnel plot showed that there was no significant publication bias in the diagnostic meta-analysis for lncRNA. Unfortunately, we failed to find other sources.
lncRNA appears to be a diagnostically valuable biomarker for BC. However, our meta-analysis has several limitations. First, most of the eligible studies were from China Meta-analysis estimates, given named study is omitted Lower CI limit Upper CI limit Estimate and Iran and included only Asian and Caucasian populations. Second, we selected 33 literatures, including 46 individual studies, which may lead to insufficient statistical sample capacity in statistics. A total of 30 lncRNAs were included in all studies, but some lncRNAs, such as SOX2OT, UCA1, USMycN, XIST, and Z38, were counted in only one study. Meanwhile, the subgroup analysis of the included studies was constrained by the limited number and size of available studies. Third, heterogeneity existed in the analysis and could not be explained by results of meta-regression and sensitivity analysis. Although the diagnosis values of lncRNAs were moderate, the performance of lncRNAs was not as satisfactory as expected based on the high accuracy criterion (NLR < 0:1, PLR > 10).
In conclusion, this meta-analysis comprehensively investigated the application of lncRNAs as a promising biomarker for diagnosis of breast carcinoma. Before the clinical use of lncRNAs as a diagnostic marker, more large-scale prospective studies are warranted to identify which lncRNAs have a better diagnostic role in BC.
## Data availability
The data used to support the findings of this study are included within the article.
## Conflicts of interest
The authors declare that they have no conflicts of interest. BioMed Research International |
Fabrication of Corona-Free Nanoparticles with Tunable Hydrophobicity
## S2
Compounds 1 to 4 were synthesized according to the reported procedure. [bib_ref] Detection and Identification of Proteins Using Nanoparticle-Fluorescent Polymer 'Chemical Nose' Sensors, You [/bib_ref]
## Compound 5 (tertiary amine derivatives):
To a solution of compound 4 (~2g) under nitrogen in dry DCM (20ml) different amines (2eq.) were added to synthesize the library of compounds. The reaction mixture was then left to stir at 40°C for 48 h and monitored using TLC (10% MeOH in DCM, ninhydrin dip). Upon completion, DCM was evaporated under vacuum and the crude product was purified by column chromatography over silica gel (flash running) using MeOH/DCM (0.5:9.5 and 1.5:8.5 v/v) as an eluent. Solvent was evaporated under vacuum to obtain compound 5 as a yellow oil.
Compound 6 (zwitterion derivatives): Compound 5 was then dissolved in ethyl acetate and 1,3propanesultone (1.2 eq) was added under nitrogen. Then the reaction mixture was stirred at 40°C for 72 hrs and monitored using TLC (10% MeOH in DCM, ninhydrin dip). Upon completion, the solvent was evaporated under vacuum and the crude product was purified by column chromatography over silica gel using MeOH/DCM (0.5:9.5 and 2:8 v/v) as an eluent. Solvent was evaporated under vacuum to obtain compound 6 as yellow oil.
## Compound 7 (final ligands):
To a solution of compound 6 (dry DCM, 5mL) an excess of trifluoroaceticacid (20eq.) was added while stirring under inert atmosphere for 10 min. As a second step, triispropylsilane (1.2eq.) was added slowly while maintaining stirring, and the reaction was left to completion for 5h. The solvent was then evaporated and the crude product was washed several times with hexanes to obtain the final product. NMRs are reported in the nanoparticle characterization section.
Ligand exchange and nanoparticle purification: To a solution of gold nanoparticle (NP) cores coated with a monolayer of 1-pentanethiol (~2nm core diameter, synthesized by the Brust-Schiffrin methodology [bib_ref] Synthesis of Thiol-Derivatised Gold Nanoparticles in a Two-Phase Liquid-Liquid System, Brust [/bib_ref] and purified to remove the phase transfer catalyst [bib_ref] Preparation of 2 nm Gold Nanoparticles for In Vitro and, Moyano [/bib_ref] in dry DCM and MeOH (9.5:0.5), a solution of compound 6 in the same solvent system was added, maintaining an inert atmosphere and constant stirring for 3 days (5:1 w/w ratio of ligand/gold cores). [bib_ref] Monolayers in Three Dimensions: Synthesis and Electrochemistry of ω-Functionalized Alkanethiolate-Stabilized Gold Cluster..., Hostetler [/bib_ref] The solvent was evaporated and the remaining dark residue was washed several times with DCM or ethyl acetate until no smell was detected (removal of 1-pentanethiol). The nanoparticles were dispersed in distilled water, and dispensed into a membrane bag (~10,000 pore size) for dialysis purification for 5 days. Water was removed by lyophilization and the final product was redispersed in MQ deionized water (or D 2 O), and the concentration of the final solution was determined using UV absorption according to the reported procedure.
[fig] S3, Figure: [NP]=20μM), MALDI-MS (Bruker Autoflex III MALDI-TOF MS, 200 shots-20 off-laser 55%, suppress up to 400Da), DLS and ZP (Malvern NanoZeta Sizer, [NP]=1 μM) to observe the chemical composition of the monolayer, the hydrodynamic size, and the surface zeta potential. The LogP values of the ligand headgroups (R groups of Figure 1) were estimated using ChemDraw Ultra 12.0. [/fig]
[fig] Figure S3: 1 HNMR spectrum of ZBu. [/fig]
[fig] Figure S5: 1 HNMR spectrum of ZDiBu. [/fig]
[fig] Figure S11: Mass spectrum of ZDiPen (MW=641.44/mol). [/fig]
[fig] Figure S12: Representative DLS profiles (% Intensity) of the series of zwitterionic NPs and controls after incubation in 1% human serum. [/fig]
[fig] Figure S13: DLS profiles (%Volume) for the different NPs in the presence and absence of serum proteins (1% serum background, spectrums of the NPs and serum reported as normalized according to the concentration of each species). [/fig]
[fig] Figure S14: Residuals of the DLS profiles in serum after removing the individual NP and serum spectrums for each NP. [/fig]
[fig] Figure S15: DLS profiles (%Volume) for the different NPs in the presence and absence of plasma (1% plasma background, spectrums of the NPs and serum reported as normalized according to the concentration of each species). [/fig]
[fig] Figure S16: Residuals of the DLS profiles in plasma after removing the individual NP and plasma spectrums for each NP. [/fig]
[fig] Figure S17: DLS profiles (% Volume) of the series of NPs after incubation in 55% human serum and successive dilutions, evidencing the absence of protein corona for ZMe-ZDiPen while NP+, NP-and TEG present NP/protein complexes. [/fig]
[fig] Figure S18, Figure S19: Agarose gel electrophoresis showing similar mobilities for NPs ZMe-ZDiPen in the presence and absence of plasma, while NP+ and NP-present a retarded band due to conjugation with proteins (a = NPs alone, p = mixture of plasma and NPs). NP concentration is 1 μM. (a) Sedimentation experiments in 10%, 55%, and undiluted plasma, including sedimentation in a continuous sucrose gradient, and in PBS. (b) UV differences (λ=506nm) of the supernatants before and after centrifugation in 10% serum evidencing the significant difference in sedimentation between TEG, NP+, NP-and ZMe to ZDiPen (p-values < 0.05). The sediment observed in the case of ZDiBu and ZDiPen were removed after washing with PBS 1-2 times indicating a reversible binding, while the positive control retained the pellet. [/fig]
[fig] Figure S20 S13, Figure S21: (a) Sedimentation assay using a sucrose cushion of 24%. (b) Gel electrophoresis of the samples obtained by the 24% sucrose sedimentation, evidencing very low level of proteins in the samples of the zwitterionic NPs (ZMe -ZDiPen, similar to control with no NP), while NP+ presented protein bands indicating corona formation. Serum lane indicates a sample with 0.1% serum directly loaded into the gel. Cellular uptake (MCF-7 cells) at 24h of incubation with NPs in the presence of serum. [/fig]
[fig] Figure S22: Viability of MCF7 cells at 24h for the different NPs. [/fig]
[fig] Figure S23: Differences in the absorbance for the sedimentation experiments between the presence and absence of RBCs. 'nns' denotates p-values > 0.05 (no statistical significance). [/fig]
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Hypofractionated stereotactic radiotherapy of limited brain metastases: a single-centre individualized treatment approach
Background: We retrospectively report treatment results of our single-centre experience with hypofractionated stereotactic radiotherapy (hfSRT) of limited brain metastases in primary and recurrence disease situations. Our aim was to find the most effective and safe dose concept. Methods: From 04/2006 to 12/2010, 75 patients, with 108 intracranial metastases, were treated with hfSRT. 52 newly diagnosed metastases (48%), without up-front whole brain radiotherapy (WBRT), received hfSRT as a primary treatment. 56 metastases (52%) received a prior WBRT and were treated in this study in a recurrence situation. Main fractionation concepts used for primary hfSRT were 6-7x5 Gy (61.5%) and 5x6 Gy (19.2%), for recurrent hfSRT 7-10x4 Gy (33.9%) and 5-6x5 Gy (33.9%). Results: Median overall survival (OS) of all patients summed up to 9.1 months, actuarial 6-and 12-month-OS was 59% and 35%, respectively. Median local brain control (LC) was 11.9 months, median distant brain control (DC) 3.9 months and intracranial control (IC) 3.4 months, respectively. Variables with significant influence on OS were Gross Tumour Volume (GTV) (p = 0.019), the biological eqivalent dose (calculated on a 2 Gy single dose, EQD2, α/β = 10) < and ≥ median of 39 Gy (p = 0.012), extracerebral activity of the primary tumour (p < 0.001) and the steroid uptake during hfSRT (p = 0.03). LC was significantly influenced by the EQD2, ≤ and > 35 Gy (p = 0.004) in both uni-and multivariate Cox regression analysis. Median LC was 14.9 months for EQD2 >35 Gy and 3.4 months for doses ≤35 Gy, respectively. Early treatment related side effects were usually mild. Nevertheless, patients with a EQD2 >35 Gy had higher rates of toxicity (31%) than ≤35 Gy (8.3%, p=0.026). Conclusion: Comparing different dose concepts in hfSRT, a cumulative EQD2 of ≥35 Gy seems to be the most effective concept in patients with primary or recurrent limited brain metastases. Despite higher rates of only mild toxicity, this concept represents a safe treatment option.
# Background
Cancer patients develop brain metastases in 10-40% [bib_ref] Management of brain metastases, Soffietti [/bib_ref] [bib_ref] Brain metastases: epidemiology and pathophysiology, Gavrilovic [/bib_ref]. The aim of treatment is to provide disease control with a good quality of life, even though prolonged survival may not always be achieved [bib_ref] Management of cerebral metastasis: evidence-based approach for surgery, stereotactic radiosurgery and radiotherapy, Jenkinson [/bib_ref]. Whole brain radiotherapy (WBRT) is considered to be the standard treatment option for multiple brain filiae [bib_ref] The palliation of brain metastases: final results of the first two studies..., Borgelt [/bib_ref] [bib_ref] Breast Cancer Expert Panel of the German Society of Radiation Oncology (DEGRO):..., Feyer [/bib_ref]. Concerns about WBRT achieving only a limited treatment response and causing side-effects like cognitive and neurological deficits, and reduced quality of life [bib_ref] Subacute brain atrophy after radiation therapy for malignant brain tumor, Asai [/bib_ref] [bib_ref] Importance of the mini-mental status examination in the treatment of patients with..., Murray [/bib_ref] [bib_ref] Effects of radiotherapy for brain metastases on quality of life (QoL), Steinmann [/bib_ref] [bib_ref] Radiation-induced dementia in patients cured of brain metastases, Deangelis [/bib_ref] [bib_ref] Memory function before and after whole brain radiotherapy in patients with and..., Welzel [/bib_ref] lead us to focus on options like stereotactic radiosurgery (SRS) or hypofractionated stereotactic radiotherapy (hfSRT) in cases of limited brain metastases. SRS is limited by the proximity to critical brain regions, the lesions' dimension and is typically restricted to tumours ≤3 cm diameter (15 ccm) [bib_ref] Management of brain metastases: the indispensable role of surgery, Al-Shamy [/bib_ref]. Large metastases and irregular contrast enhancement have been shown to correlate with an inferior outcome after SRS [bib_ref] Irradiated volume as a predictor of brain radionecrosis after linear accelerator stereotactic..., Blonigen [/bib_ref] [bib_ref] Radiosurgery for brain metastases: relationship of dose and pattern of enhancement to..., Shiau [/bib_ref] or enhanced side effects [bib_ref] Radiation-induced changes of brain tissue after radiosurgery in patients with arteriovenous malformations:..., Levegrun [/bib_ref] [bib_ref] Risk analysis of linear accelerator radiosurgery, Voges [/bib_ref]. Different study groups used hfSRT in order to overcome limitations of SRS, yielding similar tumour control and providing radiobiological advantages [bib_ref] Clinical outcome of hypofractionated conventional conformation radiotherapy for patients with single and..., Aoki [/bib_ref] [bib_ref] Phase II trial of hypofractionated stereotactic radiotherapy for brain metastases: results and..., Ernst-Stecken [/bib_ref] [bib_ref] The radiobiology of radiosurgery: rationale for different treatment regimes for AVMs and..., Hall [/bib_ref] [bib_ref] Hypofractionated conformal stereotactic radiotherapy alone or in combination with whole-brain radiotherapy in..., Lindvall [/bib_ref] [bib_ref] Tommy Bergenheim A: A comparison between surgical resection in combination with WBRT..., Lindvall [/bib_ref]. In 2006 we initiated the application of hfSRT whenever highly focussed stereotactic intracranial radiotherapy was indicated but tumour size or localization rendered a single-time treatment impossible.
HfSRT of more than one metastasis was carried out either simultaneously or successively in a primary setting or in a recurrent situation. The aim of this retrospective analysis was to evaluate an efficient and safe dose concept of hfSRT for limited (1-4) brain metastases.
# Methods
## Patient characteristics
Between April 2006 and December 2010, 75 patients with 108 brain metastases were treated with hfSRT and retrospectively analysed. After interdisciplinary discussion a surgical treatment approach had been excluded due to comorbidity, age, or localization of the tumour. Diagnosis of brain metastases was based on pre-treatment magnetic resonance imaging (MRI). Only patients with Karnofsky performance status ≥70 were included. 41 patients (55%) received a primary definitive hfSRT of 52 newly diagnosed metastases (48%). 34 patients (45%) with 56 metastases (52%) were treated with hfSRT in a recurrence situation. Patient and treatment characteristics are summarized in [fig_ref] Table 1: Patients characteristics [/fig_ref] and 2.
The need of ethical approval is waived because of the retrospective character of the study. Authorization for the use of patient data was given to the authors by the Head of the Departement of Radiooncology of the Medical School in Hannover.
## Radiotherapy
All patients were informed about radiotherapeutic alternatives, in detail. If hfSRT was chosen, repeated MRI scans were conducted after completing radiotherapy, in order to detect early intracranial progress or radiotoxicity. All patients were immobilized using a tight thermoplastic stereotactic head mask. Helical-CT images of 2 mm slice thickness were fused with axial T1 weighted contrast enhanced MR images. Gross Tumour Volume (GTV) was The chosen fractionation scheme depended on the size, number and site of the brain metastases as well as on the 're-irradiation' factor in a recurrent situation. Single doses of 5 to 6 Gy were aimed in primary setting, single lesions, small GTV (< 2 ccm) and in uncritical regions. If normal brain volume receiving more than 4 Gy (V 4Gy ) exceeded 23 ccm, the single dose was reduced to 4 Gy to prevent toxicity [bib_ref] Phase II trial of hypofractionated stereotactic radiotherapy for brain metastases: results and..., Ernst-Stecken [/bib_ref]. For recurrence, generally a more restrictive fractionation concept (Equivalent dose in 2 Gy fractions (EQD2 < 40 Gy) was used. Dose concepts and associated mean GTV are summarized in.
The EQD2 makes different radiation schedules comparable and is calculated with the equation EQD2 = D × ([d + α/β]/[2 Gy + α/β]), considering the linear-quadratic model. D is the total dose, d is the dose per fraction and the α/β ratio is an experimentally defined value of tissues. Assuming an α/β ratio of 10 Gy for tumour cell kill, the EQD2 of the radiation concepts are 40 Gy (30 Gy in 5 fractions), 44 Gy (35 Gy in 7 fractions) and 47 Gy (40 Gy in 10 fractions), exemplarily [bib_ref] The biophysical properties of 3.9-GeV nitrogen ions. VI. Interpretation of results, Kellerer [/bib_ref]. Patient positioning was checked with an on-board imaging (IGRT) before irradiation by using a cone-beam CT (XVI, Elekta) and X-ray images (Iview, Elekta) for verification of the isocenter. Radiotherapy was carried out using a Linac with 6 MVX photons in four to six beams, and a multi leaf collimator with a leaf width of 1cm. For 24 lesions Brain Lab System with individual blends was used.
Between each fraction at least one day treatment interruption was provided.
## Follow-up
Patients were monitored on a regular basis by the treating radiation oncologist. Performance status, neurological symptoms, and steroid uptake were monitored. After a planned period of 6 to 12 weeks the first MRI control was performed. Further follow-up MRI scans were carried out in intervals of 2-3 months. Treatment failure was considered as occurrence of new or increased contrast enhancement in the irradiated area (with or without increased volumes). Early side effects, i.e. alopecia, fatigue or headache, were scored with the CTC-AE 3.0 scoring system. Late effects (> 90 days after hfSRT) were not considered as these could not be analysed systematically due to retrospective data collection.
# Statistical analysis
Analysed endpoints were local control (LC), distant brain control (DC), intracranial control (IC) and overall survival (OS). Local relapse was defined as occurrence of new or increasing contrast enhancement in follow-up MRI in the irradiation area, after involvement of an experienced neuroradiologist. Distant brain failure was defined as any new brain metastases beyond local relapse, intracranial failure as any intracranial progress including local relapse. Survival rates and univariate analysis were calculated by the Kaplan-Meier log-rank test. All events were measured from the first treatment day of hfSRT. The following variables were used: sex, age (<57.5y. vs. ≥57.5y.), primary (non-small cell lung cancer vs. other), RPA (recursive partitioning analysis) prognostic group (1 vs. 2), primary vs. recurrent treatment, GTV (<2.0 vs. ≥2.0 ccm), steroid uptake (yes or no), median V 4Gy (<16.7 ccm vs. ≥16.7 ccm), EQD2 (≤35 Gy vs. >35 Gy), median EQD2 (<39 Gy vs. ≥39 Gy), median time interval between diagnosis of the primary tumour and brain metastases (<12.5 vs. ≥12.5 months). Multivariate Cox Proportional Regression Analysis was performed only with variables significant in the univariate analysis.
T-test of independent samples was used to compare GTV of different dose concepts and to compare toxicity rates. All statistical analysis was performed using IBM SPSS Statistics 20. A p-value <0.05 was considered to be statistically significant.
# Results
## Overall survival and influencing factors
Median OS summed up to 9.1 months with a median follow-up of 9.5 months (range 1.0-50.6 months). Median follow-up of patients being alive until August 2012 (n=5,7%) was 36.6 months (range 21.9-50.6 months). Actuarial 6-and 12-months-OS was 59% and 35%. Variables with significant influence on OS after multivariate analysis were the extracranial activity of primary tumour (p<0.001), the steroid uptake (p=0.03), the GTV (p=0.019) [fig_ref] Figure 1: Actuarial OS probability according to GTV calculated by Kaplan-Meier [/fig_ref] and a total dose EQD2≥39 Gy (p=0.012). Detailed OS data is presented in [fig_ref] Table 3: Actuarial overall survival according to potential factors and the result of univariate... [/fig_ref].
## Local and intracranial control
After a median of 2.2 months (range: 0.3-10.5 months) patients received the first follow-up MRI scan. Two patients (2.7%) were lost to follow-up and fourteen patients (18.7%) died within 90 days (range 13-75 days) of hfSRT and before the first follow-up imaging was carried out. 88 lesions (81.5%) were evaluated.
Imaging was done in 56 cases within 40-90 days after the end of hfSRT. 29 metastases (51.8%) showed a complete remission, 20 (35.7%) a partiell remission and 7 (12.5%) a progress.
Local control in the irradiated area after 6 and 12 months was 73% and 52%, respectively, median LC was 11.9 months. The EQD2 significantly influenced LC (p=0.004, multivariate analysis). Median LC was 14.9 months for EQD2 >35 Gy and 3.4 months for doses ≤35 Gy. Actuarial LC rates for biological irradiation doses > and ≤35 Gy were 80%/44% after 6 months and 57%/22% after 12 months, respectively [fig_ref] Figure 2: Actuarial LC probability according to EQD2 calculated by Kaplan-Meier [/fig_ref]. The median EQD2 of < and ≥39 Gy was not a significant factor influencing LC.
LC analysis of different dose concepts showed a 6-and 12-months LC of 90%/66% for the 6x5 Gy concept. LC for 5x6 Gy was 83%/63%, 73%/42% for 7x5 Gy and 64%/ 21% for 10x4 Gy, respectively. Differences in LC were not significant. Distant brain control (outside the irradiated area) after 6 and 12 months was 51% and 35%, median DC was 3.9 months. Fifteen patients with primary treated metastases suffered from a distant brain relapse, four had multiple metastases (≥3) and eleven developed limited (1-2) metastases. Two patients with distant brain relapse were treated with hfSRT, nine with WBRT, one got further chemotherapy and one had a surgical resection. Treatment of two patients was not known. Intracranial control (includes local and distant brain control) was 41% and 20%, median being 3.4 months. No other variables with significant influence on DC, IC, LC and OS could be identified.
## Survival data of recurrence hfsrt
In recurrent situations 56 metastases were treated with a median EQD2 of 38 Gy while in comparison 52 metastases in primary situation were treated with a median EQD2 of 44 Gy.
The aspect of primary or recurrence hfSRT did not have a statistically significant influence on OS or on LC. Median OS of primary hfSRT was 8.8 months, while in recurrence situations it was 10 months (p=0.217 in univariate and p=0.124 in multivariate Cox regression analysis). 6-and 12-months-OS-rates for primary hfSRT were 62%/37% and for recurrence hfSRT they were 57%/34%. 6-and 12-months-rates of LC, DC and IC for primary hfSRT were 75%/55%, 56%/51% and 46%/30% while for recurrence hfSRT they amounted 71%/49%, 46%/25% and 38%/16%, respectively.
## Toxicity
For proper interpretation of toxicity results, we would like to point out, that 36 (48.0%) patients were treated with chemotherapy during the 3-month time period before the start of hfSRT. Furhtermore, it should be considered that 40 patients (53.3%) received steroids during radiotherapy. The median dose of dexamethasone at the end of radiotherapy was 4 mg per day.
Only mild early toxicity (CTC-AE v. 3.0 grade 1) was observed: alopecia (9 patients), fatigue (8 patients), headache (6 patients), nausea (2 patients) or mucositis (2 patients). One patient with hfSRT to four brain metastases, treated with a molecular therapy (Sunitinib) until the start of radiation, showed short term memory loss and amnesic aphasia. Another patient with chemotherapy just until the start of hfSRT suffered from vision disorder.
In only 2 cases out of 24 with a radiation dose EQD2 ≤35 Gy (8.3%) a toxicity was seen compared to 26 cases out of 84 with an EQD2 >35 Gy (31%, p=0.026).
The GTV (< and ≥2 ccm) and the a brain volume treated with 4 Gy (V 4Gy) (< and ≥ median of 16.7 ccm or < and ≥ 23 ccm) had no significant influence on toxicity (29.8% vs. 21.6%, p=0.328; 30.4% vs. 21.2%, p=0.275; 31.2% vs. 17.1%, p=0.149).
One case of radiation necrosis was discussed in the follow-up analysis. This patient with small cell lung carcinoma was initially treated with a prophylactic WBRT with 30 Gy (15x2 Gy). After 14 months the MRI showed 3 metastases of the brain, each of them was treated with hfSRT, the cerebellar metastasis was treated with a total dose of 40 Gy (10x4 Gy, EQD2 = 47 Gy). This resulted in a cumulative dose of 77 Gy in the cerebellum. The suspicion of necrosis was raised after a progress of the cerebellar metastasis with additional calcification showing 6 months later. Simultaneously, the patient suffered from cephalgia, emesis, vertigo and motor dysfunction. She died 3 months later after implantation of an Omaya reservoir and subsequent treatment with chemotherapy and steroids.
# Discussion
This study retrospectively analyses therapeutic results in 75 patients treated with hfSRT to limited brain metastases in a primary, or recurrence, situation. Since 2006 we are offering this option to patients who suffer from limited brain metastases and where surgery or SRS is not a suitable treatment option. We applied different dose concepts dependent on primary or recurrence situation, localization and volume of brain lesion. Dose concepts with a total EQD2 >35 Gy achieved best local control rates with acceptable toxicity. 12-months LC rates of 52% are lower in comparison to other hfSRT studies or SRS probably because we used very different dose concepts [bib_ref] Clinical outcome of hypofractionated conventional conformation radiotherapy for patients with single and..., Aoki [/bib_ref] [bib_ref] Hypofractionated stereotactic radiotherapy for oligometastases in the brain: a single-institution experience, Marchetti [/bib_ref] [bib_ref] Outcome of moderately dosed radiosurgery for limited brain metastases, Meisner [/bib_ref] [bib_ref] Stereotactic radiosurgery alone versus resection plus whole-brain radiotherapy for 1 or 2..., Rades [/bib_ref] [bib_ref] A single institutional outcome analysis of Gamma Knife radiosurgery for single or..., Nakagawa [/bib_ref] [bib_ref] Stereotactic irradiation without whole-brain irradiation for single brain metastasis, Shirato [/bib_ref] [bib_ref] Radiosurgery for brain metastases: is whole brain radiotherapy necessary?, Sneed [/bib_ref].
The aim of our study was to find the most suitable regime with respect to disease control and side effects. Fahrig et al. also compared different dose concepts like 5x6-7 Gy, 7x5 Gy and 10x4 Gy and achieved 12 months OS of 43%, 60% and 67%, respectively [bib_ref] Hypofractionated stereotactic radiotherapy for brain metastases-results from three different dose concepts, Fahrig [/bib_ref]. They preferred the 10x4 Gy fractionation, depending on the size and localization of the metastases, and detected no adverse side effects. However, a trend towards higher rates of complete remission in patients with brain metastases was seen treated with 5x6-7 Gy or 7x5 Gy in comparison with 10x4 Gy [bib_ref] Hypofractionated stereotactic radiotherapy for brain metastases-results from three different dose concepts, Fahrig [/bib_ref]. We preferred using restrictive dose concepts (EQD2 < 40 Gy) in patients with prior WBRT as well as in patients with high tumour volume or tumour proxicity to critical structures. The median EQD2 for GTV < 2 ccm was 40 Gy and for GTV ≥ 2 ccm 38 Gy. The GTV was significantly higher for metastases treated with a single dose of 3-4 Gy (median 4.76 ccm) than with 5-6 Gy (median 1.00 ccm, p < 0.001). Recent studies yielded LC rates of 58.6% after 12 months in mean volumes of 8 ccm (24 Gy in 3 fractions) [bib_ref] Hypofractionated stereotactic radiotherapy for oligometastases in the brain: a single-institution experience, Marchetti [/bib_ref] and 76% in median volumes of 4.23 ccm (5x6 Gy after prior WBRT, otherwise 5x7 Gy) [bib_ref] Phase II trial of hypofractionated stereotactic radiotherapy for brain metastases: results and..., Ernst-Stecken [/bib_ref]. Aoyama et al. found a significant lower tumour control rate for tumours >3 ccm (35 Gy in 4 fractions) [bib_ref] Hypofractionated stereotactic radiotherapy alone without whole-brain irradiation for patients with solitary and..., Aoyama [/bib_ref]. Nevertheless, above mentioned hfSRT studies included volumes higher than 3 ccm and showed good results.
Conclusions can be drawn from our study results only to a limited extent because of the relatively small number of patients included, it being a monoinstitutional series and the potential risk of selection biases due to the retrospective study design. However, we consider the results to be valuable with regard to the objective of our analysis. Conclusions of our analysis are also limited due to high diversity of dose concepts. In our study only 48% could be classified into dose concepts defined by Fahrig et al. [bib_ref] Hypofractionated stereotactic radiotherapy for brain metastases-results from three different dose concepts, Fahrig [/bib_ref]. Therefore, we could not see significant difference for a specific dose concept for LC or OS.
Furthermore, we detected a significant influence of the EQD2 (p = 0.004) on LC (14.9 months for doses >35 Gy and 3.4 months for doses ≤ 35 Gy) in this study.
An EQD2 >35 Gy is associated with better control rates along with higher but acceptable toxcitiy. Therefore, we consider it is most effective for tumour control.
In SRS higher absolute doses (24 Gy) are associated with higher LC (85%) after 12 months (compared to 45-49% with 15-18 Gy) [bib_ref] Local control of brain metastases by stereotactic radiosurgery in relation to dose..., Vogelbaum [/bib_ref]. For brain metastases ≤2 ccm SRS studies found that a single time 20 Gy application seems to render superior results [bib_ref] Stereotatic radiosurgery of 468 brain metastases < or =2 cm: implications for..., Shehata [/bib_ref].
Rades et al. achieved higher LC rates with upfront WBRT (77%) than with SRS alone (49%), and thereby showed a benefit of up-front WBRT [bib_ref] Single brain metastasis: radiosurgery alone compared with radiosurgery plus up-front whole-brain radiotherapy, Rades [/bib_ref].
Half of our patients (45%) were treated in a recurrence situation after prior WBRT. LC was not significantly different (49% after 12 months) in comparison to patients who received primary treatment (55% after 12 months). Lindvall et al. combined WBRT and hfSRT (30 Gy in 10 fractions and 17 Gy in 1-3 fractions) in 11 patients and compared them to 44 patients with hfSRT (40 Gy in 5 fractions) alone. They showed high LC rates of 100% and 84% in a short observation time (mean 3.7 months) [bib_ref] Hypofractionated conformal stereotactic radiotherapy alone or in combination with whole-brain radiotherapy in..., Lindvall [/bib_ref].
Nevertheless, GTV significantly influenced OS in our study (< 2 ccm median 11.5 months ≥2 ccm median 5.4 months). Ernst-Stecken et al. defined GTV and PTV volume above 6 ccm and 13 ccm as negative prognostic factor for OS [bib_ref] Phase II trial of hypofractionated stereotactic radiotherapy for brain metastases: results and..., Ernst-Stecken [/bib_ref].
According to the results of Ernst-Stecken et al. brain volume receiving >4 Gy per fraction should not exceed 23 ccm [bib_ref] Phase II trial of hypofractionated stereotactic radiotherapy for brain metastases: results and..., Ernst-Stecken [/bib_ref]. Takening this into account, overall adverse effects were only mild in our study. None of our patients suffered from seizures classified as a higher grade side effect. In contrast, 11% of patients suffered of seizures within 3 months after SRS [bib_ref] Outcome of moderately dosed radiosurgery for limited brain metastases, Meisner [/bib_ref].
A recurrence situation after WBRT or hfSRT has not shown a statistically significant influence on LC, IC or OS in our and other studies [bib_ref] Hypofractionated stereotactic radiotherapy for oligometastases in the brain: a single-institution experience, Marchetti [/bib_ref]. Overall survival for hfSRT in primary and recurrence situations was median 8.8 months and 10 months, respectively. The actuarial OS of 35% one year after treatment with hfSRT is comparable to the reported SRS series (30-50%) [bib_ref] Clinical outcome of hypofractionated conventional conformation radiotherapy for patients with single and..., Aoki [/bib_ref] [bib_ref] Outcome of moderately dosed radiosurgery for limited brain metastases, Meisner [/bib_ref] [bib_ref] A single institutional outcome analysis of Gamma Knife radiosurgery for single or..., Nakagawa [/bib_ref] [bib_ref] Stereotactic irradiation without whole-brain irradiation for single brain metastasis, Shirato [/bib_ref] [bib_ref] Radiosurgery for brain metastases: is whole brain radiotherapy necessary?, Sneed [/bib_ref] and hfSRT studies (25%) [bib_ref] Hypofractionated stereotactic radiotherapy for oligometastases in the brain: a single-institution experience, Marchetti [/bib_ref]. Different studies compared SRS alone with WBRT plus a SRS boost. The omission of WBRT in the initial management of patients who underwent SRS alone did not compromise survival or intracranial control [bib_ref] Stereotactic radiosurgery alone versus resection plus whole-brain radiotherapy for 1 or 2..., Rades [/bib_ref] [bib_ref] Radiosurgery for brain metastases: is whole brain radiotherapy necessary?, Sneed [/bib_ref] [bib_ref] A multi-institutional review of radiosurgery alone vs. radiosurgery with whole brain radiotherapy..., Sneed [/bib_ref]. De Potter et al. [bib_ref] Hypofractionated frameless stereotactic intensity-modulated radiotherapy with whole brain radiotherapy for the treatment..., Potter [/bib_ref] achieved a DC at 1 year of 75%. They used 5x6 Gy as a boost in addition to WBRT. Patients with primary hfSRT treatment in our study achieved a DC of only 51% after one year and 60% of them had a WBRT as salvage therapy. Similar DC results of 36% without up-front WBRT were presented by Narayana et al. [bib_ref] Hypofractionated stereotactic radiotherapy using intensity-modulated radiotherapy in patients with one or two..., Narayana [/bib_ref].
Therefore, up-front WBRT is worth discussing and not simply expendable to avoid neurotoxicity of the normal brain tissue. Regular MR imaging is necessary to detect cerebral progress as soon as possible. Patients with limited brain metastases should be clearly informed about all possible advantages and disadvantages of the different therapy options.
# Conclusion
HfSRT to limited brain metastases is a non invasive therapy in primary and recurrence situations, and provides a reasonable tumour control and survival benefit. A total EQD2 >35 Gy is associated with better tumour control rates and with higher but acceptable toxcitiy. Therefore, it is most effective for control of limited brain metastases.
[fig] Figure 1: Actuarial OS probability according to GTV calculated by Kaplan-Meier (log-rank test; p< 0.019). GTV < 2 ccm (m=57): continuous line, GTV ≥ 2 ccm (m=51): dotted line. [/fig]
[fig] Figure 2: Actuarial LC probability according to EQD2 calculated by Kaplan-Meier (log-rank test; p=0.004). EQD2 > 35 Gy (m=73): continuous line, EQD2 ≤ 35 Gy (m=15): dotted line. [/fig]
[table] Table 1: Patients characteristics [/table]
[table] Table 3: Actuarial overall survival according to potential factors and the result of univariate and multivariate analysis [/table]
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Multiresistant bacteria and current therapy - the economical side of the story
# Abstract
severe infections with multiresistant bacteria (MRB) are a medical challenge and a financial burden for hospitals. the adequate antibiotic therapy is a key issue in multiresistant bacteria management. several major cost drivers have been identified. Remarkably drug acquisition costs are not necessarily included. Most significant are the length of stay in hospital, the hours of mechanical ventilation and the time treated on an intensive care unit.
In a systematic review of the literature the following aspects were investigated: -do generic treatment strategies contribute in cost savings? -are there specific results for recent antibiotics?
Early adequate and effective antimicrobial treatment, switch from i.v. to oral therapy, adjusted duration of therapy and adherence to guidelines have been found to be successful strategies.
looking at specific antibiotics, the best evidence for cost-effectiveness is found for linezolid in treatment of csstI as well as in HaP. daptomycin shows good economic results in bloodstream infections, so possibly being a cost-effective alternative to vancomycin. looking at tigecycline the published data show neither higher costs nor savings compared to imipeneme. doripenem as one of the newest therapy options has proven to be highly cost-saving in HaP when compared with imipenem. However, most analyses are based on pharmacoeconomic modelling rather than on directly analysing trial data or real life clinical populations. Conclusion: using modern antibiotics in whole is not more expensive than using established therapies. Modern antibiotics are cost-effective and sometimes even cost-saving. this is especially true if an effective therapy is initiated as early as possible.
abbreviations: alos = average length of stay in a given dRG, basis for determining whether a patient causes more costs than reimbursement caP = community acquired pneumonia csstI = complicated skin and soft tissue infections dRG = diagnoses related groups, systems to classify patients based on their resource consumptions HaP = hospital acquired pneumonia HMV = hours of mechanical ventilation Icu-days = treatment days on an intensive care unit los = length of stay in hospital MRB = infection with Multiresistant bacteria IntRoductIon severe MRB cause a serious burden of disease in most countries worldwide [bib_ref] Excess costs and utilization associated with methicillin resistance for patients with staphylococcus..., Filice [/bib_ref] [bib_ref] cousins d: Improving safe medication practice. nHs national Patient safety agency, Van Zanten Arh [/bib_ref]. their therapeutic management is a major cost driver in healthcare , particularly in hospitals. looking at the economical impact of antibiotic therapy of severe MRB infections, several factors were identified as the major cost drivers [bib_ref] die Bedeutung von Infektionen durch multiresistente staphylococcus aureus für das deutsche Gesundheitswesen..., Wernitz [/bib_ref] [bib_ref] costs of nosocomial pneumonia caused by meticillin-resistant staphylococcus aureus, Ott [/bib_ref] :
-prolonged hospital length of stay (los) -hours of mechanical ventilation (HMV) -duration of treatment on an intensive care unit (Icu) -cost of patient isolation (isolation) -complications, such as renal failure or infection transmission Whether any given antibiotic therapy is an economically adequate option therefore depends on its effect on one or more of these cost drivers. Interestingly, the actual daily costs of the drug itself does not significantly affect the overall costs of treatment. still in many settings, the choice of therapy is controlled by drug acquisition costs, as these data are easily available [bib_ref] Kosten einer leitli-niengerechten arzneimitteltherapie in deutschland, Dietrich Es [/bib_ref] [bib_ref] Kostenreduktion in der antibiotika-therapie, Dietrich Es [/bib_ref].
this article provides a review of the current literature on the role of recent antibiotic agents in the treatment of MRB infections from the economical perspective.
# Materials and methods
We conducted a literature review to investigate the available evidence on cost-effectiveness of antibiotic treatment strategies in the management of MRB infections. looking at the economical impact of antibiotic therapy in general, there are the following factors that have been proven to influence positively the abovementioned cost drivers: Influencing factor Effect on cost driver Ref.
Early adequate first-line antibiotic therapy shorter duration of therapy Early transfer from Icu to normal ward shorter los less complications [bib_ref] Hospital management of complicated intra-abdominal infections: pharmacoeconomic evaluation, Eandi [/bib_ref] IV-to-oral switch shorter i.v. therapy Earlier discharge and outpatient treatment [bib_ref] Bradley Js; a comparison of early versus late conversion from intravenous to..., Rt [/bib_ref] [bib_ref] Barie Ps ; does de-escalation of antibiotic therapy for ventilator-associated pneumonia affect..., Eachempati Sr [/bib_ref] [bib_ref] Bassetti s; outcomes of early switching from intravenous to oral antibiotics on..., Mertz D [/bib_ref] adjusted duration of therapy less adverse events less selection of resistance less drug cost shorter los [bib_ref] Goudie a, length of intravenous antibiotic therapy and treatment failure in infants..., Brady [/bib_ref] [bib_ref] optimizing antibiotic treatment for ventilator-associated pneumonia, St [/bib_ref] adherence to guidelines shorter duration of therapy shorter los less complications less Icu admissions less HMV [bib_ref] Kosten einer leitli-niengerechten arzneimitteltherapie in deutschland, Dietrich Es [/bib_ref] [bib_ref] Economic evaluation of adherence to treatment guidelines in nonintensive care pneumonia, Menendez [/bib_ref] With respect to these known factors affecting major cost drivers, we conducted a literature review focussed on articles dealing with recently introduced antibiotics active against MRB in the context of cost issues or cost-impacting factors.
We used the agent and 'cost-effectiveness' oR 'economical' as search terms.
# Results
We had 178 search findings in total. [fig_ref] Figure 1: Fig [/fig_ref] shows the search findings.
the number of publications referring explicitely to cost-effectiveness or economical analyses on linezolid is the highest. for closer analysis we then picked 25 articles that focussed on one or more of our above mentioned cost drivers.
In general economical analyses for drugs often differ from clinical trials. the most common method is modelling economical effects by using results from clinical trials. only rarely economical data are directly collected in the course of clinical trials. nearly no data exist on economical analyses based on clinical routine treatment. shows the results of the 25 articles closely reviewed.
In the following section we describe major findings for the agents in focus. lInEzolId Multiple economic analyses for linezolid have been published over the last decade. there are publications for both major indications (csstI, HaP) and for several countries (usa, Germany, spain, france). thus the economics of linezolid use effects appear to be well investigated. studies are either cost-effectiveness analyses based on data of clinical trials or pharmacoeconomic models [bib_ref] linezolid for treatment of ventilator-associated pneumonia: a cost-effective alternative to Vancomycin, Kollef [/bib_ref] [bib_ref] linezolid versus Vancomycin health and economic outcomes: a retrospective database study of..., Mullins Cd [/bib_ref] [bib_ref] Resch a; cost-effectiveness of linezolid versus vancomycin for hospitalised patients with complicated..., De Cock [/bib_ref]. the cost savings associated with the use of linezolid are predominantly due to a significant reduction of los. among recent antibiotics used for MRB infection, linezolid is the only drug that is available intravenous (IV) and orally (with a similar dose/exposure ratio), and the IV-to-oral switch allows earlier hospital discharge. the net effect is most significant in the treatment of csstI (2.0 -2.3 los days saved), as the overall severity of illness is lower than in pneumonia, and hospital-acquired pneumonia in particular. However, linezolid has been shown to be cost-effective in pneumonia as well. this is most likely due to earlier recovery and earlier hospital discharge. Most authors today acknowledge linezolid as a cost-effective component of the therapeutic armamentarium, particularly in MRsa infections. However, this insight has not been entirely implemented in clinical practice yet.
there are no explicit analyses available for other cost drivers. Most recently, a meta-analysissuggested that there is no specific economic rationale supporting the use of vancomycin versus linezolid for empiric therapy in settings with low MRsa prevalence.
daPtoMycIn Introduced more recently than linezolid, daptomycin already prompted a substantial number of publications on economic issues and publications referring to significant cost drivers [bib_ref] daptomycin versus standard therapy for bacteremia and endocarditis caused by staphylococcus aureus, Fowler [/bib_ref] [bib_ref] daptomycin: rationale and role in the management of skin and soft tissue..., Seaton Ra [/bib_ref] [bib_ref] Burden of methicillin-resistant staphylococcus aureus: focus on clinical and economic outcomes, Tp [/bib_ref] [bib_ref] Effectiveness and duration of daptomycin therapy in resolving clinical symptoms in the..., Krige [/bib_ref]. the most relevant indication for daptomycin is bloodstream infection with gram-positive cocci, as the overall number of patients is significantly higher than in endocarditis. similar to most of the other drugs discussed here, daptomycin is also suitable for the treatment of csstI. the main economic impact of daptomycin is associated with a reduction of los. However, earlier transfer from Icu to general ward is also described. some publications [bib_ref] daptomycin: rationale and role in the management of skin and soft tissue..., Seaton Ra [/bib_ref] [bib_ref] Effectiveness and duration of daptomycin therapy in resolving clinical symptoms in the..., Krige [/bib_ref] attribute lower costs to earlier cure and higher cure rates achieved with daptomycin versus vancomycin, which may relate to the bactericidal effect of the drug. However the trials underlying these analyses were no Rcts.
daptomycin has been shown to be associated with less occurrence of renal failure than vancomycin [bib_ref] daptomycin versus standard therapy for bacteremia and endocarditis caused by staphylococcus aureus, Fowler [/bib_ref]. It thus may be speculated that the lipopeptide antibiotic has a potential to influence positively the cost-driving duration of therapy as mentioned in one publication. However, this aspect still awaits further investigation. doRIPEnEM convincing economic analyses and articles referring to cost drivers in clinical studies have been published for doripenem, a recently approved carbapenem antibiotic. doripenem was shown to be associated with less resource use regarding los, HMV, and less complications (Psa resistance, Psa transmissions) compared to imipenem. no significant reduction in Icu days has been described so far. Remarkably, economical evaluation of this drug was included in the data and analysis of clinical trials. tIGEcyclInE among the articles retrieved on tigecycline [bib_ref] Management of complicated infections in the era of antimicrobial resistance: the role..., Dp [/bib_ref] , there were two dedicated analyses that dealt with resource utilization (i.e. cost) associated with the use of this recently introduced glycylcyclin antibiotic. tigecycline is an interesting treatment option particularly in complicated intraabdominal infections. With its very broad spectrum of activity, tigecycline can be used empirically in suspected polymicrobial infections. despite the slightly higher drug acquisition costs compared to imipenem it has been showed that first-line therapy is not more expensive than using tigecycline in second-line therapy . It also has the potential to be used as a monotherapy in this indicationand therefore would affect overall costs by abolishing the need for another drug. two publications explicitly report that los was not negatively affected when comparing tigecycline with imipenem. table 1 summarizes the data indicating a positive influence of the drugs covered in this review on cost drivers and influencing factors. dIscussIon It appears that the number of publications that evaluate the economic impact of new antimicrobial agents such as linezolid, daptomycin, tigecycline and doripenem is increasing. for every substance there are results or at least trends that indicate favourable effects on economic outcomes. the studies uniformly show that despite higher drug acquisition costs for newer antibiotics, overall treatment costs can be lower or therapy is cost-effective due to other factors (i.e. survival for linezolid in pneumonia). only a limited number of studies analyzed ‚real-life' clinical populations , whereas the greater part of the publications used pharmacoeconomic models based on data from clinical trials. While this is a well-accepted approach, it does not prove the relevance of the respective findings in the clinical routine setting. obviously, it is a most challenging task to validate data from pharmacoeconomic models in "real life", as the variance of patient characteristics is much higher than in trial population and various confounders complicate such analyses. there are some ongoing studies trying to relate dRG outcomes -i.e. the net effect on hospital reimbursement -to the chosen therapy strategies. It will be interesting to see the results in the near future. conclusIon the economic impact of antibiotic treatment choices in complicated infections with multiresistant bacteria is an increasingly important issue, as numerous publications document the enormous extra healthcare cost of these infections [bib_ref] die Bedeutung von Infektionen durch multiresistente staphylococcus aureus für das deutsche Gesundheitswesen..., Wernitz [/bib_ref] [bib_ref] Hospital management of complicated intra-abdominal infections: pharmacoeconomic evaluation, Eandi [/bib_ref] [bib_ref] Burden of methicillin-resistant staphylococcus aureus: focus on clinical and economic outcomes, Tp [/bib_ref]. the analysis of the current literature shows that therapy with modern antibiotics is generally cost-effective and may even be associated with net savings although drug acquisition costs are higher in conventional therapy regimens. currently, favourable cost effects are best documented for linezolid. a number of pharmacoeconomic analyses are also available for daptomycin, tigecyclin and doripenem, and more will certainly follow. Investigators may increasingly use economic data to document favourable cost effects of state-of-the-art therapy, thus overcoming the still wide-spread focus on ‚price-tags'. However only a few analyses use 'reallife' clinical settings for cost analyses. Modelling is the most common and most accepted approach to gain 'economical evidence'. With directly connecting economical analyses to clinical trials and/or to routine data collections the results would reach a higher level of acceptance among hospital administrators.
clinicians, pharmacists and economists in the hospital should work together to realize cost-savings with effective antibiotic therapy rather than sticking to treatment choices driven by drug acquisition cost.
## References
[fig] Figure 1: Fig. 2. literature review on 25 selected publications on new antibiotics. [/fig]
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Engineered biosynthesis of enduracidin lipoglycopeptide antibiotics using the ramoplanin mannosyltransferase Ram29
The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Grampositive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a dimannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg 11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugardependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.
# Introduction
There is an urgent need for new antibiotics to combat emerging drug-resistant pathogens. The majority of antibiotics that are used to treat infectious diseases are natural products or derivatives thereof. Natural products often require further chemical modification, to improve their biological activities or physico-chemical properties for therapeutic applications. However, many of the most promising natural products, such as the polyketides and nonribosomal peptides, are highly complex molecules that offer limited opportunity for semisynthesis and are invariably inaccessible through total synthesis on the scale required for drug development. Consequently, alternative biosynthetic engineering approaches are required that can enable the structural diversification and optimization of promising natural product scaffolds.
Biosynthetic engineering utilizes knowledge of the structure, organization and function of biosynthetic enzymes to reprogramme biosynthetic pathways at the genetic level, to produce new and potentially improved natural products. Previously, we adopted biosynthetic engineering approaches to diversify the calcium-dependent lipopeptide antibiotics (CDAs), leading to a library of 'non-natural' CDAs [bib_ref] Auxotrophicprecursor directed biosynthesis of nonribosomal lipopeptides with modified tryptophan residues, Amir-Heidari [/bib_ref] [bib_ref] Stereospecific enzymatic transformation of a-ketoglutarate to (2S,3R)-3-methyl glutamate during acidic lipopeptide biosynthesis, Mahlert [/bib_ref] [bib_ref] Biosynthesis of the (2S,3R)-3-methyl glutamate residue of nonribosomal lipopeptides, Milne [/bib_ref] [bib_ref] An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in..., Neary [/bib_ref] [bib_ref] Introduction of a nonnatural amino acid into a nonribosomal peptide antibiotic by..., Thirlway [/bib_ref] [bib_ref] Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl..., Uguru [/bib_ref]. In this work we focus on the biosynthesis of related lipoglycopeptide antibiotics: ramoplanin from Actinoplanes sp. ATCC 33076 , and the enduracidins produced by Streptomyces fungicidicus [bib_ref] The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus, Yin [/bib_ref]. Ramoplanin is a highly effective antibiotic against a number of Gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, and has entered phase III clinical trials for the treatment of Clostridium difficile infections. Enduracidin shares a similar structure with ramoplanin and is also a potent antibiotic; both antibiotics bind to lipid II, blocking the subsequent transglycosylation reactions during peptidoglycan biosynthesis of the bacterial cell wall [bib_ref] The mechanism of action of ramoplanin and enduracidin, Fang [/bib_ref]. However, enduracidin has poor aqueous solubility compared with ramoplanin . Consequently, whilst ramoplanin has entered clinical trials, enduracidin is instead utilized as an additive in animal feed [bib_ref] Structure elucidation and 3D solution conformation of the antibiotic enduracidin determined by..., Castiglione [/bib_ref] [bib_ref] The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus, Yin [/bib_ref]. The main structural difference between the two antibiotics is the presence of mannosyl groups on hydroxyphenylglycine 11 (Hpg 11 ) that contribute significantly to the solubility of ramoplanin but are absent in enduracidin. The mannosyl groups of ramoplanin are not required for antibacterial activity as the aglycone retains similar potency to the parent lipoglycopeptide. However the mannosyl groups of ramoplanin, in addition to enhancing solubility, contribute to the hydrolytic stability of the peptide core . In addition the mannosyl groups can provide a chemical handle for semisynthesis. For example, the mannosyl groups of the mannopeptimycins have been selectively derivatized to generate semisynthetic products with increased activity [bib_ref] Mannopeptimycins, a novel class of glycopeptide antibiotics active against Gram-positive bacteria, He [/bib_ref]. . Structures of the nonribosomal peptide antibiotics enduracidin, ramoplanin, mannopeptimycin and the dalbavancin precursor (A40926). All, except enduracidin, possess mannosyl substituents (in green) derived from putative polyprenyl phosphomannose-dependent glycosyltransferases Ram29, MppH/I and Dbv20 respectively.
## Antibiotic biosynthesis using ram29
Within the ramoplanin (ram) biosynthetic gene cluster there is a gene, ram29, encoding an integral membrane protein, that is absent from the enduracidin (end) cluster. Genes encoding similar proteins are also present within the gene clusters for other important mannosylated products, including the mannopeptimycins (mppH/I) [bib_ref] Biosynthetic pathway for mannopeptimycins, lipoglycopeptide antibiotics active against drug-resistant Gram-positive pathogens, Magarvey [/bib_ref] , and the commercial glycopeptide antibiotics teicoplanin (tcp15) and dalbavancin (dbv20) [bib_ref] The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by..., Sosio [/bib_ref]. The putative mannosylating enzymes have not been characterized, but are suggested to transfer mannose (Man) from a polyprenyl phosphomannose (PPM) donor within the membrane [bib_ref] Biosynthetic pathway for mannopeptimycins, lipoglycopeptide antibiotics active against drug-resistant Gram-positive pathogens, Magarvey [/bib_ref]. A recent report [bib_ref] Functional identification of the gene encoding the enzyme involved in mannosylation in..., Chen [/bib_ref] described the deletion of ram29 from the ramoplanin producer, Actinoplanes sp. ATCC 33076. The resultant deletion strain was shown to produce the ramoplanin aglycone, suggesting that Ram29 is responsible for the incorporation of the mannose sugars in ramoplanin. In this paper, we have established conditions for the heterologous expression of ram29 in the enduracidin-producing strain of S. fungicidicus, confirming the mannosyltransferase function of Ram29, and also providing access to novel mannosylated enduracidin variants.
# Methods
Bioinformatics analysis. The topology of Ram29 was predicted using bioinformatics programs which are applied widely to membrane protein analysis, namely TMPRED [bib_ref] TMBASE -A database of membrane spanning protein segments, Hofmann [/bib_ref] , HMMTOP , , SCAMPI [bib_ref] Prediction of membrane-protein topology from first principles, Bernsel [/bib_ref] , PRODIV [bib_ref] Best a-helical transmembrane protein topology predictions are achieved using hidden Markov models..., Viklund [/bib_ref] , PRO [bib_ref] Best a-helical transmembrane protein topology predictions are achieved using hidden Markov models..., Viklund [/bib_ref] , OCTOPUS ) and TOPCONS [bib_ref] Best a-helical transmembrane protein topology predictions are achieved using hidden Markov models..., Viklund [/bib_ref]. The DNA sequence was analysed using Vector NTI software V-10.1.1 (Invitrogen), and the BLAST and alignment analyses were conducted using the alignment program from the same software.
Bacterial strains, microbiological methods and genetic manipulations. The bacterial strains S. fungicidicus (ATCC 31731) and Actinoplanes sp. (ATCC 33076) were purchased from the ATCC bioresource centre (LGC Standards) and cultured in ISP Medium 2 and ISP Medium 1 broth respectively [bib_ref] Methods for characterization of Streptomyces species, Shirling [/bib_ref]. Prior to preparation of spore stocks, both strains were cultured on mannitol soya (MS) flour media for 2 weeks [bib_ref] Dispersed growth of Streptomyces in liquid culture, Hobbs [/bib_ref]. Mycelia from 1 ml of culture were used for genomic DNA extraction using a Qiagen Genomic DNA extraction kit (Qiagen) following the manufacturer's protocol for bacterial genomic DNA extraction. The genomic DNA was redissolved in 100 ml of 10 mM Tris/HCl buffer pH 8.5 and used as a template for PCR. The gene-specific primers ram29-F1, ram29-R1, ram29-F2 and ram29-R2 , available in the online Supplementary Material) were used to amplify the ram29 sequence. The resultant PCR product was digested with Bam HI alone or doubly digested with Eco RV and Nsi I and cloned into the similarly digested pIJ86 and pMS17 to yield pIJ86-ram29 and pMS17-ram29 . pIJ86 is a non-integrative vector with a constitutive ermE* promoter which has been widely used for protein expression in Streptomyces spp. [bib_ref] Cloning and analysis of the promoter region of the erythromycin resistance gene..., Bibb [/bib_ref]. pMS17 is a conjugative and integrative plasmid containing a tetracycline (Tc)-inducible promoter (kindly provided by Professor M. Smith, University of York) [bib_ref] Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a..., Wehmeier [/bib_ref]. Escherichia coli TOP10 (Invitrogen) was used for cloning procedures. The integrity of pIJ86-ram29 and pMS17-ram29 was confirmed by DNA sequencing (GATC Biotech) prior to being transformed into the dam, dcm and hsdM methylation-deficient E. coli strain ET12567 carrying the nontransmissible pUZ8002 [bib_ref] Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel..., Macneil [/bib_ref]. Intergeneric conjugation was carried out between E. coli ET12567/pUZ8002 harbouring either pIJ86-ram29 or pMS17-ram29 and S. fungicidicus. Following 3-6 days of incubation at 30 uC exconjugants were transferred to selective MS agar plates supplemented with apramycin (100 mg ml 21 ) and nalidixic acid (25 mg ml 21 ). Following 2 weeks of incubation at 30 uC, spores derived from the exconjugants were transferred to bouillon broth (1 % meat extract, 1 % peptone, 0.5 % NaCl, 0.2 % K 2 HPO 4 , pH 7.0; modified from ATCC Medium 841) and incubated at 30 uC for 2-4 days. The mycelia were pelleted by centrifugation and the genomic DNA extracted for use in sequence confirmation.
Isolation of enduracidin from S. fungicidicus fermentation media. The spores of WT or exconjugant S. fungicidicus strains (10 5 -10 7 ) were used to inoculate 75 ml of seed medium (Bouillon broth) and cultured for 3 days at 30 uC. Ten millilitres of seed culture was then transferred to 300 ml of enduracidin fermentation medium [bib_ref] Production of enduracidin and micro-organisms therefor, Nogami [/bib_ref] supplemented with the appropriate antibiotics and cultured at 30 uC with shaking for 14-21 days. For exconjugants harbouring pMS17-ram29, tetracycline was added to a final concentration of 0.1 mg ml 21 every 3 days. Following fermentation, cultures were transferred to 50 ml Falcon tubes and the mycelia pelleted by centrifugation (15 min, 10 000 g). Pellets were resuspended in 70 % acidic methanol (pH 2.0) and stirred for 3 h. Supernatants of the methanol extracts were obtained by centrifugation (10 min, 10 000 g) and proteins and lipids extracted with an equal volume of ethyl acetate. Following separation, the pH of the aqueous partition was adjusted to 8.2 and the enduracidin extracted using 300 ml (3|100 ml) of butanol. The butanol phase, containing enduracidin, was washed with water (3|100 ml), and then the enduracidin was partitioned into 300 ml (3|100 ml) of 5 mM HCl aqueous solution (pH 2.0). The pH of the aqueous layer was adjusted to 8.2 and the enduracidin extracted with 300 ml (3|100 ml) of butanol. The butanol layer containing enduracidin was separated and concentrated by evaporation under reduced pressure, leaving a crude white powder, which was redissolved in 20 % acetonitrile. Enduracidin was further purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a Gemini C18 column (10|250 mm, 5 mm; Phenomenex) using a gradient composed of water with 0.1 % formic acid (A) and acetonitrile with 0.1 % formic acid (B) as follows: 10 min linear gradient from A/B (95 : 5 %) to A/B (80 : 20 %), 30 min linear gradient to A/B (60 : 40 %), 5 min linear gradient to A/B (5 : 95 %), held at A/B (5 : 95 %) for 4 min, 3 min linear gradient to A/B (95 : 5 %) before re-equilibration at A/B (95 : 5 %) over 8 min. A flow rate of 3 ml min 21 was used and elution of enduracidin was monitored at A 230 . Fractions were collected and further analysed by liquid chromatography (LC)-MS.
Analysis of enduracidin extracts using LC-MS. LC-MS analysis was carried out on a Micromass LCT TOF mass spectrometer (Waters), equipped with an electrospray ionization source run in positive mode (scanning from m/z 700 to 2500) combined with a Waters 2790 separation module. Samples were dissolved in acetonitrile/water/formic acid (50/50/0.1). HPLC was carried out using a Gemini C18 column (4.6|150 mm, 3 mm; Phenomenex) at a flow rate of 1 ml min 21 using a gradient composed of water with 0.1 % formic acid (A) and acetonitrile with 0.1 % formic acid (B) as follows: 10 min linear gradient from A/B (80 : 20 %) to A/B (30 : 70 %), 1 min gradient to A/B (5 : 95 %) and held at A/B (5 : 95 %) for further 4 min, before re-equilibration at A/B (80 : 20 %) for 4 min.
Tandem MS. Tandem MS analysis was carried out on a Q-TOF Ultima Global instrument (Waters) by direct infusion of a purified sample dissolved in acetonitrile/water/formic acid (50/50/0.1).
Borosilicate nanoemitters (Proxeon) were loaded with sample using gel loading pipette tips. The nanoemitters were mounted onto the source and then activated at 0.4 kV capillary voltage by applying pressure against the sample cone, snapping the glass fibre. Once activated, the capillary voltage was ramped steadily until a consistent ion beam was observed, typically between 1.6 and 1.8 kV. Each nanoemitter holds approximately 20 ml and provides a spray for approximately 30 min duration, giving a flow rate of approximately 0.66 ml min 21 . The instrument was calibrated using the tandem mass spectrum of [Glu]-Fibrinopeptide B.
Once an ion beam was achieved, the MS source was tuned to the product by ramping the capillary voltage and the cone voltage until the signal intensity peaked. An MS scan was carried out to determine a suitable m/z value to fragment. An MS-MS scan was then carried out by selecting the triply charged [M+3H] 3+ ion (785 for the WT enduracidin and 839 for the mannosylated enduracidin) as the parent ion for analysis, and raising the collision energy within the collision cell until the parent ion was a minority peak on the resulting spectrum. The data were collected over 20 min, or until a sufficient spectrum was achieved for sequencing analysis. The subsequent spectrum was background subtracted (polynomial order54, 40 % below curve, tolerance50.010), smoothed (Savitzky Golay smoothing, smooth channels5+5, number of smooths52), and centred (minimum peak width at half height54, 80 % centroid top) by the MassLynx software (Version 4.0, Waters Ltd) prior to analysis.
Enduracidin sequencing. A database of theoretical m/z values was set up based on all the possible fragmentation patterns of enduracidin, assuming two cleavages yielding a linear peptide ion. Additionally, a further database was set up assuming that the first cleavage occurs at the ester bond linkage; thus additional fragmentation events yielded a, b, y and z ions at either one or both ends (as both the C-and N-termini have undergone fragmentation). Tandem MS data were then compared with these theoretical values to generate ion series that can be used to characterize the peptide, and signature losses were identified by observation.
# Results and discussion
## Bioinformatics and proposed mannosylation mechanism
Sequence similarity searches predict that Ram29 is an integral membrane protein, from the GT-C superfamily of glycosyltransferases, which is likely to be similar to other predicted mannosyltransferases including MppH/I [bib_ref] Biosynthetic pathway for mannopeptimycins, lipoglycopeptide antibiotics active against drug-resistant Gram-positive pathogens, Magarvey [/bib_ref] , Tcp15 and Dbv20 [bib_ref] The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by..., Sosio [/bib_ref]. Ram29 was further analysed using eight bioinformatics programs designed to predict the topology of membrane proteins. Depending on the algorithm used, Ram29 is predicted to possess between 10 and 14 transmembrane segments (TMS) spanning approximately 450 amino acids within the N-terminal region of the protein. Although the bioinformatics algorithms did not converge on the precise number of TMS, all of these programmes predict that the C-terminal domain, comprising approximately 150 amino acids, is extracytoplasmic. We suggest that this putative extracytoplasmic C-terminal domain, which shows little or no similarity with any other known protein sequences, is responsible for binding the ramoplanin aglycone (Figs. 2 and S2). This is consistent with earlier findings [bib_ref] Microbial de-mannosylation and mannosylation of teicoplanin derivatives, Borghi [/bib_ref] showing that the demannosylated teicoplanin aglycone can be remannosylated by incubating with the teicoplanin producing strain, possessing the putative mannosyltransferase Tcp15, demonstrating that mannosylation occurs on the outside of the cell membrane.
Other members of GT-C superfamily possess similar TMS, with acidic (DxD or DDx) motifs in either the first or (Ppm1) is located in the membrane and catalyses the synthesis of PPMs on the cytoplasmic membrane surface, before being translocated to the extracytoplasmic membrane surface. Subsequently, the membrane protein mannosyltransferase Ram29 transfers the mannose moiety from PPM to the antibiotic aglycone, which has been transported from the cytoplasm. second extracytoplasmic loop [bib_ref] Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of..., Berg [/bib_ref] [bib_ref] Three monophyletic superfamilies account for the majority of the known glycosyltransferases, Liu [/bib_ref]. The DxD motif is suggested to bind to, and activate, polyprenyl phosphosugar donors [bib_ref] The glycosyltransferases of Mycobacterium tuberculosis -roles in the synthesis of arabinogalactan, lipoarabinomannan,..., Berg [/bib_ref] , catalysing transfer of a range of different sugars, including glucose (Glu) [bib_ref] Biosynthesis of undecaprenyl phosphate-galactosamine and undecaprenyl phosphate-glucose in Francisella novicida, Song [/bib_ref] , galactosamine (GalN) [bib_ref] Biosynthetic origin of the galactosamine substituent of arabinogalactan in Mycobacterium tuberculosis, Skovierová [/bib_ref] , arabinofuranose (Araf) [bib_ref] Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of..., Berg [/bib_ref] and rhamnose (Rha), during cell wall oligosaccharide biosynthesis. Sequence alignments of Ram29 led to the identification of a putative DxD motif in the second predicted extracytoplasmic loop, containing acidic residues (E162, D163 & D175) which are conserved in other putative mannosyltransferases (Tcp15 and Dbv20) [fig_ref] Figure 3: RP-HPLC analysis of the S [/fig_ref]. Taken together, we suggest that Ram29 would use its unique C-terminal domain to recognize and bind the ramoplanin aglycone outside of the cell membrane, and the extracytoplasmic loop containing the DxD motif would recognize and transfer the mannosyl group from PPMs, which are known to be biosynthesized from GDP-mannose and polyprenyl phosphates, of varying chain length, by the PPM synthases (Ppm1) [bib_ref] Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a..., Wehmeier [/bib_ref] to form mannosylated antibiotics [fig_ref] Figure 2: Proposed two-component system for mannosylation of the glycopeptide antibiotics [/fig_ref].
Optimizing expression of ram29 in the enduracidin producer S. fungicidicus
The in vitro characterization of the putative mannosyltransferase Ram29, like other members of the GT-C superfamily enzymes, is complicated by difficulties associated with the overproduction, isolation and purification of membrane proteins as well as the lack of available PPM donor substrates with the required polyprenyl chain length. In light of this, we chose to establish the heterologous expression system for ram29 in the enduracidin producer S. fungicidicus. Given the structural similarity of enduracidin to the ramoplanin aglycone, we predicted that the endogenous enduracidin and PPM might function as substrates for Ram29 leading to mannosylated enduracidins. In addition to confirming the function of Ram29, this approach could potentially provide new mannosylated enduracidin antibiotics with improved properties, particularly those with enhanced aqueous solubility.
Accordingly, an expression cassette with the putative mannosyltransferase encoding gene ram29, including its native upstream Shine-Dalgarno (SD) sequence, was cloned into the high copy number plasmid pIJ86 under the control of the strong constitutive promoter ermE * [bib_ref] Cloning and analysis of the promoter region of the erythromycin resistance gene..., Bibb [/bib_ref]. The LC-MS analysis of cellular extracts and culture supernatants derived from S. fungicidicus pIJ86-ram29 exconjugants failed to lead to the detection of any mannosylated enduracidins. Given that Ram29 is predicted to be an integral membrane protein, the higher levels of expression afforded by pIJ86 could result in production of insoluble, non-functional Ram29. Consequently ram29 was placed under the control of a tetracycline inducible promoter by cloning the expression cassette into the integrative plasmid pMS17 [bib_ref] Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a..., Wehmeier [/bib_ref]. However, even with inducible control over the expression of the ram29 gene integrated onto the chromosome of S. fungicidicus, we still failed to detect any mannosylated enduracidin, which we rationalized may be due to a suboptimal SD sequence and GTG start codon of ram29. We therefore replaced the 59-leader sequence of ram29, including the native SD sequence and start codon, with the corresponding sequence of an eGFP expression construct from the plasmid pIJ8668 (kindly provided by Professor M. Bibb, John Innes Centre), which gives robust expression of eGFP [fig_ref] Figure 4: ESI [/fig_ref]. The resulting S. fungicidicus pMS17-ram29 exconjugants were shown to produce mannosylated enduracidins (vide infra), demonstrating the importance of the SD sequence and the distance between the SD sequence and the alternative ATG start codon for establishing functional expression of ram29.
Isolation of mannosylated enduracidins from S. fungicidicus pMS17-ram29
The optimized S. fungicidicus pMS17-ram29 was grown in liquid media for 2 weeks, the enduracidin isolated using a butanol extraction procedure and further purified by RP-HPLC [fig_ref] Figure 3: RP-HPLC analysis of the S [/fig_ref]. Fractions containing enduracidin were identified by MALDI-TOF [fig_ref] Figure 5: Tandem MS of WT and monomannosylated enduracidin A [/fig_ref] and further analysed by LC-MS [fig_ref] Figure 4: ESI [/fig_ref]. It was revealed that the strain S. fungicidicus pMS17-ram29 produced two additional new minor products differing in mass by +162 Da to enduracidins A and B, which were also evident. The +162 Da mass difference is consistent with the addition of a single mannose group to enduracidins A and B. mannosylated enduracidin A and B derivatives were absent from extracts derived from WT S. fungicidicus fermentations.
[formula] b 3 b 4 b 5 b 6 b 7 b 8 b 9 b 10 b 11 b 12 b 13 b 14 b 15 b 16 b [/formula]
The position of the putative mannose group was determined by tandem MS. The de novo sequencing of cyclic nonribosomal peptides can be non-trivial, as each linear fragment is the product of at least two fragmentation events, and the subset of possible fragments is dependent on the position of the first event. By running a series of experiments on both enduracidin A and the new glycosylated analogue in parallel, it was possible to deduce a y-ion series for both molecules under identical conditions. In the spectra, a prominent y-ion series can be observed for the WT enduracidin A peptide fragments: End 15 -Hpg 17 (y 3 , m/z 393.2), Gly 14 -Hpg 17 (y 4 , m/z 450.2), Ser 12 -Hpg 17 (y 5 , m/z 754.3), Hpg 11 -Hpg 17 (y 7 , m/z 903.3), End 10 -Hpg 17 (y 8 , m/z 1057.4), Cit 9 -Hpg 17 (y 9 , m/z 1214.4), Thr 8 -Hpg 17 (y 10 , m/z 1315.5) and Hpg 7 -Hpg 17 (y 11 , m/z 1464.5) [fig_ref] Figure 5: Tandem MS of WT and monomannosylated enduracidin A [/fig_ref]. In contrast, analysis of the monomannosylated enduracidin peptide revealed new fragment ions with m/z corresponding to: man-Hpg 11 -Hpg 17 (y 7 , m/z 1065.3), End 10 -Hpg 17 (y 8 , m/z 1219.4), Thr 8 -Hpg 17 (y 10 , m/z 1477.5) [fig_ref] Figure 5: Tandem MS of WT and monomannosylated enduracidin A [/fig_ref]. Comparison of the key observed ions y 7 , y 8 , and y 10 shows a mass difference of +162 Da between the WT and the mannosylated sample, corresponding to the addition of a single mannose group in the latter. Additional ions corresponding to non-hydrated fragment ions, and doubly charged fragments ions (both hydrated and non-hydrated) are described in the supplementary information , Figs S7 and S8). The MS-MS spectra provide strong evidence as to the regioselectivity of Ram29, with the mannosylation occurring on the Hpg 11 residue of enduracidin: the +162 Da mass difference was carried throughout the mannosylated peptide sample from y 10 to y 7 ; however once Hpg 11 was lost, thus generating the y 6 ion, this and all subsequent fragment ions were identical with those of the WT enduracidin [fig_ref] Figure 5: Tandem MS of WT and monomannosylated enduracidin A [/fig_ref]. Thus, Ram29 appears to mannosylate enduracidin at the same position as on ramoplanin. Moreover, Ram29 is evidently promiscuous and can mannosylate alternative lipopeptides to ramoplanin, such as the enduracidins A and B.
The deletion of ram29 from the ramoplanin producer, Actinoplanes sp. ATCC 33076 [bib_ref] Functional identification of the gene encoding the enzyme involved in mannosylation in..., Chen [/bib_ref] , was shown to produce the ramoplanin aglycone which was consistent with the findings presented here. Given that Ram29 is the only apparent glycosyltransferase in the ramoplanin biosynthetic gene cluster, this suggests that Ram29 may act iteratively to introduce both the mannosyl groups of ramoplanin [bib_ref] Functional identification of the gene encoding the enzyme involved in mannosylation in..., Chen [/bib_ref]. Interestingly, we show that expression of Ram29 in S. fungicidicus results in only monomannosylated enduracidins. Thus whilst Ram29 clearly recognizes the phenol group of Hpg 11 in both enduracidin and the ramoplanin aglycone as its acceptor substrate, it is possible that a second mannosyltransferase, which is not encoded from within the ramoplanin biosynthetic gene cluster, may be required to introduce a second distinct a1,2-dimannosyl glycosidic linkage. Alternatively, some Streptomyces species have been reported to have membrane-associated a-mannosidases which remove mannosyl groups from antibiotics. For example, the mycelia of Streptomyces GE 91081 can remove one mannose unit from ramoplanin , whilst the mycelia of Nocardia orientalis NRRL 2450 and Streptomyces candidus NRRL 3218 have been found to remove a mannose unit from teicoplanin [bib_ref] Microbial de-mannosylation and mannosylation of teicoplanin derivatives, Borghi [/bib_ref]. S. fungicidicus contains a putative mannosidase encoding gene (orf13) within the enduracidin gene cluster [bib_ref] The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus, Yin [/bib_ref] , which may function to remove a mannose group from any dimannosylated enduracidin that may be produced. Mannosidase activity could also potentially account for the low yields of mannosylated enduracidins produced by the S. fungicidicus pMS17-ram29 strain.
# Conclusion
In summary, we have developed protocols for the functional expression of the mannosyltransferase Ram29 in the enduracidin producer S. fungicidicus and isolated novel monomannosylated enduracidin variants. Using tandem MS, we have shown that mannosylation occurs at Hpg 11 of enduracidin, which supports the function of Ram29 in the biosynthesis of ramoplanin. Previously there have been considerable advances in the development of natural product glycodiversification strategies, which have largely focused on glycosylation pathways that involve the more common Leloir-type (sugar nucleotide-dependent) glycosyltransferases. The findings presented here provide the first example of how the less common, membrane associated PPMdependent glycosyltransferases can also be used in glycodiversification of natural product scaffolds; this could be valuable in future efforts to develop bioactive natural products, including improved enduracidin variants with enhanced aqueous solubility.
[fig] Figure 2: Proposed two-component system for mannosylation of the glycopeptide antibiotics. PPM synthase [/fig]
[fig] Figure 3: RP-HPLC analysis of the S. fungicidicus crude extract. (a) Crude extract from the fermentation broth of the S. fungicidicus pMS17-ram29 transformant. (b) Enduracidin A and B standards. [/fig]
[fig] Figure 4: ESI (electrospray ionisation)-LC-MS analysis of the S. fungicidicus pMS17-ram29 fermentation products. The spectra show the triply charged [M+3H] 3+ parent ions for WT enduracidins A (m/z: observed 785.5; expected 785.3) and B (m/z: observed 790.2; expected 790.0) and monomannosylated enduracidins A (m/z: observed 839.6, expected 839.3) and B (m/z: observed 844.3, expected 844.0). Doubly charged [M+2H] 2+ molecular ions were also observed by ESI-LC-MS(Fig. S5), and the singly charged [M+H] + ions were observed by MALDI-TOF(Fig. S6), consistent with the proposed structures. [/fig]
[fig] Figure 5: Tandem MS of WT and monomannosylated enduracidin A. (a) Localization of the mannosyl group on monomannosylated enduracidin A by MS-MS characterization. The y ions highlighted in red denote a mass difference of +162 Da from WT to mannosylated. The key y 7 ion is the last fragment to carry the +162 mannosyl group mass. (b) Structure of the monomannosylated enduracidin A analogue, based on MS-MS data. Additional MS-MS data(Tables S2-S5) and spectra (Figs S7 and S8) supporting the structure of monomannosylated enduracidin A can be found within the supplementary information. [/fig]
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Skill acquisition as a function of age, hand and task difficulty: Interactions between cognition and action
Some activities can be meaningfully dichotomised as 'cognitive' or 'sensorimotor' in naturebut many cannot. This has radical implications for understanding activity limitation in disability. For example, older adults take longer to learn the serial order of a complex sequence but also exhibit slower, more variable and inaccurate motor performance. So is their impaired skill acquisition a cognitive or motor deficit? We modelled sequence learning as a process involving a limited capacity buffer (working memory), where reduced performance restricts the number of elements that can be stored. To test this model, we examined the relationship between motor performance and sequence learning. Experiment 1 established that older adults were worse at learning the serial order of a complex sequence. Experiment 2 found that participants showed impaired sequence learning when the non-preferred hand was used. Experiment 3 confirmed that serial order learning is impaired when motor demands increase (as the model predicted). These results can be captured by reinforcement learning frameworks which suggest sequence learning will be constrained both by an individual's sensorimotor ability and cognitive capacity.
# Introduction
The study of cognition lies at the centre of psychological research. Nevertheless, there is no 'standard model' of cognition, and psychology lacks consensus on the very nature of this broad construct. Indeed, there are diametrically opposing views on how we should understand cognition. On the one hand, there is a long tradition of conceptualising 'cognition' as a closed system that is concerned purely with abstract information processing; a system which is independent of perceptual-motor functions. This entrenched view of the inconsequential nature of motor control within studies of cognition is particularly evident in the most popular approaches used to administer computerised cognitive test batteries (e.g. the CANTAB, NEP-SY-II, NIH Toolbox). These batteries either: (i) ignore the motor aspects of performance once a baseline motor task has been 'passed' or (ii) treat motor performance as a separate entity that a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
can be dissociated from other cognitive functions. The CANTAB [bib_ref] A comparative study of visuospatial memory and learning in Alzheimer-type dementia and..., Sahakian [/bib_ref] adopts approach (i) and assays visuomotor control in an initial 'induction' test before the core battery of tasks is presented. The core tests are designed to assess specific 'cognitive' abilities (e.g. attention, working memory and decision making) but necessarily require a variety of task-specific manual responses. This approach is problematic because it assumes that an individual's sensorimotor performance does not contribute to variability in any of the subsequent, explicitly 'cognitive', assessments (provided motor performance exceeds a minimum threshold on one specific 'motor' task at the beginning of the assessment). The alternative approach taken in other test batteries (e.g. the NEPSY-II [bib_ref] Effects of age on neurocognitive measures of children ages 5 to 12:..., Korkman [/bib_ref] and the NIH Toolbox [bib_ref] NIH toolbox for assessment of neurological and behavioral function, Gershon [/bib_ref] is to treat motor skill as a separate function that can be measured independently. Thus, the NEPSY-II and the NIH Toolboxincorporate comprehensive sub-tests explicitly intended to assess various aspects of motor function. However, they indicate that these are taxonomically distinct from cognitive sub-tests (e.g. the NIH Toolbox includes a sub-battery of 'Cognitive' tests and a separate sub-battery of 'Motor' tests), and this gives rise to ontological questions with no clear answers-what defines a specific sub-test with 'cognitive' but no 'motoric' demands, or vice versa?
The separation of 'cognition' from the 'sensorimotor' system is somewhat formalised in the dual-task literature where the extent to which these purportedly separate systems interfere with each other is explored by asking participants to conduct a cognitive exercise concurrently with a motor task. In contrast there are increasing numbers of researchers who argue that cognition is an open system that can only be understood in terms of its embodiment within the anatomical structures that influence and are influenced by the world [bib_ref] Six views of embodied cognition, Wilson [/bib_ref]. The extreme version of this 'embodied cognition' viewpoint posits that cognition is distributed across mind, body and environment and that "concepts, internally represented competence, and knowledge" should be removed as topics of study within cognitive psychology [bib_ref] Embodied Cognition is Not What You Think It Is, Wilson [/bib_ref].
Theorists have shown that the extreme versions of embodied cognition (i.e. where the environment is viewed as part of the cognitive system) are deeply problematic [bib_ref] Six views of embodied cognition, Wilson [/bib_ref]. Nevertheless, there is a growing consensus that "the mind must be understood in the context of its relationship to a physical body that interacts with the world" [bib_ref] Six views of embodied cognition, Wilson [/bib_ref]. Thus, there is broad support for the notion that it is both useful and appropriate to consider sensorimotor processes as being more than simple mechanisms of output. We suggest that there is a sensible and-importantly-useful middle ground that exists between the idea that cognition is encapsulated from sensorimotor function and the more extreme versions of 'embodied cognition'. We will use the term 'Cognition Action Interaction Theory' to refer to this conceptual meeting ground. Cognition Action Interaction Theory (CAIT) captures the idea that 'higher-order cognition': (i) has the sensorimotor system at its phylogenetic and ontogenetic foundations; (ii) is an emergent property of a brain originally constructed for sensorimotor processing; (iii) can operate without the 'sensorimotor control system' (and vice versa); (iv) interacts with the 'sensorimotor system' in ontogeny and, throughout life, in numerous everyday activities. Thus, CAIT recognises cognition and sensorimotor processing as somewhat separate evolutionary entities (worthy of study in their own right) whilst acknowledging that they are synergistically interdependent.
One implication of this viewpoint is that many complex human activities cannot be meaningfully reduced to their component cognitive and motor parts. We would suggest that it can be meaningful to study cognition in the context of tasks that have minimal motor input (e.g. abstract reasoning about the potential outcomes of a chess move) and also to examine sensorimotor control in the context of tasks that have minimal cognitive input (e.g. picking a fallen chess piece off the floor). Even so, a full understanding of complex skilled behaviours in humans (e.g. playing a game of chess) requires a consideration of both the motor and cognitive systems, and how these systems interact. In other words, the impressive repertoire of skills possessed by human adults is not just a testament to the extraordinary neurophysiological organisation that underpins the sensorimotor system. It is the more recently evolved higherorder cognitive architecture that allows Homo Sapiens to display skills that go far beyond the quantity and diversity of skills observed in any other animal species. Thus, the acquisition and production of complex skills in humans rests upon the motor and cognitive apparatus working together in unison [bib_ref] A Test of Motor (Not Executive) Planning in Developmental Coordination Disorder and..., Van Swieten [/bib_ref]. This fact is particularly important to recognise when understanding and supporting individuals who show deficits in learning skilled behaviours, because improved understanding of the interaction between motor and cognitive processes can allow for the development of more tailored treatment regimens within a rehabilitation context.
The focus of the present paper is on older adults who often show impaired skill acquisition relative to their younger counterparts. An extensive review of the literature on learning in older adults concluded that complex tasks have a greater likelihood of revealing age differences [bib_ref] Motor-skill learning in older adults-a review of studies on age differences, Voelcker-Rehage [/bib_ref]. Age-related decline in cognitive ability might provide one explanation as to why older adults have particular difficulties with the acquisition of novel skills [bib_ref] Skill learning in the elderly: diminished implicit and explicit memory for a..., Harrington [/bib_ref] [bib_ref] Multidimensional Motor Sequence Learning IIs Impaired in Older But Not Younger or..., Boyd [/bib_ref]. This is because the learning of a new skill is likely to require storage and/or attentional resources associated with working memory [bib_ref] Working memory: Theories, models, and controversies, Baddeley [/bib_ref] [bib_ref] Functional mapping of sequence learning in normal humans, Grafton [/bib_ref] [bib_ref] Transition of brain activation from frontal to parietal areas in visuomotor sequence..., Sakai [/bib_ref] [bib_ref] Individual differences in working memory capacity and learning: Evidence from the serial..., Unsworth [/bib_ref]. It is well established that older age is associated with a decline in such cognitive functions (e.g. cognitive slowing, poorer working memory and reduced attention [bib_ref] Working-memory capacity, age, and memory for discourse, Light [/bib_ref] [bib_ref] Meta-Analyses of Age-Cognition Relations in Adulthood: Estimates of Linear and Nonlinear Age..., Verhaeghen [/bib_ref]. Moreover, the literature suggests that there are age differences in how new skills are acquired. One specific difference is the way in which serial order information is encoded. Studies show that younger adults store parts of a sequence in 'chunks', which are internal representations of groups of elements that feature in a given sequence. Encoding sequences in this way saves limited processing resources-instead of recalling each element of a sequence individually, integrated sections of the array (typically three-to-five elements) can be combined and recalled together [bib_ref] Visuospatial Working Memory Capacity Predicts the Organization of Acquired Explicit Motor Sequences, Bo [/bib_ref] [bib_ref] Age-Related Declines in Visuospatial Working memory Correlate With Defecits in Explicit Motor..., Bo [/bib_ref] [bib_ref] Chunking during human visuomotor sequence learning, Sakai [/bib_ref] [bib_ref] Buffer Loading and Chunking in Sequential Keypressing, Verwey [/bib_ref] [bib_ref] Evidence of a multi-stage model of practice in a sequential movement task, Verwey [/bib_ref] [bib_ref] Evidence for Lasting Sequence segmentation in the Discrete Sequence-Production Task, Verwey [/bib_ref] [bib_ref] Segmentation of short keying sequences does not spontaneously transfer to other sequences, Verwey [/bib_ref] [bib_ref] Diminished motor skill development in elderly: Indications for limited motor chunk use, Verwey [/bib_ref]. Older adults do not always benefit from a chunking encoding strategy and show limited chunking compared to younger adults. Even when chunking is used, the chunks are comprised of fewer elements [bib_ref] Diminished motor skill development in elderly: Indications for limited motor chunk use, Verwey [/bib_ref] [bib_ref] Age-Related Effects in Sequential Motor Learning, Shea [/bib_ref]. There is evidence that older adults have generalised difficulties in memorising chunks in tasks involving immediate serial recall [bib_ref] Age-related differences in immediate serial recall: Dissociating chunk formation and capcity, Naveh-Benjamin [/bib_ref] and long-term association formation [bib_ref] Aging and memory for new associations: Direct versus indirect measures, Howard [/bib_ref] [bib_ref] Feature memory and binding in young and older adults, Chalfonte [/bib_ref] [bib_ref] Adult-age differences in memory performance: Tests of an associative deficit hypothesis, Naveh-Benjamin [/bib_ref] [bib_ref] The effects of aging and divided attention on memory for item and..., Castel [/bib_ref] [bib_ref] Aging and visual feature binding in working memory, Allen [/bib_ref]. Bo et al. [bib_ref] Age-Related Declines in Visuospatial Working memory Correlate With Defecits in Explicit Motor..., Bo [/bib_ref] found that older adults had reduced visual working memory capacity and produced shorter chunk lengths in a movement sequence learning task. Positive correlations between working memory and chunk length and between chunk length and sequence learning were also observed (though no direct relationship between working memory and learning rate in older adults was found.).
The decline of working memory in older adults certainly provides a plausible explanation for the problems encountered by older people when faced with the task of learning a new skill. But this does not rule out other factors contributing to a reduction in learning ability. In the past, studies have consistently reported an age-related decline in motor performance, whereby older adults exhibit reduced motor speed and accuracy across a range of manual control and coordination tasks [bib_ref] Kinematics of arm movement in elderly humans, Cooke [/bib_ref] [bib_ref] Upper-extremity motor coordination of healthy elderly people, Desrosiers [/bib_ref] [bib_ref] The Locus of Age-Related Movement Slowing: Sensory Processing in Continuous Goal-Directed Aiming, Pohl [/bib_ref] [bib_ref] Elderly subjects are impaired in spatial coordination in fine motor control, Contreras-Vidal [/bib_ref] [bib_ref] Movement control in older adults: does old age mean middle of the..., Raw [/bib_ref] [bib_ref] Reduced motor asymmetry in older adults tracing paths, Raw [/bib_ref] [bib_ref] Structural learning predicts the 'goldilocks zone' needed for getting the measure of..., Raw [/bib_ref] [bib_ref] Motor Sequence Learning in Healthy Older Adults is Not Necessarily Facilitated by..., Raw [/bib_ref]. This can be explained by age-related physiological factors such as limited joint flexibility and reduced muscle strength in the limbs [bib_ref] Effects of age upon the mobility of human finger joints, Barnett [/bib_ref] [bib_ref] Neural control of aging skeletal muscle, Delbono [/bib_ref] , neural changes [bib_ref] Age-related changes in motor cortical properties and voluntary activation of skeletal muscle, Clark [/bib_ref] [bib_ref] Neurophysiological correlates of age-related changes in human motor function, Mattay [/bib_ref] [bib_ref] Neural correlates of age-related changes in cortical neurophysiology, Talelli [/bib_ref] [bib_ref] Age-related changes in the neural correlates of motor performance, Ward [/bib_ref] , increased susceptibility to diseases that affect movement (e.g. stroke, arthritis) and compensatory changes in motor strategy [bib_ref] Movement control in older adults: does old age mean middle of the..., Raw [/bib_ref] [bib_ref] Age-related motor slowness: simply strategic?, Morgan [/bib_ref]. Little is known about how this decline in underlying motor performance can impact on learning a novel skill. While motor learning undoubtedly relies on the higher-order processes of the cognitive system (i.e. the reasoning and memory processes that allow a new skill to be retained and retrieved [bib_ref] Learning and production of movement sequences: Behavioral, neurophysiological, and modeling perspectives, Rhodes [/bib_ref] [bib_ref] Physical and motor fitness are both related to cognition in old age, Voelcker-Rehage [/bib_ref] , the CAIT perspective would suggest that the motor processes that underlie a particular skill (i.e. the functions of the motor system that allow one to physically move and coordinate one's fingers to type out a memorised password, for example) must also be considered as an integral part of the learning process.
The aim of the present series of experiments is to better understand the relationship between motor performance and the learning of a skill that requires 'visual serial order learning'. We capture the features of a complex skill by considering a movement sequence containing a series of sub-goals that need to be achieved in a specific order to produce a given outcome (i.e. the goal). In classic motor learning theory, a central component of learning a complex skill is the 'cognitive phase', which entails memorising the requisite movement sequence. Following the cognitive phase is the 'associative phase', which involves linking the 'component parts' into one smooth perceptual-motor action. Hence, learning a complex skill requires the memorisation of a series of individual movements (i.e. lower-order elements) so that these components can be linked into a smooth action and ultimately become an automated, single, higher-order behaviour. Incidentally there may well be difficulties in defining the lower-order elements for many complex movements; but this issue goes beyond the scope of this manuscript. The modelling of motor learning has been greatly refined over the last four decades, but most motor theorists accept the basic insights provided by Fitts and Posnerregarding the key stages involved in movement learning. Indeed, neuroimaging methods have revealed that the release of cognitive control hubs in frontal cortex and cingulate cortex can predict individual differences in rate of learning [bib_ref] Learning-induced autonomy of sensorimotor systems, Bassett [/bib_ref].
The CAIT perspective suggests that motor performance is likely to impact on the memory processes responsible for learning the serial order of the lower-order elements within the cognitive phase. We therefore hypothesised that sequence learning would deteriorate as the elements increased in motor difficulty. It is widely accepted that task difficulty is well indexed by movement duration, so that as an individual's skill level increases so movement duration decreases. One major reason for decreased movement duration at higher skill levels is because there is less need to make online error corrections. Therefore, movements can be faster when the demands on error correcting mechanisms are reduced (i.e. bandwidth availability decreases when more error corrections are required and vice versa). The lawful nature of this speed-accuracy relationship was first formalised by Fitts [bib_ref] The information capacity of the human motor system in controlling the amplitude..., Fitts [/bib_ref] , though empirical findings led to 'Fitts' law' being reformulated by Welford [bib_ref] Speed and accuracy of movement and their changes with age, Welford [/bib_ref]. There is still some controversy over the precise nature of the function that relates movement duration (time) to task parameters, but Welford's formulation captures most of the variance in a wide range of experiments (see [bib_ref] Speed/accuracy trade-offs in target-directed movements, Plamondon [/bib_ref] for a discussion). Welford's formulation is given in Eq 1 below:
[formula] MT ¼ a þ b Log 2 A þ c Log 2 Wð1Þ [/formula]
Where MT is movement time; A is the amplitude of the movement; W is the size of the target; and a, b and c are constants for a particular individual performing a particular task. It is apparent that increasing the need for error corrections by decreasing the size of the target (W), results in increased movement time (MT). Furthermore, raising the values of a, b and c captures the increase in movement time associated with motor performance decline (i.e. because of increased task difficulty or decreased skill level).
Whilst Eq 1 captures most of the variance associated with a single movement from one target to another, a storage buffer with properties akin to that of working memory is required to model serial learning processes. It has been established that working memory has a limited capacity [bib_ref] Individual differences in working memory capacity and learning: Evidence from the serial..., Unsworth [/bib_ref] , thus it follows that increasing the level of difficulty of the lower-order elements in a sequence learning task may well decrease the total number of elements that can be held in working memory (i.e. because increased difficulty increases the informational load associated with each element). Accordingly, we can model sequence learning as a process with a storage buffer (working memory) that has a limited storage capacity C (Eq 2). The limited capacity means that the buffer only stores elements when the condition Q of sufficient storage space is met. For all conditions where Q � 0, the elements (e) are held in serial order:
[formula] Q ¼ C À P n i¼1 e ið2Þ [/formula]
The buffer can then write the stored element sequence to a longer-term memory store (as a 'chunk'), and the process repeats iteratively until the whole sequence has been learned. Eq 1 shows that the difficulty associated with any element (measured in terms of information) is directly related to individual skill level and task difficulty, in any activity that involves movement. Thus, increasing the task difficulty increases the informational load of each element and thereby decreases the number of elements that can be stored in any iteration of the learning process. In combination, Eqs 1 and 2 can be used to model sequence learning under circumstances of increased task difficulty or reduced working memory capacity. In the discussion we show how this can be implemented as a model-free reinforcement learning algorithm (though our findings have implications for current computational approaches to modelling learning which typically lack a 'working memory' component). While this model is obviously a gross simplification of the learning process, it does capture known features of the cognitive and perceptual-motor system. It also makes clear predictions. First, smaller working memory capacity (or larger element size) will decrease recall on any single iteration and lengthen the time taken to learn the whole sequence , respectively). Combinations of smaller elements and larger capacity will also improve recall, whereas larger elements and smaller capacity will impair recall . Secondly, smaller capacity (or larger element informational load) will decrease the number of items within the 'chunks' written to longer-term storage . Third, an individual with worse motor skill (indexed by the constants in Eq 1) will show slower learning. Finally, increased task difficulty (as defined in Eq 1) will result in a reduced number of elements being learned per iteration (resulting in a longer learning time constant, i.e. more iterations to achieve the same learning).
We tested this model empirically by measuring the effects of age and motor performance on skill learning in healthy adults. We chose to study aiming movements that were made to discrete target locations because this allowed us to readily define the 'lower-order' elements (i.e. the individual movements that underpinned the complete sequence). It is well established that older populations prefer to perform fine motor coordination tasks more slowly [bib_ref] Reduced motor asymmetry in older adults tracing paths, Raw [/bib_ref] [bib_ref] Structural learning predicts the 'goldilocks zone' needed for getting the measure of..., Raw [/bib_ref] [bib_ref] Motor Sequence Learning in Healthy Older Adults is Not Necessarily Facilitated by..., Raw [/bib_ref] [bib_ref] Effects of age upon the mobility of human finger joints, Barnett [/bib_ref]. A control experimentsee S1 File for experimental details using methods reported in [bib_ref] A new tool for assessing human movement: The Kinematic Assessment Tool, Culmer [/bib_ref] confirmed that the quicker movements of younger adults were more directthan the longer movement of older adults which were associated with increased error correction compared to a younger group. To test the relationship between motor performance and learning the first experiment used a novel sequence learning task, suitable for both younger and older adults. The task consisted of training trials that prompted participants to move a mouse cursor to one of eight targets on a screen in a sequence of aiming movements. Following each training trial, learning was assessed by asking participants to attempt to recall the sequence without prompts or feedback. The second experiment employed this learning task in a new set of younger and older participants, with participants using both the preferred (right) or non-preferred (left) hand. Because motor performance is usually worse with the non-preferred hand, our model predicted that this would impact negatively on sequence learning for both age groups. The final experiment compared learning under two levels of motor difficulty using only the preferred hand (to avoid possible confounds linked with hemispheric specialisation): a computer mouse was either used in a standard fashion or oriented sideways on an unfamiliar vertical surface-where reduced motor performance was expected.
## Experiment 1
# Method
Participants. Twenty healthy individuals with no previous history of ophthalmological or neurological problems formed an opportunistic sample. All participants were right-handed as indexed by the Edinburgh Handedness Inventory (EHI), with an average score of 96.5 (SD = 9.88) out of the maximum 100 (Nb. Scores of 40+ indicate right-handedness). Participants were split into two age groups. The 'Younger' group (6 females, 4 males) were aged between 18 and 40 years (mean age = 24.9, SD = 7.45) and the 'Older' group (6 females, 4 males) were aged between 60 to 75 years (mean age = 69.60, SD = 4.12). All participants gave their written informed consent, and the experiment complied with ethical guidelines Recall performance when iteratively using Eqs 1 and 2 to model storage of a sequence of movements in the correct order: (A) A smaller working memory capacity will decrease recall on any iteration and lengthen the time taken to learn the whole sequence; (B) A larger element size has a similar effect; (C) The combination of smaller elements and larger capacity will increase recall, whereas larger elements and smaller capacity will decrease recall; (D) A smaller capacity and/or larger element size will decrease the 'chunk size'-the number of items within a unit written to the longer term storage system.
https://doi.org/10.1371/journal.pone.0211706.g001
Interactions between cognition and action approved by the University of Leeds ethical committee, in accordance with the Declaration of Helsinki. Procedure and apparatus. A motor learning task was created using 'KineLab' [bib_ref] A new tool for assessing human movement: The Kinematic Assessment Tool, Culmer [/bib_ref]. Participants used a tablet PC and standard computer mouse to learn a sequence of movements each made between the centre and one of eight target locations on the screen (see [fig_ref] Fig 3: Screen shots of the learning task as it appeared to participants in... [/fig_ref]. The task consisted of a series of 'training' and 'test' trials that alternated to allow 14 opportunities each for participants to practice and then reproduce the sequence (i.e. training trial, test trial x 14 repetitions = 28 trials in total). The same sequence was used across all the participants to ensure that there were no confounding effects created by sequences of differing difficulty. Interactions between cognition and action 3A shows the screen as it appeared to participants in the training trial, where there was one central white box (height = 25mm; width = 25mm), encircled by eight identical target location boxes. In the training trials, a black arrow appeared in the central box as a cue for participants to move the circular cursor to the target location adjacent to the direction of the arrowhead (e.g. the correct response would be to move the dot to the top left box for the example given in [fig_ref] Fig 3: Screen shots of the learning task as it appeared to participants in... [/fig_ref]. After each individual move to a target location, participants returned to the centre at their own pace, at which point the next arrow in the sequence would appear (no mouse clicks were required). There were 30 moves per sequence to learn, which followed an irregular pattern (see example traces from a participant completing the training trial in [fig_ref] Fig 3: Screen shots of the learning task as it appeared to participants in... [/fig_ref]. All participants received the same training sequence, but the timing of the training was driven by the preferred movement speed of the participant. After each training trial, participants were required to attempt to reproduce the sequence of moves by moving the cursor back-and-forth between the central box and target locations as quickly and as accurately as possible. No feedback was given to participants about whether these movements were correct. Recall trials continued until participants no longer wanted to make movements (either they felt they had completed the sequence or no longer knew where to move). Participants were not instructed when to stop-they could either keep on guessing or stop when they felt unsure about the sequence. Trials were terminated by the participant when they returned and stayed over the centre square for more than 4s. Examples of a training and test trials are shown in [fig_ref] Fig 3: Screen shots of the learning task as it appeared to participants in... [/fig_ref] and [fig_ref] Fig 3: Screen shots of the learning task as it appeared to participants in... [/fig_ref]. To ensure participants' complete understanding of the task, standardised instructions were presented in a series of slides, which included screen shots of the two trial types (similar to those pictured in [fig_ref] Fig 3: Screen shots of the learning task as it appeared to participants in... [/fig_ref]. Participants were twice given practice of training and test trials, which featured a 16-move sequence different to that used in the experimental task.
Analyses. The following outcome measures were calculated to examine speed and accuracy of recall (i.e. motor learning) in the test trials, and level of motor performance in the training trials.
i. Recall during test trial measures: Number of moves recalled in the correct sequential order (Correctly Recalled; CR), with a maximum score of 30 (points were not deducted for incorrect moves but in the majority of cases the first error in the sequence marked the end of successful recall); Recall Movement Time (MT r ) was the mean time (s) taken to move the mouse from the centre to a target box when recalling the sequence (i.e. a measure of recall speed).
ii. Training trial measures: Path Length (PL) indicated the length of the path (mm) taken by participants throughout an entire training trial, thus providing a marker of movement accuracy (i.e. straight paths will be shorter) with longer paths suggesting the presence of error corrections; Training Movement Time (MT t ) was the time (s) taken to complete a training trial from start to finish.
For the analyses of data from the test trials, mean values for CR and MT r across the first five trials (F5) and last five trials (L5) were calculated. These data were input into two separate mixed-model ANOVAs to compare speed and accuracy of sequence recall between the beginning and end trial blocks (i.e. to identify progression of learning from the first to second half of the task), and between the older and younger age groups. For the training trials, mean values for PL and MT t across the L5 trials were used as a baseline measure of motor performance.
# Results
Recall during test trials. The ANOVA for number of moves recalled in the correct sequential order (CR) revealed a significant effect of age (F (1, 18) = 15.70, p < 0.05, η 2 p = .47), whereby the Younger adults learned a greater number of moves than the Older adults (see [fig_ref] Fig 4: Measurements of motor learning recorded in the test trials of Experiment 1... [/fig_ref]. A main effect of trial block (F (1, 18) = 50.08, p < 0.001, η 2 p = .74) shows that all participants had learned a greater number of moves by the end of the task (mean CR for L5 = 15 items or 50% of the sequence) compared to the first half (mean CR for F5 = 8 items or 27% of the sequence). The interaction between age and trial block (F (1, 18) = 9.93, p < 0.01, η 2 p = .36), suggests that the Younger group learned disproportionally more than the Older group by the end of all the training.
We also measured speed of recall for correct responses during the task, hence [fig_ref] Fig 4: Measurements of motor learning recorded in the test trials of Experiment 1... [/fig_ref] shows the mean Recall Movement Time (MT r ) for older and younger participants on the F5 and L5 blocks of the test trials. Analyses of the MT r data showed shorter duration movements in the L5 trials compared to the F5 trials (F (1, 18) = 17.06, p < 0.05, η 2 p = .49; mean MT r for the F5 = 2.40s, compared to L5 = 1.89s), and the younger adult group had shorter movement durations than the older adults (F (1, 18) = 11.46, p < 0.05, η 2 p = .39; mean MT r for Old = 2.46s, compared to Young = 1.83s). There was no age ρ trial block interaction (F (1, 18) = .06, p > 0.05).
Training trials. The younger adults demonstrated superior motor performance in the training trials, whereby Training Movement Time (MT t ) was significantly shorter in the Younger group (t (18) = 2.54, p < 0.05). There was, however, no reliable age difference in accuracy of aiming movements, as indicated by PL (t (18) = 1.25, p > 0.05), presumably because the Older adults moved at a slower pace, thus allowing them to maintain comparable accuracy to the Younger adults (i.e. through speed-accuracy trade-offs [bib_ref] Reduced motor asymmetry in older adults tracing paths, Raw [/bib_ref].
# Discussion
The results of Experiment 1 show that the selected task provided a useful measure of movement sequence learning in Younger and Older adults. All participants showed evidence of learning the movement sequence over the set of training trials. The task was neither too Interactions between cognition and action difficult (i.e. too little learning), nor too easy (i.e. the sequence learned too quickly), hence it provides a useful metric of learning ability. During test trials participants were free to keep producing movements (including incorrect movements) since there was no explicit feedback for when they started getting the sequence wrong. It was, therefore, possible that younger and older age groups adopted different strategies during recall (it has sometimes been observed that older groups adopt more conservative strategies). [fig_ref] Fig 5: The number of moves recalled during each of 14 test trials by... [/fig_ref] shows how many movements were produced relative to the total number of moves in the sequence (as a proportion) for each test trial. It can be seen that both groups adopted similar strategies, making a number of incorrect moves (producing many more Total moves than Correct moves). Initially both age groups produce similar numbers of moves, but by the end of the test trials the Younger group were producing approximately double the number of moves as the Older group. Note that the Older group produce a similar number of Total moves as the number of Correct moves produced by the Younger group, suggesting that there wasn't a simple physical limitation preventing the production of that number of moves. This experiment also reinforces previous reports of reduced learning in the Older adults [bib_ref] Motor-skill learning in older adults-a review of studies on age differences, Voelcker-Rehage [/bib_ref]. There are a few possible reasons why the older adults may have shown a reduced ability to learn the sequence. One plausible reason is that older adults have poorer cognitive capabilities (it has been reliably demonstrated that WM declines with age [bib_ref] Working-memory capacity, age, and memory for discourse, Light [/bib_ref] [bib_ref] Meta-Analyses of Age-Cognition Relations in Adulthood: Estimates of Linear and Nonlinear Age..., Verhaeghen [/bib_ref]. However we were predominantly interested in whether there might be a relationship between the reduced motor performance of the Older participants and their reduced learning. In other words, we wanted to see whether the model shown in Eq 2 might capture an important feature of sequence learning (the interaction between cognitive and motor constraints). Between-group studies cannot address this question satisfactorily because it is often difficult to disentangle the influence of cognitive differences on learning rates. The next experiment therefore varied the motor performance of Younger and Older adults, within individuals, to examine whether sequence learning would be affected.
## Experiment 2
The results of the first experiment are consistent with the hypothesis that there is a relationship between motoric performance level and sequence learning-older participants were found not only to recall fewer moves (than the younger adults) at test, but also moved more slowly during the training trials. Two possible explanations are: (i) that encoding a movement sequence into memory has an influence over the speed of movement (i.e. learning alters motor performance, in this case movement duration), or (ii) that less skilled movements have a causal role in impairing motor sequence learning (i.e. movement performance level affects learning). To distinguish between these explanations a second experiment was conducted on a new set of Older and Younger participants, this time measuring learning when using both the preferred and non-preferred hands. Explanation (i) would predict impaired recall in the Older adults compared to Younger, but no differences between which hand was used to perform the task. Explanation (ii) would predict impaired recall in the Older adults, but also for both age-groups when using the non-preferred hand (i.e. superior motor performance is usually expected in the preferred hand when completing basic motor coordination tasks; see Raw et al. [bib_ref] Reduced motor asymmetry in older adults tracing paths, Raw [/bib_ref]. The same motor learning task was used as in Experiment 2, but because testing needed to be carried out with each hand, the number of movements to be learnt was reduced to keep overall experiment testing time equivalent, to avoid participant fatigue.
# Method
## Participants.
A new group of thirty-seven right-handed healthy individuals with no history of ophthalmological or neurological problems was selected from an opportunistic sample (mean EHI score = 87.40, SD = 15.20). We had planned to test forty participants but three did not attend their scheduled testing session. The 'Younger' group consisted of 18 participants (11 female, 7 males) aged between 20 and 25 years (mean age = 20.83, SD = 1.12), and 19 participants (14 female, 5 males) aged between 61 and 80 years (mean age = 70.79, SD = 6.09) formed the 'Older' group. The Addenbrooke's Cognitive Examination Revised (ACE-R) [bib_ref] The Addenbrooke's Cognitive Examination Revised (ACE-R): a brief cognitive test battery for..., Mioshi [/bib_ref] was administered to older participants as a measure of basic cognitive ability and the mean score indicated no cognitive deficit at 91.53 out of 100 (SD = 5.54). The University of Leeds ethics and research committee approved this study and all participants gave written, informed consent in accordance with the Declaration of Helsinki.
Procedure and apparatus. KineLab [bib_ref] A new tool for assessing human movement: The Kinematic Assessment Tool, Culmer [/bib_ref] was used to create two new versions of the task deployed in Experiment 1, each with a different 16-move sequence. Participants completed version one of the task using their preferred (right) hand and version two with their non-preferred (left) hand. The order of which hand/version was used first was counterbalanced across participants (though counter-balancing was incomplete as three participants did not attend). Instructions were the same as for Experiment 1 and participants were given two opportunities to practice the training and test trials, (which included a different 16-move sequence to those used in the experimental tasks). The same hand was used to complete the training and test trials. Each task had 10 training and test trials, resulting in a total of 20 trials per task.
Analyses. Outcome measures were identical to those used in Experiment 1 (CR, MT r , PL and MT t ). For the test trial analyses, mean scores across the L5 trials were calculated and two separate mixed-model ANOVAs applied in order to examine age and hand differences in motor learning (CR and MT r ). Two further ANOVAs were carried out to identify the effects of hand and age on motor performance during the L5 trials of training (PL and MT t ).
# Results
Recall during test trials. Experiment 1 showed that participants became more accurate at recalling the serial order as the trials progressed [fig_ref] Fig 4: Measurements of motor learning recorded in the test trials of Experiment 1... [/fig_ref]. In Experiment 2, a similar improvement in serial order recall was apparent [fig_ref] Fig 6: Measurements of motor learning recorded in Experiment 3 for the preferred [/fig_ref] but particularly for the Younger adults, and the preferred hand condition. To formally analyse these differences, data from the L5 trials were examined [fig_ref] Fig 7: Measurements of motor learning and performance for the preferred [/fig_ref]. The ANOVA for CR identified a main effect of age group (F (1, 35) = 135.5, p < 0.001, η 2 p = .79), a main effect of hand (F (1, 35) = 9.13, p < 0.01, η 2 p = .21) and a hand ρ age group interaction (F (1, 35) = 4.73, p < 0.05, η 2 p = .12). Planned comparisons showed that the main effect of hand was driven by a strong effect in the Younger group (F (1, 17) = 10.64, p < 0.01, η 2 p = .39) but no significant effect in the Older adults (F (1, 18) = .47, p = .5, η 2 p = .026). This indicates that the Younger adults recalled a greater number of moves in the correct sequential order than the Older adults, and more moves were recalled by the preferred hand than the non-preferred hand in the Younger adults (see [fig_ref] Fig 6: Measurements of motor learning recorded in Experiment 3 for the preferred [/fig_ref]. The hand difference in the Younger group amounts to 2 items fewer recalled by the non-preferred hand. As a proportion of the overall items remembered this is~12.5% drop off in performance (mean performance for preferred hand over last 5 trials was 14 items). If a similar scale change between preferred and non-preferred hands was to be expected for the older adults then we would predict 0.5 fewer items to be recalled when using the left hand (a 12.5% drop off from the 4.5 items recalled with the preferred hand). It is therefore not surprising that we were unable to measure a hand difference in correct recall for the Older group, given the low level of recall even with the preferred hand. The ANOVA for MT r also revealed a significant main effect of age group (F (1, 35) = 34.74, p < 0.001, η 2 p = .50) and hand used (F (1, 35) = 37.73, p < 0.001, η 2 p = .52) but there was no interaction (F (1, 35) = .17, p > 0.05). Hence younger participants showed decreased movement duration (faster movements) compared to the older group, and the preferred hand was quicker than the non-preferred hand (see [fig_ref] Fig 6: Measurements of motor learning recorded in Experiment 3 for the preferred [/fig_ref].
Training trials. It is possible that recall accuracy in test trials could be explained by movement performance during training. To determine whether movement patterns were similar during test and training an ANOVA was run on PL and MT t measures. The PL analysis found main effects of age group (F (1, 35) = 19.72, p < 0.001, η 2 p = .36) and hand condition (F (1, 35) = 12.68, p < 0.001, η 2 p = .27) as well as an age ρ hand interaction (F (1, 35) = 6.12, p < 0.05, η 2 p = .15), with the PL difference between the hands being greater in the Older group (see [fig_ref] Fig 7: Measurements of motor learning and performance for the preferred [/fig_ref]. Similarly, the ANOVA for MT t also revealed effects of age group [fig_ref] Fig 3: Screen shots of the learning task as it appeared to participants in... [/fig_ref]. Whilst the general patterns were similar between test and training the increased manual asymmetries for the Older group (i.e. a greater difference in movement duration between the preferred and non-preferred hand in the Older adults; see [fig_ref] Fig 7: Measurements of motor learning and performance for the preferred [/fig_ref] was not what was observed at test.
Chunk length as a function of age and hand. To determine whether there were age differences in the encoding strategies used by participants, a measure of chunking was calculated. We took the average increase in the number of moves recalled in the correct sequential order for each test trial (see . It is apparent that older adults did not encode chunks of multiple movements on each trial, and instead they generally only increased the number of moves recalled from one trial to the next by a single item. In contrast, the younger adults seem to have stored the motor sequence in chunks of three or four items, certainly over the first three trials (where most of the required moves were stored). Interestingly the chunk size appears larger for the preferred hand in the Younger group, suggesting that motor performance during training can perhaps interact with strategic encoding.
# Discussion
The second experiment confirmed the results of experiment 1, whereby the Older adults showed reduced sequence learning relative to the Younger adults. It is clearly the case that there are often cognitive differences between younger and older adults [bib_ref] Working-memory capacity, age, and memory for discourse, Light [/bib_ref] [bib_ref] Meta-Analyses of Age-Cognition Relations in Adulthood: Estimates of Linear and Nonlinear Age..., Verhaeghen [/bib_ref] , and these cognitive differences could explain age discrepancies in an individual's ability to learn a complex sequence of movements. However, we hypothesised that the reduced baseline level of motor performance typically observed in older adults (i.e. slower, less accurate movements) might also be a contributing factor. This hypothesis was tested in Experiment 2 by asking participants to use both their preferred and non-preferred hand to complete the sequence learning task. The underlying cognitive capabilities of an individual should remain constant regardless of which hand is used to undertake the task, hence differences in learning between the hands would support the prediction (i.e. that reduced motor performance, as typical when using the non-preferred hand, affects motor learning). The data showed that Younger participants learned more of the sequence, and recalled it at a faster pace, when using their preferred hand. These results support the idea that reduced motor performance will impact on complex sequence learning over and beyond any difficulties caused by cognitive decline. It is notable that age and hand significantly interacted in different directions for the training and test trials. At test, the hand used had an impact on recall in the Younger group, especially during the latter half of the trials [fig_ref] Fig 6: Measurements of motor learning recorded in Experiment 3 for the preferred [/fig_ref]. In later trials the Younger adults were producing many more movements, suggesting that this effect may have been cumulative: the more movements that had to be stored and subsequently implemented from memory using the non-preferred hand, the greater the impact of relative motor inefficiency. In contrast, the Older group were only able to produce slightly longer sequences with each new trial (due to limited chunking), thus reducing the opportunity for hand preference to impact on performance. When participants were trying to learn the whole motor sequence in the training trials, however, the effects of reduced working memory capacity and/or processing speed were compounded by reduced motor performance in the non-preferred hand, thus leading to larger effects of hand in the Older group than the Younger group during this phase.
The results of the first two experiments are consistent with the model presented in the introduction. Eq (1) shows that task duration is a function of the informational demand associated with the movement, so further and smaller targets have higher demands and thus result in longer movement times. Eq (1) also shows that the duration of the task is a function of the individual and the task difficulty. Older adults find the given motor task more difficult than their younger counterparts and this can be interpreted as the task having higher informational demands for the older adults. This interpretation is consistent with the presence of errors within the movements made by the older adults-errors that needed to be corrected. The correction of errors requires the allocation of attentional resources and this can explain why these movements use more bandwidth. Eq [bib_ref] Effects of age on neurocognitive measures of children ages 5 to 12:..., Korkman [/bib_ref] shows that increasing the information load of individual elements will decrease the number of elements that can be stored in working memory and thus decrease the rate of serial order learning. This suggests that older adults are being affected by both a reduced working memory capacity and the higher information demands created by their reduced motor capabilities.
## . the average number of additional items recalled in each test trial (over and above those recalled in the previous trial) for older and younger participants when using the preferred (right) and non-preferred (left) hand to complete the sequence learning task in
## Experiment 3
The findings of Experiment 2 suggest that there is a relationship between an individual's baseline level of motor performance and their ability to learn a novel sequence. When Older and Younger participants used their non-preferred hand to complete the task, we found reduced quality of movements (i.e. longer trajectories and slower movements, during training), and poorer learning (i.e. moves recalled at a slower pace at test, and for the young at least fewer items recalled). This suggests that reduced motor performance can directly inhibit motor sequence learning, over and above the influences of cognitive capacity. An alternative explanation for our results, however, is that learning may be influenced at a neurological level by hemispheric specialisation. According to hemispheric specialisation theory, the left side of the brain is predominant in analytical skills such as tasks that involve breaking down problems into parts, reasoning, and logical thinking-the hemisphere said to possess "sequential, analytic, time-dependent mechanisms" [bib_ref] The nature of hemispheric specialization in man, Bradshaw [/bib_ref]. In contrast, the right hemisphere is said to specialise in subjective functions such as intuition, creativity and emotion [bib_ref] Putting Our Whole Brain to Use: A Fresh Look at the Creative..., Garrett [/bib_ref] [bib_ref] Asymmetry of Hemispheric Functions and Creativity: An Empirical Examination, Harpaz [/bib_ref]. It might therefore be argued that because participants in Experiment 2 were right-handed, and the contralateral side of the brain that controls right-side movement specialises in skills that are vital for motor sequence learning, participants could have been at an advantage when completing the task with their preferred hand. There are hence two alternative plausible explanations for the finding of reduced motor sequence learning in the non-preferred hand; (i) participants were able to learn more of the sequence when using the hand that is controlled by the left hemisphere because it specialises in the processes involved in motor sequence learning; or (ii) a poor baseline level of motor performance inhibits motor sequence learning.
To tease apart these hypotheses we ran a final experiment using the same sequence learning task, but with two conditions that varied the motor demands placed on the preferred hand. Specifically, participants used their preferred hand to complete the task once when controlling a PC mouse in its regular position flat down on the table (as in Experiments 1 and 2) and then again using the mouse rotated sideways and placed against an inverted T-shaped stand (see [fig_ref] Fig 8: Young adult completing the learning task in Experiment 3, using the mouse... [/fig_ref]. The task of using the mouse in a sideways orientation is reminiscent of when an individual is required to carry out a familiar task (e.g. writing) with the non-preferred hand-the action is possible, but less skilled.
# Method
## Participants.
A new opportunistic sample of 15 healthy Young adults (7 female, 8 males) aged between 21 and 40 years (mean age = 29.13, SD = 5.64), were recruited. All participants were right-handed (mean EHI score = 87.33, SD = 13.87), and reported no history of ophthalmological or neurological problems. The University of Leeds ethics and research committee approved this study and participants gave written, informed consent in accordance with the Declaration of Helsinki.
Procedure and apparatus. The sequence learning task from Experiment 2 (created in 'KineLab' [bib_ref] A new tool for assessing human movement: The Kinematic Assessment Tool, Culmer [/bib_ref] was administered under two blocked conditions; (i) participants used their preferred hand to learn the 16-move sequence with a PC mouse in its Regular (R) orientation, flat down on the table (see [fig_ref] Fig 4: Measurements of motor learning recorded in the test trials of Experiment 1... [/fig_ref] ; (ii): participants used their preferred hand to learn a new 16-move sequence with the PC mouse rotated Sideways (S), and placed up against an inverted-t-shaped stand [fig_ref] Fig 8: Young adult completing the learning task in Experiment 3, using the mouse... [/fig_ref]. The stand was made of wood (width from left to right of base = 620mm; height = 340mm), with an off-centre vertical partition (width = 340mm; height = 310mm). The tablet PC (same as Experiments 1-3) was placed on the left side of the partition, and a mouse mat (width = 230mm; height = 200mm) was secured vertically to the surface of the opposite side. Participants were verbally reminded that when using the mouse in Interactions between cognition and action the S orientation, they would need to adapt their movements to account for the change in orientation (i.e. a forward-back motion to produce up-down movements; an up-down motion to make left-right movements). The order in which participants completed the S and R conditions was counterbalanced. At the start of the experiment, participants practiced using the mouse in both orientations, completing one training trial (i.e. whereby participants were shown the 16 targets to move to by centrally placed directional arrows) and one test trial (i.e. free recall of the sequence practiced in the training trial) with the mouse in the R or S orientations (NB. the practice sequence used was different to the sequences used in the experimental tasks). The experimental task comprised 10 training and test trials, hence 20 trials in total for each mouse orientation condition. The same mouse orientation was used to complete the training and test trials.
Analyses. Outcome measures were identical to those used in Experiments 1 and 2 (CR, MT r for the recall at test analyses; PL and MT t for the training trial analyses). For recall during test trials, mean scores for CR and MT r across the last five (L5) trials were calculated, and a One-Way ANOVA was performed on each recall measure, to examine the effect of mouse orientation (R vs. S) on motor learning. A One-Way ANOVA was also completed to identify the effects of mouse orientation on motor performance during the L5 training trials (one for each training measure: PL and MT t ).
# Results
Recall during test trials. As previously observed in Experiments 1 and 2, participants were able to recall more of the motor sequence, and at a faster pace, as the task progressed. It can be seen in [fig_ref] Fig 9: Measurements of motor learning of the preferred [/fig_ref] differences in motor learning (indexed by CR) began to materialise between the mouse orientation conditions in the second half of the task. We therefore analysed data from the last five (L5) test trials to formally explore these differences.
One-Way ANOVAs of the CR data revealed a main effect of mouse orientation (F (1, 14) = 4.99, p < 0.05, η 2 p = .26; [fig_ref] Fig 10: Measurements of motor learning and performance of the preferred [/fig_ref] , whereby participants were able to recall significantly more moves when using the mouse in the R orientation (mean CR = 13 items or 81% of the sequence), compared to the S orientation (mean CR = 11 items or 69% of the sequence). The speed at which participants were able to recall the moves (MT r ) was similar for the S and R orientation conditions (mean MT r for S = 1.75s; mean MT r for R = 1.70s; [fig_ref] Fig 10: Measurements of motor learning and performance of the preferred [/fig_ref] , hence there was no main effect of mouse orientation on MT r (F (1, 14) = .28, p > 0.05; [fig_ref] Fig 10: Measurements of motor learning and performance of the preferred [/fig_ref]. To ensure that the sideways conditions did indeed cause motoric difficulties we next looked at performance during the Training Trials. Training trials. To confirm a significant reduction in motor performance in the S condition we analysed speed (MT t ) and accuracy (PL) of movements recorded during the training trials (using separate One-Way ANOVAs). Analyses revealed main effects of mouse orientation for both PL [fig_ref] Fig 4: Measurements of motor learning recorded in the test trials of Experiment 1... [/fig_ref] = 34.84, p < 0.001, η 2 p = .71; [fig_ref] Fig 10: Measurements of motor learning and performance of the preferred [/fig_ref] and MT t [fig_ref] Fig 4: Measurements of motor learning recorded in the test trials of Experiment 1... [/fig_ref] = 91.32, p < 0.001, η 2 p = .87; [fig_ref] Fig 10: Measurements of motor learning and performance of the preferred [/fig_ref] whereby PLs were significantly longer, and movements slower (mean MT t , for S = 54s; mean MT t , for R = 48s), when the mouse was used in the S orientation. It seems that it is the increased motor difficulty exhibited during training that is having the Interactions between cognition and action detrimental effect upon recall performance, because there were only small differences in movement times at recall (MT r ).
# Discussion
In Experiment 2 we observed reduced sequence learning in the non-preferred hand. This effect may have been due to differences in hemispheric specialisation (i.e. the left hemisphere, which controls the right hand, could have been dominant in the processes vital for sequence learning). Alternatively, this effect could be caused by a reduced baseline level of motor performance (i.e. as typical of the non-preferred hand) that directly inhibits motor sequence learning. Experiment 3 aimed to distinguish between these explanations by controlling task difficulty of just the preferred hand-comparing learning when using the mouse in its Regular (R) orientation and in an unfamiliar sideways (S) orientation (i.e. hemisphere use and individual differences in cognitive capacity remain stable between conditions). The findings of Experiment 3 confirmed our original hypothesis-sequence learning was reduced when participants used the mouse in the S orientation (i.e. as indexed by fewer moves recalled in the correct sequence order at test), and this was coupled with significantly poorer accuracy and decreased speed of movements (i.e. both markers of poor motor performance) during training. General discussion. The Cognition-Action Interaction Theory (CAIT) suggests that the acquisition of many skills will involve a synergistic interplay between the cognitive and motor systems. We hypothesised that increasing the difficulty of the motor elements within a complex sequence learning task would decrease the number of elements that could be held in working memory, and thereby reduce the rate of learning. Our hypothesis was based on the observation that increasing motor difficulty increases the informational load. For example, increased difficulty is likely to require more error corrections, with such corrections consuming attentional resources and thus decreasing the system's available bandwidth. We created a simple model (see Eq 2) that captured the increased informational load associated with task difficulty and the limited capacity of working memory. The model predicts reduced learning with decreased cognitive capacity as well as with reduced motor abilities (both established as being present in older adults). We tested the model by exploring whether increased task difficulty impaired sequence learning. Experiment 1 revealed reduced movement speed and increased path length (indicating error corrections) in older adults when making similar aiming movements during the training phase of a sequence learning task (i.e. reduced motor performance, as indicated by increased movement duration). Moreover, the older adults showed poorer learning outcomes. Experiment 2 examined whether motor performance directly affected movement sequence learning (as predicted by our model) by having a different group of older and younger participants perform the learning task with their preferred or non-preferred hands. The results confirmed that the older participants learned less of the sequence than the younger participants, and that use of the non-preferred hand reduced learning compared to the preferred hand. At recall, movements with the non-preferred hand were slower in both age groups and the number of correctly recalled items was significantly impaired for the younger adults. This suggests that sequence learning can be influenced by the underlying motor performance as predicted by CAIT. Overall, the present findings support the conjecture that the motor and cognitive systems both play essential but interacting roles in skill learning. This raises the question of whether motor performance could have been influenced by the quality of the memory formed for the movement sequence itself. While the acquisition of accurate memory representations does support subsequent skilful execution of movements, within the context of the current task it appears that motor performance affected the task learning, rather than the reverse. This was most clearly observed in Experiment 3 where slower and less accurate movements during training led to worse recall despite there being no apparent motor performance differences when recalling at test.
Our model (see demonstrates that motor performance will not explain all the group differences that were observed in Experiments 1 and 2. the predicted change in performance when working memory capacity is set to a lower value which effectively models the capacity of older adults. These predictions match our findings: for example, motor performance during the training phase of Experiment 2 (MT t ) was similar for the non-preferred hand of the younger adults and the preferred hand of the older adults [fig_ref] Fig 7: Measurements of motor learning and performance for the preferred [/fig_ref] , but there was a 45% reduction in the number of correct movements recalled by the older adults compared to the younger adults with these hands [fig_ref] Fig 7: Measurements of motor learning and performance for the preferred [/fig_ref]. Not only was this difference predicted by the model, but the model captured a reduction in chunk size for the older group (because chunk size is limited by working memory capacity as described in . To examine the group differences, chunk sizes used by the older adults and younger adults when recalling the sequence were calculated and are displayed in . Younger adults used a standard chunk size of 3-5 items (particularly when recalling the first 10 items), which is comparable with previous research [bib_ref] Age-Related Declines in Visuospatial Working memory Correlate With Defecits in Explicit Motor..., Bo [/bib_ref]. Such chunking during sequence learning improves processing efficiency and is thought to be critical in representing lengthy sequences [bib_ref] Chunking during human visuomotor sequence learning, Sakai [/bib_ref]. In contrast, the older group did not effectively add chunks to their overall representation of the sequence on each trial. Instead, the number of items recalled tended to increase by a single item at a time. This pattern fits with previous research showing age-related impairments in chunking [bib_ref] Age-Related Declines in Visuospatial Working memory Correlate With Defecits in Explicit Motor..., Bo [/bib_ref] [bib_ref] Buffer Loading and Chunking in Sequential Keypressing, Verwey [/bib_ref] and association-formation [bib_ref] Adult-age differences in memory performance: Tests of an associative deficit hypothesis, Naveh-Benjamin [/bib_ref]. The study by Bo et al. [bib_ref] Age-Related Declines in Visuospatial Working memory Correlate With Defecits in Explicit Motor..., Bo [/bib_ref] showed impaired learning of sequences in older adults; specifically, visuo-spatial working memory ability was found to indirectly predict learning in older adults via a mediating effect on the size of chunks that could be constructed. It therefore appears that age differences in learning some skills are at least partly the result of reduced working memory capacity constraining the size of chunks that can be built on each trial-but our results show that this effect will be magnified when there are higher motor demands.
The model we present is consistent with reinforcement learning frameworks and can be implemented within such schemes. Q-Learningis a model-free reinforcement learning algorithm, through which an agent learns how to act in an environment. In Q-learning, tasks are modelled as a Markov Decision Process (MDP), which is a tuple: M = hS,A,p,ri where S is the set of states, A is the set of actions, and p(s t+1 ,r t+1 |s t ,a t ) is the joint distribution over the next state and reward given the current state and action, and r : S � A � S ! R is the reward function. The interaction proceeds in discrete steps: the agent chooses an action based on the current state according to a policy represented by a probability distribution π(a t |s t ), and receives the perception of the next state s t , and immediate reward r t . The agent's goal is to maximize the long-term cumulative reward, called the return G t ¼ P T t¼1 r t , where T is the maximum number of actions in the task. Q-learning is used to learn an estimate of the value function q p � ðs t ; aÞ ¼ Ej p � ½G t � from the information provided by the samples of states and rewards-that is, the expected value for taking action a in state s t when acting according to the optimal policy. The estimateq p � of q p � represents the long-term memory of the agent, and is used to determine the policy of the agent during learning. Following learning (i.e. once the estimate of the value function is close to the true optimal value function), the optimal policy can be retrieved simply by choosing the action that maximizes the q-value in every state. An agent is normally naïve with respect to the environment, so starts with all state-action pairs having the same estimated value. Typically, after an action the state-action valuation is immediately updated, but the work reported within this manuscript suggests that the working memory process could be represented at a basic level by adding new information to a fixedcapacity queue. When a new piece of information is generated, a check would be conducted to only add the item to working memory if it would "significantly" change the existing value function. A test of recall would then be a measure of the agent's attempt to use the current estimate of the optimal value function,q p � to complete the task. [fig_ref] Fig 11: Schematic of the model implementation within a Q-learning framework [/fig_ref] the postulated process within a simple grid world where the state space is constituted by the cells of the grid, and the actions are to move in one of the four cardinal directions. The environment is deterministic, so each action always leads to the state in the corresponding direction, except for when it would lead the agent outside of the grid in which case the agent stays in its current state. The reward is -1 per action, and an episode ends when the agent reaches a target state, or has executed the maximum number of actions. If we apply this system to the sequence learning task reported in Experiments 1-3, each target in the sequence has its own 2D grid and q-function that needs training. The working memory system is then simply implemented by a queue that rejects new items once it is full and updates longterm memory at the end of an iteration (note this can be implemented in a number of possible ways to suit an application or to capture features like recency effects). Under this system, only early goals in the sequence are initially trained, but once these goals have been 'learned' then new information for these goals does not need to be committed to working memory, so the next goals in the sequence can be trained until all goals are learned via an iterative process. In this implementation, task difficulty can be represented by the number of steps required to reach a target (which captures the notion that more difficult tasks require more information). The differences we found between preferred and non-preferred hand use (Experiment 2) can then be explained by differences in the accuracy of the agent's existing models of the environment.
It should be highlighted that the notion of a synergistic interaction between cognition and action in complex tasks is not equivalent to the hypothesised interference that occurs in dual tasking. Dual tasking can be a useful conceptual framework when considering activities that are readily dichotomised as being either distinctly motor or cognitive in nature. The classic example is maintaining posture whilst undertaking mental arithmetic. In this situation, the goal of the motor task (not falling over) can be argued to be separate from the goal of the cognitive task (answering the maths question), and hence serves as a useful tool for exploring the extent to which these separate processes share neural resources. A problem, however, is that success in numerous human activities (including the task used within the present study) requires a combination of both motor and cognitive elements, and it becomes meaningless to conceptualise the outcome of one system as being separate to the goal of the other system. Thus, our model does not depict the motor system as interfering with the cognitive system, but rather demonstrates that these two systems are mutually dependent when learning a complex skill (and sometimes share neural resources-as explored in some dual-task experiments). It is also interesting to note that the research literature suggests that complex motor behaviours become increasingly 'cognitive' (i.e. less reliant on automated motor routines and requiring grater cognitive resource) as adults become older.
The ability for procedural memory to inform the necessary sequence of actions to achieve a goal is crucial for many activities of daily living (e.g. tying a shoe-lace), but it also underpins highly skilled and risky activities such as driving or carrying out complex surgical procedures. Such highly practiced abilities, eventually stored as procedural knowledge, must initially be acquired through learning processes that are much more resource-intensive and controlled [bib_ref] Right hemisphere lateralization for emotion in the human brain: interactions with cognition, Schwartz [/bib_ref] [bib_ref] Attention to action: willed and automatic control of behaviour, Norman [/bib_ref] , and require construction and temporary storage in working memory. It is this initial learning phase that was examined in the present experimental work. Temporary storage and control processes are likely to become less critical once the initial stages of learning have progressed and procedural memory develops. In line with this, Sakai, Hikosaka & Miyauchi et al. [bib_ref] Transition of brain activation from frontal to parietal areas in visuomotor sequence..., Sakai [/bib_ref] suggested a shift in the importance of certain brain regions during the transition from declarative to procedural memory in visual-motor sequence learning; whereby early learning primarily activates frontal areas, particularly the dorsolateral prefrontal cortex (DLPFC) and presupplementary motor area (pre-SMA), with a shift to parietal areas as sequences become consolidated. The observed age and hand effects in Experiment 2 may reflect the potential roles of the DLPFC and pre-SMA in initial visuo-motor learning.
The findings of our experiments have important implications within applied settings. For example, CANTAB sells "cognitive assessment and data collection software" (Cantab Connect Research Alzheimer's) that purports to test cognitive function in older adults. The software has a test for paired associate learning which requires participants to touch boxes displayed around the screen. This test clearly has a motor component (the task is not dissimilar to the one reported in our experiments) and is advertised as providing "insight into individuals' episodic memory abilities". The present findings show that such tests need to consider the motor abilities of the participants-and these 'cognitive' measures absolutely must be interpreted in terms of their interaction with the motor system.
While the present work examined some of the deficits associated with old age, we would argue that the conclusions drawn from our results can also be generalized to other groups that experience motor deficits. One example is children with developmental coordination disorder (DCD), who make up approximately 5% of the population [bib_ref] A Test of Motor (Not Executive) Planning in Developmental Coordination Disorder and..., Van Swieten [/bib_ref]. Children with DCD experience a host of related problems that often become particularly apparent in mainstream education. Movement difficulties could lead to greater demands on working memory within many school learning tasks (e.g. following a set of instructions relating to the placement of objects within the classroom). Working memory itself provides a good predictor of scholastic ability [bib_ref] Working memory assessments at school entry as longitudinal predictors of National Curriculum..., Gathercole [/bib_ref] but given the co-morbidity of DCD with other developmental problems there could well be complex interactions between memory and motor deficits (resulting in poorer educational outcomes for these groups of children). We note that numerous classroom activities require a complex interaction between cognitive and motor systems (e.g. handwriting involves producing words which must be mentally manipulated whilst concurrently controlling the forces applied to a handheld stylus). In line with this, it has been found that children with poor working memory have problems in following and implementing instructions within the classroom, and that working memory tasks measuring this ability may have an important motoric component.
The present experiments show that increased motor demands can impair sequence learning and better motor performance is associated with improved sequence learning. We conclude by highlighting that the interactions between cognition and action may have numerous positive effects that can be exploited within clinical settings. For example, information can be stored within the motor system and thereby decrease the storage and retrieval demands placed on cognition. Recent findings indicate that the motor component of complex movement tasks may have such facilitating effects, with recall being improved when miming an action, for example recalling the digit sequence of a PIN number when the spatial layout of the keypad is present [bib_ref] Modality specificity and integration in working memory: Insights from visuospatial bootstrapping, Allen [/bib_ref]. A key priority for future research should be to determine how action and memory interact, to fully understand the performance of skilled actions. This will also help to identify what causes errors in critical tasks, such as driving or surgery, and how to best support individuals who show deficits in learning skilled behaviours.
Supporting information S1 File. Control experiment establishing impaired motor performance by older adults performing perceptual-motor aiming tasks similar to those used in Experiments 1-3. (DOCX)
[fig] Fig 2: (A) Older adult completing an aiming task using a tablet PC and handheld stylus pen (Nb. Not to scale. Arrow for illustrative purposes only). Examples of kinematic traces (speed against time) made in an aiming task by (B) a younger adult, and (C) an older adult. The spatial paths made by the older participant are much more variable and show more evidence of corrections (which results in quantifiable decreases in smoothness as can be seen in Eq 3 within the text). https://doi.org/10.1371/journal.pone.0211706.g002 [/fig]
[fig] Fig 3: Screen shots of the learning task as it appeared to participants in Experiments 1 and 2 (Nb. not to scale). (A) Training trial whereby participants moved the dot into the box corresponding to the direction indicated by an arrow in the central box (e.g. top left in the example pictured). (B) Test trial in which participants recalled the pattern of movements previously displayed in the training trial. (C) Older adult completing the learning task using a standard computer mouse. Example traces of one participant's movements during (D) a training trial and (E) a test trial. https://doi.org/10.1371/journal.pone.0211706.g003 [/fig]
[fig] Fig 4: Measurements of motor learning recorded in the test trials of Experiment 1 for younger (dark grey bars) and older (light grey bars) groups, averaged across the first five trials (F5) and last five trials (L5). (A) Proportion (%) of movements recalled in the correct sequential order at test (CR) (B) Mean time taken between moves during free recall (MT r ). Bars = Standard Error of the Mean. https://doi.org/10.1371/journal.pone.0211706.g004 [/fig]
[fig] Fig 5: The number of moves recalled during each of 14 test trials by the older (open symbols, dashed lines) or younger (filled symbols, solid lines) groups as a proportion of the total moves in the sequence (proportion of movements). Square symbols show the Total number of moves made, and Triangular symbols show the Correct number of moves made. https://doi.org/10.1371/journal.pone.0211706.g005 [/fig]
[fig] Fig 6: Measurements of motor learning recorded in Experiment 3 for the preferred (right) hand (empty symbols) and non-preferred (left) hand (filled symbols) in the older (dashed line and circles) and younger (solid line and squares) groups for each of the 10 test trials. (A) Mean number of moves recalled in the correct sequential order (CR). (B) Mean time taken between moves during free recall (MT r ). https://doi.org/10.1371/journal.pone.0211706.g006 Interactions between cognition and action PLOS ONE | https://doi.org/10.1371/journal.pone.0211706 February 7, 2019 hand ρ age interaction (F (1, 35) = 8.03, p < 0.01, η 2 p = .19) [/fig]
[fig] Fig 7: Measurements of motor learning and performance for the preferred (right) hand (white bars) and non-preferred (left) hand (black bars) for older and younger participants averaged across the last five (L5) test trials (A,B) and training trials (C,D) in Experiment 2. (A) Proportion (%) of movements recalled in the correct sequential order at test (CR) (B) Mean time taken between moves during free recall (MT r ). (C) Length of entire path taken throughout a training trial (PL). (D) Time taken to complete a training trial from start to finish (MT t ). Bars = Standard Error of the Mean. https://doi.org/10.1371/journal.pone.0211706.g007 [/fig]
[fig] Fig 8: Young adult completing the learning task in Experiment 3, using the mouse in the Sideways (S) orientation. https://doi.org/10.1371/journal.pone.0211706.g008 [/fig]
[fig] Fig 9: Measurements of motor learning of the preferred (right) hand with the mouse in the Regular (R) position (empty symbols), and the Sideways (S) position (filled symbols) for each of the 10 test trials in Experiment 3. (A) Mean number of moves recalled in the correct sequential order (CR). (B) Mean time taken between moves during free recall (MT r ). https://doi.org/10.1371/journal.pone.0211706.g009 [/fig]
[fig] Fig 10: Measurements of motor learning and performance of the preferred (right) hand with the mouse in the Regular (R) position (white bars), and the Sideways (S) position (black bars), averaged across the first five (F5) and last five (L5) test trials (A, B) and training trials (C,D) in Experiment 3. (A) Proportion (%) of movements recalled in the correct sequential order (CR) at test (B) Mean time taken between moves during free recall (MT r ). (C) Length of entire path (PL) taken throughout a training trial. (D) Time taken to complete a training trial from start to finish (MT t ). Bars = Standard Error of the Mean. https://doi.org/10.1371/journal.pone.0211706.g010 [/fig]
[fig] Fig 11: Schematic of the model implementation within a Q-learning framework. https://doi.org/10.1371/journal.pone.0211706.g011 [/fig]
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A traveling SARS-CoV-2 laboratory as part of a pandemic response among vulnerable Brazilian populations
Background Brazil has been dramatically hit by the SARS-CoV-2 pandemic and is a world leader in COVID-19 morbidity and mortality. Additionally, the largest country of Latin America has been a continuous source of SARS-CoV-2 variants and shows extraordinary variability of the pandemic strains probably related to the country´s outstanding position as a Latin American economical and transportation hub. Not all regions of the country show sufficient infrastructure for SARS-CoV-2 diagnosis and genotyping which can negatively impact the pandemic response.Methods Due to this reason and to disburden the diagnostic system of the inner São Paulo State, the Butantan Institute established the Mobile Laboratory (in Portuguese: LabMovel) for SARS-CoV-2 testing which started a trip of the most important "hotspots" of the most populous Brazilian region. The LabMovel initiated in two important cities of the State: Aparecida do Norte (an important religious center) and the Baixada Santista region which incorporates the port of Santos, the busiest in Latin America. The LabMovel was fully equipped with an automatized system for SARS-CoV-2 diagnosis and sequencing/genotyping. It also integrated the laboratory systems for patient records and results divulgation including in the Federal Brazilian Healthcare System.ResultsCurrently,16,678 samples were tested, among them 1,217 from Aparecida and 4,564 from Baixada Santista. We tracked the delta introductio in the tested regions with its high diversification. The established mobile SARS-CoV-2 laboratory had a major impact on the Public Health System of the included cities including timely delivery of the results to the healthcare agents and the Federal Healthcare system, evaluation of the vaccination status of the positive individuals in the background of exponential vaccination process in Brazil and scientific and technological divulgation of the fieldwork to the most vulnerable populations.
# Conclusions
The SARS-CoV-2 pandemic has demonstrated worldwide the importance of science to fight against this viral agent and the LabMovel shows that it is possible to integrate researchers, clinicians, healthcare workers and patients to take rapid actions that can in fact mitigate this and other epidemiological situations. Keywords Brazil, COVID-19, SARS-CoV-2, LabMovel, Real-time detection, Molecular surveillance Background Brazil has been dramatically affected by the COVID-19 pandemic, resulting in some of the highest rates of COVID-19 morbidity and mortality worldwide. From the confirmation of the first SARS-CoV-2 infection case in Brazil in February 2020 in the city of São Paulo [bib_ref] Importation and early local transmission of COVID-19 in Brazil, De Jesus [/bib_ref] , São Paulo State has been considered a national epicenter of the pandemic, showing the highest indexes of COVID-19 incidence, morbidity, and mortality (Brazilian Ministry Health, 2021). The city is the largest national and international hub with the most important international airports and bus stations in Brazil and South America and is the economic and industrial heart of the country. The state is the most populous and highly industrialized area in Brazil, with the largest and busiest port complex in Latin America, the port of Santos. These specific geographic and economic characteristics of the state have shaped the COVID-19 pandemic in Brazil, contributing not only to the highest SARS-CoV-2 infection indexes in Brazil but also to the extensive circulation of variants of concern (VOCs) and interest (VOIs), many of which have been uniquely described in the state [bib_ref] Genomic monitoring unveil the early detection of the SARS-CoV-2 B.1.351 (beta) variant..., Slavov [/bib_ref]. The Brazilian COVID-19 pandemic has also been a continuous source of new VOCs and, more specifically, the gamma VOC (P.1), which emerged in the Brazilian state of Amazonas (North Brazil) during late 2020 but rapidly spread throughout the whole country [bib_ref] Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil, Faria [/bib_ref] and was responsible for the second pandemic wave, which also affected São Paulo State in terms of contingency and diagnostic policies. Currently, this scenario has been shaped by the delta VOC, which was the dominant lineage in the state and is now being replaced by the omicron VOC (https:// butan tan. gov. br/ covid/ bolet im).
During this alarming scenario at the beginning of 2020, Instituto Butantan, located in the city of São Paulo, was in charge of coordinating the molecular diagnosis for the state territory. A network of 27 public laboratories (mostly from universities) was established, which received daily nasopharyngeal swabs (NPSs) from the 17 Regional Health Departments of the State for routine SARS-CoV-2 diagnosis. The functioning of this network relies on a complex logistic organization providing diagnostic reagents and clinical samples from hospitals and institutions throughout the state and delivering results an average of four days after sample collection. At the beginning of 2021, when SARS-CoV-2 variants were threatening public health, the Butantan Network for Pandemic Alert of SARS-CoV-2 Variants was established to trace VOC and VOI dissemination in São Paulo State.
The Butantan network provides rapid information about SARS-CoV-2 variants circulating in the 17 Regional Health Divisions of the State composed of the regions of Metropolitan São Paulo, Baixada Santista, Registro, Taubate, Sorocaba, Campinas, São João da Boa Vista, Piracicaba, Bauru, Ribeirão Preto, Araraquara, Marilia, Araçatuba, Presidente Prudente, São José do Rio Preto, Barretos, and Franca. This initiative allowed us to fully characterize the second gamma wave in the state, circulating VOCs such as the beta variant (B1.351) [bib_ref] Genomic monitoring unveil the early detection of the SARS-CoV-2 B.1.351 (beta) variant..., Slavov [/bib_ref] , and the introduction of the deltaand omicron VOCs (https:// butan tan. gov. br/ covid/ bolet im), among others. Although the highest SARSCoV-2 diagnostic potential is attributed to the city of São Paulo, this could not be extrapolated to the whole state territory, which represents an uneven distribution of SARS-CoV-2 detection capabilities, such as time to diagnosis and a timely manner of obtaining SARS-CoV-2 VOC/VOI information. Moreover, after 20 months of the pandemic in Brazil, it is clear that efforts from scientists, researchers, clinicians, and healthcare workers need to be integrated for a rapid and efficient response to the actual and ever-changing challenges imposed by the COVID-19 pandemic.
# Methods
## Sample collection, transportation, labeling
Between August 5, 2021, and September 16, 2021, NPS samples were collected from symptomatic patients suspected of COVID-19 in the Basic Health Units of each municipality. After collection, all samples were sent to LabMovel for SARS-CoV-2 diagnosis and genome sequencing. Briefly, NPS samples were collected in 15 mL Falcon tubes. Tubes were labeled individually with a number that was used in the whole process for sample tracking. They were then transported in refrigerated boxes to LabMovel. Upon receipt, tubes were visually inspected to meet the processing requirements (tube type, proper tag position, undisrupted barcode).
## Nucleic acid extraction and sars-cov-2-rt-pcr
SARS-CoV-2 RNA was isolated from 100 µL of liquid in which the NPS was immersed. Samples were delivered in swabs immersed in 3 mL of phosphate-saline buffer solution (PBS).
Sample inactivation was performed using a hermetic automated tool for sample isolation and handling, such as airflow and HEPA filters that works as biosafety level 2. This automation was especially conceived to enforce the lab biosafety capabilities. RNA extraction was then carried out automatically using an Extracta Kit (Loccus) following the manufacturer's instructions. RT-PCR was carried out automatically using a liquid handler (Extracta Prep, Loccus). SARS-CoV-2 RNA detection was performed using a Gene FinderTM COVID-19 Fast RealAmp Kit (OSang Healthcare Co., Ltd.) targeting the viral RdRp, E, and N genes. Amplification was performed in a QuantStudio 5 (Thermo Fisher Scientific). Positive samples with a cycle threshold (Ct) for at least 2 viral genes of Ct < 30 and a positive RNAse P test were submitted to viral genotyping.
## Genomic library preparation and sars-cov-2 next-generation sequencing
All positive samples with Ct values < 30 were submitted to SARS-CoV-2 variant identification. Genomic libraries were prepared using COVIDseq (Illumina) in an automated SP-960 system (Loccus/MGI). To evaluate the input concentration of the generated libraries, they were quantified using the FluorQuant system with a FluorQuant High Sensitivity Detection Kit (Loccus). Pooled libraries were sequenced using iSeq (Illumina).
## Decontamination of labmovel and discard of the biohazardous waste
As no manual sample handling (tube opening, pipetting, etc.) was performed outside the equipment, our main concern was related to always maintain all analogic or automated equipment decontaminated. Considering that our stay in each city had a timespan of 3 weeks, we established two types of decontamination protocols: decontamination on arrival and daily decontamination protocol. The first one contemplates a general decontamination. As the container is transported between the cities, initially non-specific cleaning was performed on the whole LabMovel composition using common disinfectant reagents. After this, the laboratory contaminants were eliminated by daily protocols contemplating use of isopropyl alcohol applied on all surfaces followed by application of RNaseZAP ™ (Sigma Aldrich) and UV light in between the testing of each sample plate. To certify decontamination efficiency, after these protocols we also run blank plates or known positive/negative SARS-CoV-2 samples to control the whole process from extraction to sequencing.The Daily protocol was performed twice a day, at the beginning and ending of the shift. Partnership with cities government was established and a team of trained professionals collected the waste every day, transporting it to the correct disposal sites according to legislation.
## Sequence assembly
The raw sequence data obtained were submitted to quality control analysis using FastQCsoftware version 0.11.8. Trimming was performed using Trimmomatic [bib_ref] Trimmomatic: A flexible trimmer for Illumina sequence data, Bolger [/bib_ref] version 0.3.9 to select the sequences with the best quality. Only sequences with quality scores > 30 were used. We mapped the trimmed sequences against the SARS-CoV-2 reference (GenBank RefSeq NC_045512.2) using BWA [bib_ref] Aligning sequence reads, clone sequences and assembly contigs with, Li [/bib_ref] software and SAMtools [bib_ref] The Sequence Alignment/Map format and SAMtools, Li [/bib_ref] for read indexing. The mapped files were submitted to refinement using Pilon [bib_ref] An integrated tool for comprehensive microbial variant detection • fast, convenient online..., Walker [/bib_ref] to obtain the correct indels and insertions. The trimmed sequences were subjected to remapping against the genome refined by Pilon. Finally, we used bcftools [bib_ref] BCFtools/RoH: A hidden Markov model approach for detecting autozygosity from next-generation sequencing..., Narasimhan [/bib_ref] for variant calling and seqtk [bib_ref] SeqKit: A cross-platform and ultrafast toolkit for FASTA/Q file manipulation, Shen [/bib_ref] for the assembly of the consensus SARS-CoV-2 genomes. Positions covered by fewer than 10 reads (DP < 10) and bases of quality lower than 30 were considered gaps in coverage and converted to Ns. Coverage values for each genome were calculated using SAMtools version 1.12. We assessed the consensus genome sequence quality using Nextclade version 0.8.1 (https:// clades. nexts train. org). Furthermore, only sequences with more than 20,000 bases representing > 10 × depth were selected for analysis.
# Phylogenetic analysis
For the phylogenetic analysis, we sequenced 79.03% of all samples positive for SARSCoV-2. After assembly and quality assessment, 820 sequences from LabMovel were used. Complete SARS-CoV-2 genome sequences from delta and gamma strains were downloaded from the GISAID database, including sequences with collection dates from July 26, 2021, to September 30, 2021. In this way, 2,862 sequences from Brazil were obtained, and 38,047 sequences were obtained from the global database to perform subsampling. Brazilian sequences were grouped by state, except those from São Paulo, and 7% from each state was retrieved; sequences from São Paulo State were grouped by city, and 3% of each city was recovered; this resulted in a Brazilian background dataset of 360 sequences. Global samples were grouped by continent, and 0.7% from each continent was retrieved, resulting in 258 sequences. Additionally, one alpha sample and the Wuhan-Hu-1 sequences were added to the dataset, resulting in a total of 1,440 sequences [fig_ref] Table 1: Crude and adjusted prevalence ratio of factors associated with positive results for... [/fig_ref].
For phylogenetic analysis, sequence alignment was performed using MAFFT version 7.475 [bib_ref] MAFFT multiple sequence alignment software version 7: Improvements in performance and usability, Katoh [/bib_ref] and the alignment was manually curated to remove artifacts using Aliview [bib_ref] AliView: A fast and lightweight alignment viewer and editor for large datasets, Larsson [/bib_ref]. Maximum likelihood (ML) phylogenetic trees were estimated using FastTree MP version 2.1.11, applying the ML algorithm with statistical support of bootstrapping with 1000 replicates.
# Statistical analysis
Poisson regression with robust variance was performed to identify factors associated with positive results for COVID-19. The dependent variable was the positive result for COVID-19, and the independent variables were vaccine schedule, age, sex, comorbidities, and presence of symptoms at the time of the test. Independent variables with p < 0.20 in the bivariate analyses were tested in the multivariate model, in decreasing order of statistical significance (stepwise forward technique). In the final model, the significance level was 5%. We used Stata 14.0 (Stata Corp., Texas, USA) for statistical analyses.
# Results and discussion
## Establishing a traveling laboratory for sars-cov-2 diagnosis and sequencing: the labmovel
In response to the importance of reducing the interval between sample collection and test result and the need to unify scientists and healthcare agents as well as provide effective communication regarding the importance of science for the general population, we idealized a traveling laboratory [fig_ref] Figure 1: LabMovel and its organization [/fig_ref] that could reach (i) cities with a shortage of diagnostic tests; (ii) cities with a high influx of people; and (iii) cities with a lack of sample delivery logistics. This traveling laboratory was named in Portuguese LabMovel (Mobile Lab) and was a result of a partnership between a public institution (Instituto Butantan) and a private company (Loccus). The equipment was provided by Loccus and Illumina. The consumables were funded by Loccus, the Butantan Foundation and FAPESP. All of the diagnostic and sequencing testes were free of charge for the general population.
The LabMovel laboratory infrastructure could be easily visualized from outside (glass windows), and it has an analysis capacity of 7000 samples/day for viral diagnosis and generation of 288 complete SARS-CoV-2 genomes per week. It was equipped with infrastructure allowing a complete workflow from NPS arrival to SARS-CoV-2 diagnosis, next-generation sequencing (NGS) library preparation, SARS-CoV-2 sequencing and variant tracking. The sample processing, SARS-CoV-2 diagnosis and sequencing were performed by four technicians who received training for key processes like extraction, qPCR and sequencing. Additionally, we established the Tayna system for patient registration, which consists of a remote laboratory management system allowing registration, collection of clinical data, and delivery of test results. In the first two cities visited by LabMovel, the diagnostic results were delivered in an interval of 24 h by this system directly to the individual's cell phone/email or health unit, significantly reducing the risk of circulation of positive SARS-CoV-2 individuals. Additionally, the median time between sample collection and SARS-CoV-2 variant identification was 8 days due to the necessity of the accumulation of enough samples to complete the NGS plate.
## Labmovel route and diagnostic and sequencing actions
Initially, LabMovel was positioned in two cities of São Paulo State: i) Aparecida do Norte (also receiving samples from the adjacent cities Guaratingueta, Potim, and Roseira) and ii) the city of Santos (also receiving clinical samples from 7 adjacent cities that compose the Baixada Santista region: Guarujá, Itanhaém, Mongaguá, Peruíbe, Praia Grande, São Vicente, and Cubatão). Aparecida do Norte city was chosen since this region is located on the principal highway that links the cities of São Paulo and Rio de Janeiro, and by this period, Rio de Janeiro was the epicenter of the delta VOC outbreak in Brazil. Additionally, Aparecida do Norte city is the principal religious and pilgrimage center in Brazil and experiences a significant human influx. The largest and busiest port in South America, the port of Santos, is located in the city of Santos. During the stay of LabMovel in the city of Aparecida do Norte (between the 5th and 26th of August 2021), the median SARS-CoV-2 incidence (number of positives/ total number of tests applied) in the region was 33.9%. By this period, the vaccine coverage with only one dose was 69.1%, and 27.7% of the tested individuals were completely vaccinated (54.40% received Coronavac and 40.22% received AstraZeneca). The median SARS-CoV-2 incidence in the region of Santos city between August 27th, 2021, and September 16th, 2021, was 13.8%. The incomplete vaccine coverage was 74.4% (only one vaccination dose), and 41.1% of the tested individuals had received a complete vaccination schedule (48.47% of them received Coronavac and 39.02% received Astra-Zeneca). Positioned at Aparecida do Norte, LabMovel performed 1217 tests, and 351 genomes were sequenced (84.1% of the positive samples), while in Santos, Lab-Movel performed 4564 tests, and 463 genomes were sequenced (75.28% of the positive samples).
The performed phylogenetic analysis was consistent with the dissemination of two SARS-CoV-2 VOCs, the gamma and delta VOCs. The period when molecular surveillance was performed coincided with the gradual replacement of the gamma VOC with the delta VOC, which can be observed in [fig_ref] Figure 2: Genomic surveillance of SARS-CoV-2 variants by LabMovel [/fig_ref]. The phylogenetic analysis of the delta [fig_ref] Figure 2: Genomic surveillance of SARS-CoV-2 variants by LabMovel [/fig_ref] VOCs revealed that both of them were randomly interspersed with other reference strains obtained from Brazil and worldwide, highlighting that multiple independent In contrast, the delta VOCs obtained from the Baixada Santista region were highly diverse and interspersed with other delta strains obtained from Brazil and worldwide, which can be related to multiple introductions and reintroductions, probably due to the presence of the largest port in the country. Considering all the positive samples, only 16.4% of the total number of strains was classified as gamma. This can be related to the substitution of the gamma VOC by the delta VOC in these Brazilian regions [fig_ref] Figure 2: Genomic surveillance of SARS-CoV-2 variants by LabMovel [/fig_ref]. Therefore, LabMovel was responsible for providing information on the genetic diversity of variants in the tested regions, revealing the gradual substitution [fig_ref] Figure 2: Genomic surveillance of SARS-CoV-2 variants by LabMovel [/fig_ref]. The obtained sequencing results show the importance of molecular surveillance for the detection of novel SARS-CoV-2 variants that helps elucidate SARS-CoV-2 dynamics and dispersion [bib_ref] Sars-cov-2 variants, rbd mutations, binding affinity, and antibody escape, Yang [/bib_ref]. It is challenging for Brazilian scientists to link the metadata of patients with detected SARSCoV-2 variants. For this reason, we established a questionnaire that was completed by each patient tested in LabMovel. This information permitted us to link the positive SARS-CoV-2 results with the vaccination status of the patients. To validate this approach, we used bivariate and multivariate models to analyze the factors associated with SARS-CoV-2 positivity. Using a bivariate model, we observed that incomplete and complete vaccination protected against COVID-19 and was related to negative diagnostic results. Moreover, male individuals had a greater risk than female individuals for positive SARS-CoV-2 PCR results [fig_ref] Table 1: Crude and adjusted prevalence ratio of factors associated with positive results for... [/fig_ref]. The same result was obtained with the multivariate model [fig_ref] Table 1: Crude and adjusted prevalence ratio of factors associated with positive results for... [/fig_ref]. The observed findings were similar to those of an American study that observed a reduced attack rate in completely vaccinated individuals, especially elderly individuals [bib_ref] The Impact of Vaccination on Coronavirus Disease 2019 (COVID-19) Outbreaks in the..., Moghadas [/bib_ref] , and to those of studies [bib_ref] On the effect of age on the transmission of SARSCoV-2 in households,..., Goldstein [/bib_ref] that demonstrated a higher susceptibility to SARS-CoV-2 in individuals above 60 years of age.
# Conclusions
In conclusion, the most important advantages offered by LabMovel were the following: (i) timely delivery of the results that used to be 5-7 days, was was achieved considering the period between sample collection and the result report. The SARS-CoV-2 diagnosis results obtained within 24 h allowed workers to stay away for a minimum possible time (in the case of a negative result) and facilitated rapid contact tracing; (ii) the testing of SARS-CoV-2 in the study locations by the LabMovel increased with 25% compared to one week before the arrival of Labmovel; (iii) the LabMovel software system delivered the diagnostic data directly to the Federal Healthcare System (SUS, Sistema Único da Saúde), which exempts the public health agents from the submission of these results (20-30 min per positive case); (iv) Realtime information of the circulating SARS-CoV-2 variants was provided; (v) Implementation of the questionnaire allowed the relation of metadata with the diagnostic and sequencing results; (vi) Easy visualization of LabMovel allowed the population to be familiarized with the diagnostic and sequencing procedures, which had a scientific and educational impact. Scientific concepts such as "genome", "PCR", "mutation" and "variant" were largely explained to the most vulnerable Brazilian population. Due to the importance of the actions of LabMovel, it is currently being continued. LabMovel disrupted paradigms in public health care, showing the importance of the collaboration made up of scientists, health professionals, logistics, and government bodies responsible for public policy measures. [fig_ref] Figure 3: Current itinerary of LabMovel in São Paulo State, Brazil [/fig_ref] shows the itinerary that LabMovel covered until December 2021, reaching the regions of Piracicaba, Araçatuba, Marilia, Olímpia, and Ribeirão Preto and traveling from the starting point (Aparecida Norte) to the most recent destination (Ribeirão Preto) for a total distance of ~ 1,573 km in 135 days. In the near future, we will expand the activities of LabMovel by extending the real-time surveillance for important viral agents, such as influenza A and B, dengue, Zika, and yellow fever viruses, in São Paulo State and probably throughout Brazil.
[fig] Figure 1: LabMovel and its organization. A) Picture of LabMovel at Ribeirão Preto, an inner São Paulo State city. B) Internal organization of LabMovel, showing the pipetting robot system that allows the manipulation of samples at LabMovel (MGISTP7000), the pipetting robot that performs RNA extraction (Extracta-96) and PCR preparation (MGISP-960), RT-PCRs machines (Quat-studio), and PCR (TC-9639) and sequencing (iSeq 100) machines. c. Workflow of diagnosis and sequencing of SARS-CoV-2. Figure of own authorship introduction events occurred over time. However, we observed a large monophyletic cluster composed of AY.4 strains, 75.5% of which were obtained from the city of Aparecida do Norte, which may be related to the sustained transmission of the AY.4 sublineage in this region. Moreover, 59.7% of all delta VOC sequences obtained from the region of Aparecida do Norte belonged to AY.4. [/fig]
[fig] Figure 2: Genomic surveillance of SARS-CoV-2 variants by LabMovel. A-B. Frequency of SARS-CoV-2 variants in the tested population according to the position of LabMovel (for information about the prevalence of positive cases, see Fig. 3). C-D.Genomic characterization of SARS-CoV-2 variants of concern (VOCs; delta-gamma) circulating in the location where LabMovel was positioned. Time-stamped maximum likelihood (ML) phylogenetic tree, including the 820 newly sequenced isolates obtained in this study as well as a representative dataset obtained by subsampling containing 360 Brazilian gamma and delta strains and 258 worldwide obtained from GISAID (https:// www. gisaid. org) as of September 2021. The sequences obtained in this study are presented as green (Baixada Santista) and red (Aparecida) dots. The delta VOCs obtained from the Baixada Santista region are randomly interspersed within the phylogenetic tree, while the ones from Aparecida do Norte form a monophyletic cluster (see the box) [/fig]
[fig] Figure 3: Current itinerary of LabMovel in São Paulo State, Brazil. The map on the right side shows the position of São Paulo State in Brazil. On the left side, the map shows the itinerary of LabMovel across the territory of São Paulo State (red line). Blue regions show municipalities that were attended by LabMovel with the respective prevalence of SARS-CoV2 infection in the tested population. The size of circles is related to the number of tests. The map was plotted using GeoPandas (v 0.10.2), data from IBGE (Instituto Brasileiro de Geografia e Estatística https:// geoftp. ibge. gov. br/ organ izacao_ do_ terri torio/ malhas_ terri toria is/ malhas_ munic ipais/ municipio_2021/UFs/SP/SP_Municipios_2021.zip) and edited using Adobe Photoshop Version 14.1.2 (https:// www. adobe. com/) [/fig]
[table] Table 1: Crude and adjusted prevalence ratio of factors associated with positive results for COVID-19 [/table]
|
Anti‐envelope antibody responses in individuals at high risk of hepatitis C virus who resist infection
Injection drug users uninfected by hepatitis C virus (HCV) despite likely repeated exposure through high-risk behaviour are well documented. Factors preventing infection in these individuals are incompletely understood. Here, we looked for anti-HCVenvelope antibody responses in a cohort of repeatedly exposed but uninfected subjects. Forty-two hepatitis C diagnostic antibody-and RNA-negative injection drug users at high risk of exposure were studied and findings compared to healthy controls and cases with chronic HCV infection. Purified IgGs from sera were tested by ELISA for binding to genotype 1a and 3a envelope glycoproteins E1E2 with further testing for IgG and IgM reactivity against soluble E2. Virus-neutralizing activity was assessed using an HCV pseudoparticle system. Uninfected subjects demonstrated significantly greater IgG and IgM reactivities to envelope glycoproteins than healthy controls with IgG from 6 individuals additionally showing significant neutralization. This study is the first to describe humoral immunological responses targeting the HCV envelope, important for viral neutralization, in exposed uninfected individuals. A subset of these cases also had evidence of viral neutralization via anti-envelope antibodies. In addition to confirming viral exposure, the presence of specific anti-envelope antibodies may be a factor that helps these individuals resist HCV infection.K E Y W O R D SE1E2, exposed uninfected (EU), injection drug user (IDU), neutralization, neutralizing antibodies 1
# | introduction
Hepatitis C virus (HCV) is a major cause of liver morbidity and mortality worldwide.Approximately 75% of infected individuals proceed to chronic infection. [bib_ref] Adaptive immune responses in acute and chronic hepatitis C virus infection, Bowen [/bib_ref] In developed countries, the major route of transmission remains the use of injection drugs and sharing of related paraphernalia by injection drug users (IDUs). [bib_ref] Modelling hepatitis C virus incidence, prevalence and long-term sequelae in Australia, Law [/bib_ref] Symptomatic acute infection is rare, and HCV has the potential to spread undetected within these populations. While effective antiviral medication is now available, 4 treatment remains costly and ineffectively implemented with the prevalence of HCV in IDUs rising in England. [bib_ref] Hepatitis C infection among injecting drug users in England and Wales (1992-2006):..., Sweeting [/bib_ref] It is clear that global eradication of HCV will not be possible unless robust preventative strategies are also developed. [bib_ref] Best strategies for global HCV eradication, Hagan [/bib_ref] Studying individuals who resist infection can help inform prevention strategies and vaccine design.
In natural infection, successful clearance of HCV infection has been associated with the activation of elements of both the adaptive and the innate immune system (e.g. HCV-specific B and T cell, NK cell, dendritic cell and interferon responses [bib_ref] Failure of innate and adaptive immune responses in controlling hepatitis C virus..., Thimme [/bib_ref] [bib_ref] Adaptive immune responses to hepatitis C virus: from viral immunobiology to a..., Thimme [/bib_ref] [bib_ref] A polymorphism in IL28B distinguishes exposed, uninfected individuals from spontaneous resolvers of..., Knapp [/bib_ref] [bib_ref] Genetic variation in IL28B and spontaneous clearance of hepatitis C virus, Thomas [/bib_ref]. There is growing evidence that a robust antibody response targeting HCV virion *Both authors contributed equally to the work and are joint first authors.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. envelope glycoproteins (E1 and E2) responsible for virus entry into host cells can contribute significantly to viral clearance in acute infection. [bib_ref] Spontaneous clearance of chronic hepatitis C virus infection is associated with appearance..., Raghuraman [/bib_ref] [bib_ref] Spontaneous control of primary hepatitis C virus infection and immunity against persistent..., Osburn [/bib_ref] [bib_ref] Cell entry of hepatitis C virus, Bartosch [/bib_ref] [bib_ref] Claudin-1 and its potential role in HCV entry: another piece of the..., Hamilton [/bib_ref] [bib_ref] Hepatitis C virus entry: beyond receptors, Meredith [/bib_ref] [bib_ref] Hepatitis C virus host cell entry, Ploss [/bib_ref] Rapid onset of anti-HCV envelope antibodies 17 capable of neutralizing diverse strains of HCV is associated with acute clearance. Furthermore, broadly neutralizing antibodies may contribute to resolution of infection even once HCV has become established. [bib_ref] Spontaneous clearance of chronic hepatitis C virus infection is associated with appearance..., Raghuraman [/bib_ref] [bib_ref] Broadly neutralizing antibodies abrogate established hepatitis C virus infection, De Jong [/bib_ref] In the search for a prophylactic vaccine, it is especially relevant to study individuals who appear to have natural resistance to HCV infection. Individuals who regularly inject drugs and share injecting equipment are at very high risk of HCV exposure with seroprevalence rates of up to 90% reported in long-term users.
Resistance to HCV infection is increasingly well documented in IDUs who remain uninfected despite a long history of unsafe injection practices. [bib_ref] Resistance to hepatitis C virus: potential genetic and immunological determinants, Mina [/bib_ref] Individuals at high risk of exposure with no evidence of past or current infection have been termed exposed but uninfected (EU).
EU cohorts are immunologically distinct from both healthy controls and those who spontaneously clear HCV infection (see . [bib_ref] Hepatitis C virus (HCV) specific immune responses in anti-HCV positive patients without..., Cramp [/bib_ref] [bib_ref] Hepatitis C virus (HCV)-specific T cell responses in injection drug users with..., Thurairajah [/bib_ref] [bib_ref] Interleukin 12B gene polymorphism and apparent resistance to hepatitis C virus infection, Hegazy [/bib_ref] Up to 60% of EU individuals display HCV-specific adaptive T-cell responses. Furthermore, they display enhanced NK cell, IL6/IL8 and TNF-α activity compared to HCV-infected IDUs. [bib_ref] Increased natural killer cell cytotoxicity and NKp30 expression protects against hepatitis C..., Golden-Mason [/bib_ref] [bib_ref] Cytokine profiles in high risk injection drug users suggests innate as opposed..., Warshow [/bib_ref] They are also genetically distinct from spontaneous resolvers and those with chronic HCV 9 with the combination of KIR2DL3, HLA-C1 over represented in both the EU and spontaneous resolver groups while the prevalence of the IL28B polymorphism is similar in EU to chronically infected cohorts. [bib_ref] Interleukin 12B gene polymorphism and apparent resistance to hepatitis C virus infection, Hegazy [/bib_ref] [bib_ref] Cytokine profiles in high risk injection drug users suggests innate as opposed..., Warshow [/bib_ref] [bib_ref] Exploration of genetically determined resistance against hepatitis C infection in high-risk injecting..., Sugden [/bib_ref] [bib_ref] Consistent beneficial effects of killer cell immunoglobulin-like receptor 2DL3 and group 1..., Knapp [/bib_ref] While this EU cohort is defined by the lack of antibody response to HCV core and nonstructural proteins in a diagnostic assay, the role that anti-envelope neutralizing antibodies might play in their protection from infection has not yet been explored. Given the upregulation of neutralizing antibodies in those who acutely clear HCV infection on multiple exposures, it is possible that such antibodies may have a significant protective effect in the IDU EU population. 12 Therefore, we aimed to determine the presence of functional anti-HCV envelope antibody responses in a cohort of IDUs who remain uninfected despite their high risk of repeated exposure to HCV. variety of locations in Plymouth, UK, as previously described. [bib_ref] Hepatitis C virus (HCV)-specific T cell responses in injection drug users with..., Thurairajah [/bib_ref] [bib_ref] Interleukin 12B gene polymorphism and apparent resistance to hepatitis C virus infection, Hegazy [/bib_ref] All individuals completed a confidential structured questionnaire to collect demographic data and a detailed injecting history. This included age at first injection, duration of injecting behaviour, frequency of injecting episodes, current injecting behaviour, frequency of sharing intravenous paraphernalia (needles, syringes, filters, spoons and water), frequency of sharing with a contact known to have HCV infection and risk of non-IDU HCV exposure. For this study, we included only those judged to be at substantial risk of HCV exposure based on a >1 year history of injecting drug use and regular sharing of needles and related paraphernalia. All individuals had serum and whole blood samples drawn for laboratory analysis and clinical details entered in respective databases. Where possible, follow-up clinical information and clinical samples from recruited individuals were obtained.
# | materialsandmethods
## | theeuandcontrolcohorts
The exposed group was screened for the evidence of current or previous HCV infection using diagnostic tests for antibodies to core and nonstructural proteins (third-generation ELISA by Abbot IMx) and HCV RNA by qualitative PCR (Amplicor, Roche Diagnostics). Those individuals negative on both tests were termed exposed uninfected (EU) and were included in the study. Two individuals who were found to have developed HCV infection on subsequent testing were excluded. In addition, we separately recruited positive and negative control cohorts of individuals with chronic HCV infection (CHCV) and non-IDU healthy controls (HC) with no liver disease or history of HCV infection, respectively. The full details of these cohorts have been described elsewhere. [bib_ref] Elevated interferonstimulated gene transcription in peripheral blood mononuclear cells occurs in patients..., Robinson [/bib_ref] Study protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by regional ethical committees. Informed written consent was obtained from each subject prior to entry in the study.
T A B L E 1 Immunological characteristics of exposed uninfected individuals KIR2DL3 genotype Increased frequency of KIR2DL3:HLA-C1 homozygosity Chronically infected individuals [bib_ref] Consistent beneficial effects of killer cell immunoglobulin-like receptor 2DL3 and group 1..., Knapp [/bib_ref] No difference in frequency of KIR2DL3:HLA-C1 homozygosity Pre-infection samples from IDUs with subsequent seroconversion [bib_ref] Exploration of genetically determined resistance against hepatitis C infection in high-risk injecting..., Sugden [/bib_ref] IL12B CC allele (rs3213113) Increased frequency compared to healthy controls Healthy controls [bib_ref] Interleukin 12B gene polymorphism and apparent resistance to hepatitis C virus infection, Hegazy [/bib_ref] Serum cytokine levels Elevated IL6, IL8 and TNF-α Healthy controls, spontaneous resolvers and chronically infected individuals [bib_ref] Cytokine profiles in high risk injection drug users suggests innate as opposed..., Warshow [/bib_ref] HCV-specific T cells Evidence of IFN-γ production and T-cell proliferation Healthy controls and chronically infected individuals [bib_ref] Hepatitis C virus (HCV)-specific T cell responses in injection drug users with..., Thurairajah [/bib_ref] [bib_ref] Hepatitis C virus (HCV)-specific immune responses of long-term injection drug users frequently..., Mizukoshi [/bib_ref] Natural Killer Cells Enhanced IL-2-mediated cytotoxicity. Increased NKp30 expression
Pre-infection samples from IDUs with subsequent seroconversion [bib_ref] Increased natural killer cell cytotoxicity and NKp30 expression protects against hepatitis C..., Golden-Mason [/bib_ref] | 875
# | statisticalanalysis
Statistical analysis was conducted using GraphPad Prism 6 Software (GraphPad Software, California, USA) and SPSS v. [bib_ref] Resistance to hepatitis C virus: potential genetic and immunological determinants, Mina [/bib_ref].09 (IBM, New York, USA).
## | generationofhcvpseudoparticle(hcvpp)and e1e2lysate
As genotypes 1 (gt 1) and 3 (gt 3) account for the vast majority of HCV infections in the UK, envelope glycoprotein sequences from a standard HCV gt 1a and a UK derived gt 3 strain were used. HCV pseudoparticles bearing envelope proteins from gt 1a (strain H77c, accession number AF011751.1) and gt 3a (sequence UKN3a1.28 F4/2-35;
closely related to accession number AY734984.1) were generated in HEK-293T cells as described previously. [bib_ref] In vitro assay for neutralizing antibody to hepatitis C virus: evidence for..., Bartosch [/bib_ref] Further details are available in Supplementary Methods. Following harvesting of pseudoparticles, cells were lysed in 1 mL of lysis buffer and centrifuged and the supernatant used for E1E2 ELISA assays as described below.
## | gnacapturee1e2elisa
IgG was purified from EU, CHCV and HC samples using Protein G IgG-specific spin columns (Thermo Scientific, UK; see Supplementary Methods) including 3 wash steps prior to elution ensuring removal of nonspecific serum factors that may interfere with the assay. Purified
IgG was tested in an ELISA assay to detect antibodies to the E1 and E2
glycoproteins contained in the HEK-293T lysate supernatant. These ELISAs were performed as described previously with further detail in Supplementary Methods. [bib_ref] Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of..., Patel [/bib_ref] Absorbance readings were normalized to a multiple of the mean of the HC readings. Significant binding by ELISA was determined as absorbance values ≥2 times HC mean.
Reactivity to both gt 1a and gt 3a E1E2 was tested.
## | solublegt1ae2bindingelisas:iggandigm
As IgM is difficult to purify, diluted serum was used to detect binding of IgG and IgM to purified HCV soluble gt 1a E2 (sE2). EU and control serum samples were diluted 1:50 in PBSTM and tested for binding to sE2 by ELISA. Absorbance readings were normalized to healthy control mean and analysed as above with further detail in the Supplementary Methods.
## | pseudoparticleneutralizationassays
In the subset of EU individuals displaying significantly elevated E1E2 binding compared to HC on ELISA, pseudoparticle (pp) neutralization assays were conducted as previously described. [bib_ref] Analysis of a highly flexible conformational immunogenic domain a in hepatitis C..., Keck [/bib_ref] Briefly, purified subject IgG was added to 40 μL of HCVpp-containing medium prepared as above. Purified IgG was added at a concentration of 400 μg mL −1 for screening of the EU cohort, for some individuals where serum was scarce, this was reduced to 200 μg mL −1 . The mouse monoclonal anti-E2 antibody AP33 31 and HC IgG were included as positive and negative controls, respectively. After incubation for 1 hour, the IgG-HCVpp mixture was added to a 96-well plate preseeded with Huh-7 cells. Following incubation for 3 hour, the inoculum was replaced with fresh medium, and after a further 72-hour incubation, luciferase activity in infected cells was detected as a marker of HCVpp infectivity using the Bright-Glo Luciferase kit (Promega, UK). Virus neutralization was defined as 50% reduction in pseudoparticle infectivity as measured in relative light units (RLU) using a Chameleon II plate reader.
Ability to neutralize both gt 1a and gt 3 was tested. For those samples with apparent neutralizing activity, further neutralization assays were conducted using serial dilutions of subject IgG.
## | e2competitionelisa
For EUs with neutralizing activity and sufficient IgG available, competition ELISAs were performed using a panel of well-characterized monoclonal antibodies targeting known CD81-binding epitopes . CHCV samples with known neutralizing activity were used as positive controls.
Purified IgG was incubated on E2-coated Immulon II plates at a concentration of 200 μg mL −1 in PBSTM. Subsequently, biotinylated antibodies to known conformational epitopes were added to these wells in addition to control wells with no EU IgG. Streptavidin-HRP was added and binding measured as previously. The reduction in relative binding of each biotinylated antibody (calculated as percentage reduction in absorbance) on addition of EU IgG compared to control was determined. PBSTM and neutralizing IgG from the CHCV cohort were used as negative and positive controls, respectively.
# | results
## | clinicalcohorts
Forty-two EU subjects, 8 gt 1 chronically infected patients (CHCV) and 8 healthy controls (HC) were studied. By definition, all EU cases consistently tested negative for HCV RNA and anti-HCV antibodies using standard diagnostic tests as described. In 5 EU individuals, serial samples were tested. Demographics of the cohorts are detailed in .
## | reactivityofiggtohcvenvelope
The median absorbance readings for the HCV gt 1a E1E2 capture ELISA were significantly higher in the EU cohort than in the HC group (P=.038) . While there was a trend towards overall higher absorbance readings against gt 3a E1E2 in the EU group than in controls, this did not reach significance (P=.067, . However, three samples showed levels of binding to gt 3a lysate comparable to chronically infected controls.
## | hcvexposureriskande1e2reactivity
EU IgG absorbance levels to gt 1a E1E2 and gt 3a E1E2 were analysed for association with reported injection behaviour with a significant correlation between IgG reactivity against gt 1a E1E2 and greater lifetime injecting episodes (Spearman R=.38, P=.02, , although this association was not seen with gt 3a reactivity .
## | iggandigmse2elisa
To eliminate reactivity against HEK antigens and medium components, diluted serum samples from all individuals were tested for IgG reactivity to gt 1a sE2. For two of the subjects, serum from 2 separate time points was tested. There was significantly higher absorbance of IgG from EU to sE2 than HC (P<.01, . Serum was also tested for IgM binding to sE2. The EU cohort showed significantly higher quantities of IgM binding to sE2 than the HC group (P=.001 Wilcoxon rank sum, with several individuals displaying levels comparable to the CHCV group (data not shown).
## | positivityacrossmultipleassays
We set a cut-off of absorbance ≥2 times the HC mean value as indicat- displaying absorbance readings ≥2 times the HC mean , with 2 more showing above average binding to sE2 but below this cutoff value. Seven further individuals with significant responses against sE2 showed elevated reactivity against gt 1a lysate, but the absorbance attained did not reach the cut-off value. Only four samples showed significant binding to sE2 alone. It should be noted that for the vast majority of assays the value of 2 times the HC mean was higher (therefore a more stringent cut-off) than HC mean + 2 times standard deviations.
## | abilitytoneutralizehcvpp
Of those EU subject sera with significant binding to E1E2 by ELISA, 6 (257, 306, 307, 315, 331-1 and 458) showed ability to neutralize gt 1a
HCVpp by 50% or more at a purified IgG concentration of 400 μg mL −1 . For subjects 257, 306 and 307, this level of neutralization was achieved at a concentration of 200 μg mL −1 , while IC 50 value for the remaining samples was between 200 μg mL −1 and 400 μg mL −1 .
Of those able to neutralize gt 1a HCVpp, [bib_ref] Modelling hepatitis C virus incidence, prevalence and long-term sequelae in Australia, Law [/bib_ref]. Two further individuals (461, 301) showed evidence of a weaker neutralization effect against both gt 1a and gt 3a HCVpp, consistently reducing infectivity by 40%.
## | competitionwithantibodiesto knownepitopes
IgG from EU individuals with evidence of neutralizing activity was tested in a competition assay with a small panel of well-characterized conformation-sensitive anti-E2 antibodies. Three of these (HC1, HC11 and CBH7) recognize amino acid residues/regions that are critical for the interaction of E2 to CD81, the host entry factor essential for virus entry into target cells [bib_ref] Cell entry of hepatitis C virus, Bartosch [/bib_ref] [bib_ref] Claudin-1 and its potential role in HCV entry: another piece of the..., Hamilton [/bib_ref] [bib_ref] Hepatitis C virus entry: beyond receptors, Meredith [/bib_ref] [bib_ref] Hepatitis C virus host cell entry, Ploss [/bib_ref]. Although chronically infected samples with neutralizing activity tended to compete with antibodies to these regions, we did not see any evidence of significant competition in the EU samples . While there was evidence of binding to pure E2 in these samples at >2× control samples ,
it was considerably weaker than in individuals with chronic infection which bound to viral glycoproteins at >20 times the strength of HC samples (data not shown).
## | durationofantibodydetectionovertime
For 5 EU individuals, serial samples from time points separated by a period of at least 1 year were studied. Their E1E2 reactivity values relative to HC samples were consistent over the time periods studied, either remaining at HC levels or being consistently elevated .
In the one individual with significant E1E2 and neutralizing responses studied serially, these responses remained detectable over time but with a diminution in strength at later time points.
# | discussion
Long-term injection drug users who recurrently, persistently and frequently share injection equipment but who remain HCV antibody negative with undetectable HCV RNA represent an optimal cohort for advancing knowledge and understanding of mechanisms of natural protection from HCV infection. These individuals appear resistant to infection. Unlike HIV where resistance may be conferred by mutations in host entry proteins, no such variants have been identified in HCV exposed uninfected cases. 32-34 Therefore, it is likely that these subjects resist infection through immune-mediated mechanisms.
This study is the first to report evidence of humoral immunological responses targeting the HCV envelope, potentially a key area for viral neutralization, in exposed uninfected individuals. In those who resolve acute infection, detectable neutralizing antibody responses are seen to reduce over a period of months to years F I G U R E 4 EU IgG fails to compete with conformational antibodies to known epitopes on gt 1a E2. IgGs from individuals with neutralizing ability in the HCV pp system were selected for testing by competition ELISA to determine competitive binding with monoclonal antibodies to known conformational epitopes on E2 . Samples from chronically infected individuals (CHCV) with known neutralizing activity were also included as positive controls. Percentage reduction in absorbance of the monoclonal antibodies was calculated and plotted. Significant competition would be expected at a level of 50% inhibition | 879
following viral clearance although they may be restored in subsequent episodes of infection to aid more rapid viral control. [bib_ref] Spontaneous control of primary hepatitis C virus infection and immunity against persistent..., Osburn [/bib_ref] The presence of potentially protective humoral responses in our cohort is seen most strikingly in cases with the greatest likelihood and frequency of HCV [bib_ref] Use of an anti-hepatitis C virus (HCV) IgG avidity assay to identify..., Gaudy-Graffin [/bib_ref] it is also conceivable that the EU antibody responses are directed at CD81 binding regions in E2 but do not bind with sufficient avidity to avoid being 'competed off' by the conformational antibodies derived from chronically infected individuals. It is also possible that these individuals, in common with most acutely infected cases, develop antibodies which predominantly target the Highly Variable Region-1 region of the E2 protein or regions of E1 essential for entry. An alternative explanation may be that due to high levels of diversity in E2 amino acid sequences, these individuals may raise antibodies to a local E2 sequence which only provides weak cross-reactivity with the H77 sequence at CD81 binding epitopes, and future work should aim to explore the breadth of E1E2 reactivity within these individuals.
In conclusion, this is the first report of HCV envelope-specific humoral immune responses in a cohort of exposed uninfected injection drug users who remain HCV PCR and EIA negative despite repeated risk of exposure. In addition to adaptive humoral immune responses to envelope proteins, some individuals produce neutralizing anti-envelope antibodies which may contribute to host immunity.
These neutralizing antibodies do not appear to compete with commonly targeted CD81 binding sites and may therefore recognize novel epitopes. Our data complement previous reports of HCV-specific T-cell responses [bib_ref] A polymorphism in IL28B distinguishes exposed, uninfected individuals from spontaneous resolvers of..., Knapp [/bib_ref] [bib_ref] Consistent beneficial effects of killer cell immunoglobulin-like receptor 2DL3 and group 1..., Knapp [/bib_ref] and upregulated innate immune responses in exposed uninfected cases. [bib_ref] Exploration of genetically determined resistance against hepatitis C infection in high-risk injecting..., Sugden [/bib_ref] Together, these studies provide robust evidence that such individuals have been exposed to HCV, but are resistant to developing established infection. Whether any one of these responses is central to resisting infection, or whether a combination of upregulated innate immunity together with adaptive T-and B-cell responses is needed is not yet known. Further study of the antibody responses present in EU individuals will enhance our understanding of the role of antibodies in HCV infection and inform future preventative vaccine design. |
Indacaterol improves daily physical activity in patients with chronic obstructive pulmonary disease
Background:The current mainstay of therapy for chronic obstructive pulmonary disease (COPD) is long-acting bronchodilators. To date, the effect of indacaterol, a β2-agonist, on activities of daily living in COPD patients is not well understood. The aim of this study was to evaluate the efficacy of indacaterol with regard to activities of daily living in patients with COPD.Methods:In this nonrandomized open-label study, 23 patients with COPD were instructed to carry an accelerometer for 4 weeks without indacaterol therapy and then for another period of 4 weeks while receiving indacaterol therapy. Results: The number of steps, duration of moderate or greater physical activity, and energy expenditure were significantly increased after treatment with indacaterol compared with baseline data in all patients with COPD; the metabolic equivalent of task was also significantly enhanced after treatment with indacaterol. Conclusion: This study provides early evidence that indacaterol improves daily physical activity in patients with COPD.
# Introduction
Chronic obstructive pulmonary disease (COPD) results from a chronic inflammatory process of the airways. [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary..., Rabe [/bib_ref] The prevalence of COPD is very high, and the disease affects more than 10% of subjects aged over 40 years worldwide, and it is predicted that COPD will be the third leading cause of death by 2020. [bib_ref] Epidemiology of chronic obstructive pulmonary disease: a literature review, Rycroft [/bib_ref] The main cause of the disease is cigarette smoking. [bib_ref] Epidemiology of chronic obstructive pulmonary disease: a literature review, Rycroft [/bib_ref] The inflammatory process predominantly affects the small airways, is characterized by infiltration of neutrophils, macrophages, and CD8+ T cells, and leads progressively to fibrosis of the small bronchioles and destruction of the alveolar walls and lung parenchyma architecture. [bib_ref] Immunology of asthma and chronic obstructive pulmonary disease, Barnes [/bib_ref] The functional consequences of these pathological changes are airflow limitation with air-trapping and dynamic hyperinflation, leading to reduced inspiratory capacity and increased end-expiratory lung volumes. [bib_ref] Immunology of asthma and chronic obstructive pulmonary disease, Barnes [/bib_ref] Therefore, patients with COPD complain generally of dyspnea, particularly during exercise. [bib_ref] Epidemiology of chronic obstructive pulmonary disease: a literature review, Rycroft [/bib_ref] This shortness of breath limits physical activity and worsens quality of life in these patients.
At present, there is no curative therapy for COPD, and the aim of treatment is to reduce symptoms and exacerbations and to improve quality of life. [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary..., Rabe [/bib_ref] Treatment includes bronchodilators, rehabilitation, and home oxygen therapy. Recent studies have shown that the inhaled long-acting bronchodilators are very effective for improving symptoms, exercise capacity, and quality of life, as well as for prevention of exacerbations. [bib_ref] Long-acting beta-agonists in the management of chronic obstructive pulmonary disease: current and..., Tashkin [/bib_ref] The β2-agonist, indacaterol, is an ultralong-acting bronchodilator with an established clinical efficacy and safety profile which needs to be administered only once daily. [bib_ref] Long-acting beta-adrenoceptor agonists in the management of COPD: focus on indacaterol, Beier [/bib_ref] [bib_ref] Indacaterol: a review of its use as maintenance therapy in patients with..., Mckeage [/bib_ref] Indacaterol was approved for use in the clinic by the European Medicines Agency in 2009 and by the US Food and Drug Administration in 2011. Several clinical trials performed in patients with moderate to severe COPD have shown that indacaterol significantly improved symptoms, pulmonary function test results, and quality of life, and markedly reduced the use of rescue medication compared with a placebo control group. [bib_ref] Efficacy of once-daily indacaterol 75 mug relative to alternative bronchodilators in COPD:..., Cope [/bib_ref] [bib_ref] Comparative efficacy of indacaterol in chronic obstructive pulmonary disease, Ribeiro [/bib_ref] [bib_ref] Acute effects of indacaterol on lung hyperinflation in moderate COPD: a comparison..., Rossi [/bib_ref] Recent studies suggest that indacaterol may also improve exercise endurance in patients with COPD, but its effect on daily physical activity in these patients is not well understood as yet. [bib_ref] Indacaterol add-on therapy improves lung function, exercise capacity and life quality of..., Mroz [/bib_ref] [bib_ref] Effect of indacaterol on exercise endurance and lung hyperinflation in COPD, O'donnell [/bib_ref] Physical activity refers to movement of the body produced by skeletal muscle contraction leading to energy expenditure. [bib_ref] Activity monitoring in assessing activities of daily living, Casaburi [/bib_ref] The level of physical activity during routine daily life has recently been recognized to be an important outcome parameter when evaluating the therapeutic response in patients with COPD. [bib_ref] Energy expenditure and impact of bronchodilators in COPD patients, Cazzola [/bib_ref] [bib_ref] Physiologic limitations during daily life activities in COPD patients, Lahaije [/bib_ref] [bib_ref] Characteristics of physical activities in daily life in chronic obstructive pulmonary disease, Pitta [/bib_ref] [bib_ref] Physical activity monitoring in COPD: compliance and associations with clinical characteristics in..., Waschki [/bib_ref] [bib_ref] Physical activity in patients with COPD, Watz [/bib_ref] Limited physical activity has been reported to be associated with more hospitalizations because of exacerbations, and markers of physical activity have been shown to predict mortality in patients with COPD. [bib_ref] Physical activity, health status and risk of hospitalization in patients with severe..., Benzo [/bib_ref] [bib_ref] Prognostic value of the objective measurement of daily physical activity in COPD..., Garcia-Rio [/bib_ref] [bib_ref] Physical activity and hospitalization for exacerbation of COPD, Pitta [/bib_ref] [bib_ref] Physical activity is the strongest predictor of all-cause mortality in patients with..., Waschki [/bib_ref] Several studies have validated the use of accelerometers for objective measurement of physical activity in patients with the disease. [bib_ref] Characteristics of physical activities in daily life in chronic obstructive pulmonary disease, Pitta [/bib_ref] [bib_ref] Physical activity and hospitalization for exacerbation of COPD, Pitta [/bib_ref] [bib_ref] Physical activity monitoring in COPD: compliance and associations with clinical characteristics in..., Waschki [/bib_ref] In the present study, the effect of indacaterol on daily physical activity was evaluated in patients with COPD using a uniaxial accelerometer.
## Materials and methods subjects
This study included 23 patients with COPD attending the outpatient department at Matsusaka Municipal Hospital. [fig_ref] Table 1: Baseline demographic data for patients with chronic obstructive pulmonary disease [/fig_ref] shows the anthropometry, clinical staging, and results of pulmonary function testing in these subjects. All of the patients were ex-smokers and not on active smoking cessation pharmacotherapy, and had stable disease with no exacerbations before the beginning or during the study. Patients who were physically unable to move and those receiving home oxygen therapy or pulmonary rehabilitation were excluded. Three patients had comorbid arterial hypertension and three had hyperuricemia. In total, 24 patients were included in the study but one was excluded because of contusions and a sprained ankle following a traffic accident. Among the 23 eligible patients, 14 were receiving tiotropium 18 µg/day (by Handihaler) while nine were receiving no therapy; thus, during the entire study period, 14 cases received treatment with tiotropium together with indacaterol and nine with indacaterol alone. None of the patients received steroids or rescue with short-acting β-agonists during the study.
The diagnosis of COPD was made according to the criteria of the American Thoracic Society. [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary..., Rabe [/bib_ref] Pulmonary function tests were performed using a volume-type spirometer (Super Spiro, Discom-21FX III; Chest Corporation, Tokyo, Japan). The 6-minute walk test (6MWT) and the COPD Assessment Test (CAT) were performed based on a protocol and criteria previously described. [bib_ref] Usefulness of the Chronic Obstructive Pulmonary Disease Assessment Test to evaluate severity..., Mackay [/bib_ref] Informed consent was obtained from all subjects before entry into the study.
## Study design
This was an open-label clinical trial with no randomization, no placebo group, and no blinding. To record physical activity, the patients were asked to carry a Lifecorder, a uniaxial accelerometry sensor (Suzuken Corporation, Nagoya, Japan), [bib_ref] The use of uniaxial accelerometry for the assessment of physical-activity-related energy expenditure:..., Kumahara [/bib_ref] fixed to their belts for 4 weeks without receiving indacaterol therapy and then for another period of 4 weeks while receiving 150 µg of inhaled indacaterol . Physical activity before and after treatment with indacaterol was then compared. Before the trial was started, there was a period of adaptation during which each patient received instructions on how to fix and use the device correctly. Patients were instructed to fix the device every day upon waking and to remove it before going to sleep.
# Statistical analysis
All data are expressed as the mean ± standard error of the mean. The difference between the means of two variables was assessed using the nonparametric Wilcoxon's rank test. Statistical analyses were performed using the StatView 4.5 package for Macintosh (Abacus Concepts, Berkeley, CA). A P value less than 0.05 was considered to be statistically significant.
# Results
## Pulmonary function, 6mwt, and cat
Forced expiratory volume in one second (FEV 1 ) and 6MWT are important parameters when assessing the response to therapy in patients with COPD. [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary..., Rabe [/bib_ref] Comparing these parameters before and after treatment showed a significant increase in FEV 1 and in maximum inspiratory capacity after 4 weeks of treatment with indacaterol . Similarly, the distance walked during the 6MWT was significantly increased after treatment compared with that before starting indacaterol . The CAT score also tended to decrease after treatment with indacaterol but this did not reach a statistically significant level .
## Physical activity parameters
The Lifecorder was carried by all patients, and the number of steps, duration of moderate or higher degree exercise, energy expenditure, and metabolic equivalent of task were measured. The number of steps [fig_ref] Figure 3: Effect of indacaterol on physical activity parameters [/fig_ref] , time in seconds of moderate or more activity [fig_ref] Figure 3: Effect of indacaterol on physical activity parameters [/fig_ref] , and energy expenditure expressed in kilocalories [fig_ref] Figure 3: Effect of indacaterol on physical activity parameters [/fig_ref] were significantly increased after treatment compared with baseline in all patients. The metabolic equivalent of task was also significantly improved after treatment with indacaterol compared with baseline data [fig_ref] Figure 3: Effect of indacaterol on physical activity parameters [/fig_ref].
# Discussion
The results of this study show that the long-acting β2-agonist, indacaterol, significantly and effectively improved daily physical activity in patients with COPD and that the physical activity parameters recorded using the uniaxial accelerometry The mainstay of treatment for symptomatic improvement in patients with COPD is inhaled bronchodilators. [bib_ref] Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary..., Rabe [/bib_ref] Of these, the ultra-long-acting β2-adrenoreceptor agonist, indacaterol, and the long-acting antimuscarinic, tiotropium bromide, have been shown to be very effective for reducing symptoms, to increase exercise tolerance, to reduce the frequency of exacerbations, and to improve quality of life test scores. [bib_ref] Long-acting beta-agonists in the management of chronic obstructive pulmonary disease: current and..., Tashkin [/bib_ref] These drugs have a further advantage over previous long-acting bronchodilators, in that they can be used once daily, so are associated with fewer side effects. [bib_ref] Long-acting beta-agonists in the management of chronic obstructive pulmonary disease: current and..., Tashkin [/bib_ref] [bib_ref] Efficacy of a new once-daily longacting inhaled beta2-agonist indacaterol versus twice-daily formoterol..., Dahl [/bib_ref] [bib_ref] Efficacy and safety of indacaterol 150 microg once-daily in COPD: a double-blind,..., Feldman [/bib_ref] In the present study, we focused on the β2-agonist, indacaterol, and evaluated whether it can improve exercise capacity and FEV 1 in patients with COPD treated for 4 weeks, and our results showed that this agent significantly improved the results of lung function testing and exercise endurance as measured by the 6MWT.
One hurdle that needs to be overcome in the management of patients with COPD is the assessment of response to therapy in terms of physical activity during routine daily life. Recent studies have validated the use of several accelerometers in the assessment of the level of activity in COPD patients. [bib_ref] Characteristics of physical activities in daily life in chronic obstructive pulmonary disease, Pitta [/bib_ref] [bib_ref] Physical activity is the strongest predictor of all-cause mortality in patients with..., Waschki [/bib_ref] The results reported have shown a significant association between a low level of physical activity as measured by accelerometers and increased mortality and morbidity from COPD. [bib_ref] Physical activity is the strongest predictor of all-cause mortality in patients with..., Waschki [/bib_ref] In addition, less active patients are more likely to be hospitalized as a result of exacerbations. [bib_ref] Physical activity and hospitalization for exacerbation of COPD, Pitta [/bib_ref] To the authors' knowledge, the efficacy of indacaterol in daily life activity has not been evaluated until now. In the present study, we used a novel accelerometer, the sensitivity and efficacy of which has been previously validated. [bib_ref] The use of uniaxial accelerometry for the assessment of physical-activity-related energy expenditure:..., Kumahara [/bib_ref] This novel accelerometer can measure several indicators of daily physical activity, including the number of steps, energy expenditure, and metabolic equivalent of task. [bib_ref] The use of uniaxial accelerometry for the assessment of physical-activity-related energy expenditure:..., Kumahara [/bib_ref] The device is easy to use, cost-effective, and can record data on a real-time basis every 4 seconds and over a lengthy period of time. We carried out a prospective and open clinical trial to assess whether indacaterol can improve daily physical activity in patients with COPD. The number of steps, energy expenditure, and metabolic equivalent of task were significantly increased in all patients after treatment with indacaterol, suggesting that this β2-agonist improves routine daily activity in patients with COPD.
Our study has several limitations, including its open nature, a small patient population, lack of a placebo control group, and no randomization. In addition, although the patients were The International Journal of COPD is an international, peer-reviewed journal of therapeutics and pharmacology focusing on concise rapid reporting of clinical studies and reviews in COPD. Special focus is given to the pathophysiological processes underlying the disease, intervention programs, patient focused education, and self management protocols.
This journal is indexed on PubMed Central, MedLine and CAS. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.
International Journal of COPD 2013:8 not asked or motivated to perform any exercise other than their normal or routine daily physical activity, they knew that they were receiving the drug during the second period of the study and therefore they could have been psychologically motivated or encouraged to perform better during their daily physical activity. However, to avoid some of these confounding factors, the patients were allowed to have an adaptation period during which they received training about how to use the device and about the performance of lung function tests. In brief, this study provides early evidence that indacaterol improves daily activity in patients with COPD. However, a larger patient population and a placebo control group should be included in future studies to validate the present findings.
[fig] Figure 1, Figure 2: Study design. Notes: Patients fixed the Lifecorder to their belts for a period of 4 weeks without receiving indacaterol therapy and then for another period of 4 weeks while receiving 150 µg of inhaled indacaterol. Response to indacaterol. Maximum inspiratory capacity (A), FEV 1 (B), and 6MWT (C) improved significantly after 4 weeks of treatment with indacaterol. The CAT score (D) tended to decrease after treatment with indacaterol. Notes: Horizontal bars indicate the means. Statistical analysis was performed using the nonparametric Wilcoxon's rank test. Abbreviations: 6MWT, six-minute walk test; CAT, COPD Assessment Test; FEV 1 , forced expiratory volume in one second. submit your manuscript | www.dovepress.com Dovepress Dovepress sensor are useful for monitoring the therapeutic response in this group of patients. [/fig]
[fig] Figure 3: Effect of indacaterol on physical activity parameters. Number of steps (A), duration of moderate or more exercise (B), energy expenditure (C) and metabolic equivalent of task (D) improved significantly after treatment with indacaterol in all patients. Notes: Horizontal bars indicate the means. Statistical analysis was performed using the nonparametric Wilcoxon's rank test. submit your manuscript | www.dovepress.com Dovepress Dovepress International Journal of COPDPublish your work in this journalSubmit your manuscript here: http://www.dovepress.com/international-journal-of-copd-journal [/fig]
[table] Table 1: Baseline demographic data for patients with chronic obstructive pulmonary disease [/table]
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Risk Factors for Ebola Exposure in Health Care Workers in Boende, Tshuapa Province, Democratic Republic of the Congo
Background. Health care workers (HCW) are more likely to be exposed to Ebola virus (EBOV) during an outbreak compared to people in the general population due to close physical contact with patients and potential exposure to infectious fluids. However, not all will fall ill. Despite evidence of subclinical and paucisymptomatic Ebola virus disease (EVD), prevalence and associated risk factors remain unknown.Methods. We conducted a serosurvey among HCW in Boende, Tshuapa Province, Democratic Republic of Congo. Human anti-EBOV glycoprotein IgG titers were measured using a commercially available ELISA kit. We assessed associations between anti-EBOV IgG seroreactivity, defined as ≥2.5 units/mL, and risk factors using univariable and multivariable logistic regression. Sensitivity analyses explored a more conservative cutoff, >5 units/mL.Results. Overall, 22.5% of HCWs were seroreactive for EBOV. In multivariable analyses, using any form of personal protective equipment when interacting with a confirmed, probable, or suspect EVD case was negatively associated with seroreactivity (adjusted odds ratio, 0.23; 95% confidence interval, .07-.73).Discussion. Our results suggest high exposure to EBOV among HCWs and provide additional evidence for asymptomatic or minimally symptomatic EVD. Further studies should be conducted to determine the probability of onward transmission and if seroreactivity is associated with immunity.
Ebola virus disease (EVD) is a severe, often lethal disease that has led to substantial morbidity and mortality in sub-Saharan Africa. Case fatality rates range from 50% to 90%, depending on the species and have historically been highest for Ebola virus (EBOV). EBOV is considered to be a classic zoonosis and bats are the suspected reservoir, though this is unconfirmed. Since 2000, the number of outbreaks and cases of EVD have increased substantially across the continent, due in part to rapid human population growth and increased contact with wildlife host species in previously untouched forest environments [bib_ref] Impacts of environmental and socio-economic factors on emergence and epidemic potential of..., Redding [/bib_ref].
Since the first reported outbreaks of EVD in humans in 1976, nosocomial infections have been an important driver of transmission, particularly among health care workers (HCWs) [bib_ref] Ebola virus disease cases among health care workers not working in Ebola..., Matanock [/bib_ref] [bib_ref] Ebola hemorrhagic fever associated with novel virus strain, Uganda, Wamala [/bib_ref] [bib_ref] Ebola outbreak in Kikwit, Democratic Republic of the Congo: discovery and control..., Muyembe-Tamfum [/bib_ref]. Nosocomial infections can easily occur in the absence of stringent protective measures, as human-to-human EBOV transmission generally occurs through contact with body fluids of symptomatic or deceased individuals [bib_ref] Emergence of Zaire Ebola virus disease in Guinea, Baize [/bib_ref]. HCWs are an estimated 21 to 32 times more likely to be infected with EBOV during an outbreak compared to people in the general adult population, due to close physical contact with patients and potential exposure to infectious fluids. A survey conducted in and around Kikwit, in the Democratic Republic of the Congo (DRC), during the 1995 EBOV outbreak, found a 9% attack rate among HCWs [bib_ref] Serologic survey among hospital and health center workers during the Ebola hemorrhagic..., Tomori [/bib_ref]. During the 2014-2015 EVD outbreak in West Africa, at least 3% of EVD cases were among HCWs and of those, two-thirds died [bib_ref] Ebola outbreak in rural West Africa: epidemiology, clinical features and outcomes, Dallatomasina [/bib_ref]. The outbreak in North Kivu and Ituri in 2018-2020 resulted in 171 (5%) HCW infections, of which 44% died.
The difficulties associated with clinical recognition, lack of diagnostic capabilities, inadequate supply of personal protective equipment (PPE) such as gloves, gowns, and face shields, inadequate training, and poor public health infrastructure has led to challenges in the implementation of universal precautions to prevent exposure in resource-limited settings [bib_ref] Protecting health care workers from Ebola: personal protective equipment is critical but..., Fischer Wa 2nd [/bib_ref]. Moreover, the consequences of EVD among HCWs can be significant and can lead to increased viral spread, particularly in the early stages. Additionally, significant losses in the workforce can lead to closure of health facilities and loss of routine services [bib_ref] Ebola virus disease cases among health care workers not working in Ebola..., Matanock [/bib_ref].
Although HCWs have represented a significant portion of EVD cases in past outbreaks, not all HCWs fall ill, despite frequent exposure to infectious patients. There is evidence for asymptomatic or paucisymptomatic EVD, that is few or mild symptoms, although the extent to which this occurs is unknown and estimates vary widely, ranging from 1.0% to 45.8% depending on the population, sampling method, location, time, and assay [bib_ref] A systematic review and meta-analysis of seroprevalence surveys of ebolavirus infection, Bower [/bib_ref] [bib_ref] Serologic prevalence of Ebola virus in equatorial Africa, Steffen [/bib_ref]. A high prevalence of asymptomatic infection could have significant epidemiologic consequences, particularly if subclinical infections confer protective immunity [bib_ref] Ebola control: effect of asymptomatic infection and acquired immunity, Bellan [/bib_ref].
The DRC has experienced 11 documented EVD outbreaks since its discovery in 1976 and is currently experiencing an outbreak in the province of Equateur, which was announced on 1 June 2020. In July 2014, during the massive West African EVD outbreak, DRC's seventh EVD outbreak was confirmed near Boende, DRC. The etiologic agent was EBOV [bib_ref] Ebola virus disease in the Democratic Republic of Congo, Maganga [/bib_ref]. Between 26 July and 7 October 2014, a total of 68 EVD cases were reported (suspected, probably, and confirmed) and 38 were confirmed [bib_ref] Ebola virus disease in the Democratic Republic of the Congo, Rosello [/bib_ref]. Among the 68 cases, 11.6% (8) occurred in HCWs [bib_ref] Ebola virus disease in the Democratic Republic of Congo, Maganga [/bib_ref].
Following this outbreak, we conducted a serosurvey in November 2015 among HCWs providing care in Boende to improve our understanding of EBOV transmission dynamics. This study expands on previous work in a subset of this population to explore the seroprevalence EBOV using multiple assays [bib_ref] Serologic markers for ebolavirus among healthcare workers in the Democratic Republic of..., Hoff [/bib_ref]. We further investigated the occurrence of asymptomatic or paucisymptomatic forms of EVD and associated risk factors among HCW.
# Methods
## Study location
Detailed methods are described elsewhere [bib_ref] Serologic markers for ebolavirus among healthcare workers in the Democratic Republic of..., Hoff [/bib_ref]. The serosurvey was conducted in Boende town, Tshuapa province (formerly Équateur province), in the northwestern part of the DRC [fig_ref] Figure 1: Location of the serosurvey in Boende town, Tshuapa province, Democratic Republic of... [/fig_ref]. Boende has an estimated population of 45 000 and lies 70 km from Inkanamongo village, the location of the suspected index case, and 700 km northeast of Kinshasa, the capital city. Boende is surrounded by tropical rainforests and 2 large rivers.
## Study population
Individuals were invited to participate if they were 18 years or older and were providing care to the local population during the time of the study. Additionally, all participants were healthy, that is presenting with no fever or illness at the time of enrollment, and not previously diagnosed with EVD. HCWs included those involved in both clinical and nonclinical care at health facilities, as well as informal care givers such as traditional healers and pastors who may not work in a standard health facility location.
## Study procedures
All consenting individuals were interviewed and asked to provide a blood specimen by venipuncture in red-top vacutainer tubes (BD Biosciences) and undergo a basic physical assessment to obtain height, weight, and blood pressure measurements. A structured questionnaire was administered by trained interviewers in the participant's preferred local language (French or Lingala). Data were collected using Open Data Kit Collect on a standard android tablet [bib_ref] Open data kit: tools to build information services for developing regions, Hartung [/bib_ref]. Interviewers documented demographics and potential exposure to EBOV virus in the community, health care facility, and via animals. Participants were compensated for transportation costs to and from the study site. After processing blood samples, aliquots of serum were frozen and shipped to the Institut National de Recherche Biomedicale for serological testing. Results were not provided to participants. Ethics approval was obtained from University of California Los Angeles Fielding School of Public Health and the Kinshasa School of Public Health.
## Serological testing
Human anti-EBOV glycoprotein immunoglobulin G (GP IgG) titers were measured using a commercially available ELISA kit (Alpha Diagnostic International) following the manufacturer's protocol. The methodology has been described elsewhere [bib_ref] Serologic markers for ebolavirus among healthcare workers in the Democratic Republic of..., Hoff [/bib_ref] [bib_ref] Ebola virus neutralizing antibodies detectable in survivors of the Yambuku, Zaire outbreak..., Rimoin [/bib_ref]. Participants were considered seroreactive for this analysis if titers were greater than 2.5 units/mL. we chose a higher cutoff than the manufacturers cutoff of 1.0 units/ mL as other studies have suggested the utility of a more conservative cutoff [bib_ref] Minimally symptomatic infection in an Ebola 'hotspot': a cross-sectional serosurvey, Richardson [/bib_ref] [bib_ref] Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immunoassay requires..., Logue [/bib_ref].
## Statistical analyses
Basic descriptive statistics were calculated as frequencies and continuous variables were expressed as median and interquartile range (IQR). We assessed associations between anti-EBOV IgG seroreactivity, defined as ≥2.5 units/mL and risk factors using univariable logistic regression. Multivariable logistic regression analyses were used to identify community and occupational predictors for seroreactivity and were adjusted for age and sex. A 95% confidence interval (CI) that did not cross the null was considered to be evidence of an association. Variables of interest included receiving a blood transfusion, attending a funeral, having contact with human remains, participating in funeral rites, touching dead animals, traveling outside the province, frequenting markets, receiving an injection, visiting a health facility, receiving medication, and participating in active research or case finding activities. Occupational exposure data were gathered among HCWs who had interacted with confirmed, suspected, or probable Ebola cases. HCWs were classified based on their potential exposure to patients by reported occupation. Classifications were based on the World Health Organization (WHO) system of classification and have been described elsewhere [bib_ref] Serologic markers for ebolavirus among healthcare workers in the Democratic Republic of..., Hoff [/bib_ref] [bib_ref] Epidemiology of Ebola virus disease transmission among health care workers in Sierra..., Olu [/bib_ref]. Direct contact was defined as those with close contact to sick patients (eg, doctors, nurses, and traditional healers). Indirect contact was defined as contact with biological specimens, patient materials, or family members of sick patients (eg, laboratory technicians and room cleaners). Unlikely contact was defined as any position not directly related to clinical patient care (eg, hospital guards, administrators). We also considered use of PPE and hand washing as predictors for seroreactivity. Sensitivity analyses were explored using a more conservative definition of seroreactivity defined as ≥5 units/ mL. All statistical analyses were carried out using SAS software, version 9.4 (SAS Institute).
# Results
Among the 611 HCWs interviewed, 4 were EVD survivors and 25 had missing serologic data and were therefore excluded from the analysis. Demographics characteristics are presented in [fig_ref] Table 1: Sample Characteristics of 582 Health Care Workers from Boende Health Zone in... [/fig_ref]. When stratifying by current position, administrators had 2.42 times the odds of being seroreactive (95% CI, 1.03-5.69) and traditional healers/pastors had 3.14 times the odds of being seroreactive (95% CI, 1.58-6.25) compared to nurses.
All community and occupational exposures were age-and sex-adjusted. Using any form of PPE when interacting with a confirmed, suspected, or probable EVD patient was negatively associated with seroreactivity (adjusted odds ratio [aOR], 0.23; 95% CI, .07-.73) [fig_ref] Table 3: Adjusted Odds Ratios of Seroreactivity [/fig_ref]. When stratifying by type, only a face mask (aOR, 0.29; 95% CI, .09-.94) and gloves (aOR, 0.23; 95% CI, .07-.73) were associated with a decrease in odds of seroreactivity.
In the sensitivity analyses, similar trends were observed when a more conservative cutoff for seroreactivity (≥5 units/mL) was used. Results are presented in [fig_ref] Table 1: Sample Characteristics of 582 Health Care Workers from Boende Health Zone in... [/fig_ref].
# Discussion
Our results suggest high exposure to EBOV among HCWs in Boende without a history of diagnosed EVD. Using a ≥2.5 units/ mL cutoff, 22.5% of surveyed participants were seroreactive, which is consistent with other studies [bib_ref] Minimally symptomatic infection in an Ebola 'hotspot': a cross-sectional serosurvey, Richardson [/bib_ref] [bib_ref] The serology of ebolavirus-a wider geographical range, a wider genus of viruses..., Formella [/bib_ref] [bib_ref] High prevalence of IgG antibodies to Ebola virus in the Efe pygmy..., Mulangu [/bib_ref]. A 2016 meta-analysis estimated proportions of asymptomatic Ebola infections at 27.1% (95% CI, 14.5%-39.6%) [bib_ref] Transmissibility and pathogenicity of Ebola virus: a systematic review and meta-analysis of..., Dean [/bib_ref]. Individual serostudies in areas surrounding EVD outbreaks commonly find seroprevalence of Ebola antibodies in individuals who have no history of EVD diagnosis. In 1976, in Sudan, the WHO found that 19% of contacts of individuals with EVD had antibodies to the virus. Estimates from other studies are lower; for example, a serologic survey conducted during the 1995 EVD outbreak in Kikwit found an average seroprevalence of 9.3% in surrounding villages [bib_ref] Prevalence of IgG antibodies to Ebola virus in individuals during an Ebola..., Busico [/bib_ref]. In addition to areas surrounding EVD outbreaks, this phenomenon has also been observed in areas with no record of EVD cases. A serostudy in the Sankuru province in DRC found that 11% of the study population was seropositive for EBOV despite no known outbreaks having occurred in the area [bib_ref] Serologic evidence of ebolavirus infection in a population with no history of..., Mulangu [/bib_ref]. Finally, a study in the northeastern region of the DRC reported an EBOV seroprevalence of 18.7% in the indigenous Efe (pygmy) population [bib_ref] High prevalence of IgG antibodies to Ebola virus in the Efe pygmy..., Mulangu [/bib_ref]. Collectively, despite varying study methods and serological tests, the results imply high seroprevalence of EBOV antibodies among individuals with no recollection of an EVD-like illness in various parts of the DRC and sub-Saharan Africa.
We would expect to see higher rates of exposure in an HCW population such as the one surveyed here when compared to the general population. HCWs are considered at higher risk of EVD due to their close contact with patients and many in our sample reported involvement in the 2014 EVD response [bib_ref] Serologic markers for ebolavirus among healthcare workers in the Democratic Republic of..., Hoff [/bib_ref] [bib_ref] We are despised in the hospitals': sex workers' experiences of accessing health..., Scorgie [/bib_ref]. While it is likely that some of the participants were exposed to EBOV while working during the outbreak, we cannot confirm when and where exposure may have occurred. It is possible that exposure could have happened outside of the workplace during the 2014 outbreak or be unrelated to the 2014 EVD outbreak. Other studies suggest that filoviruses may circulate in the environment without any severe clinical manifestations [bib_ref] Human asymptomatic Ebola infection and strong inflammatory response, Leroy [/bib_ref] [bib_ref] Ebola and Marburg virus antibody prevalence in selected populations of the Central..., Gonzalez [/bib_ref] [bib_ref] Risk factors associated with Ebola and Marburg viruses seroprevalence in blood donors..., Moyen [/bib_ref]. Boende and the surrounding areas are rural and the population mostly subsists on hunting and gathering. These characteristics make Boende at risk for zoonotic spillover of EBOV, which may have contributed to our observed seroreactivity outside of the official 2014 outbreak. Additionally, we cannot rule out the possibility of the presence of an unidentified EBOV strain with low pathogenicity and cross-reactivity with our antibody assay. However, serologic response to other EBOV proteins, including nucleoprotein, viral protein 40, and EBOV GP-bearing human immunodeficiency virus pseudotype viruses was documented in a subset of this population. Approximately 11.7% of the population was considered reactive to more than one test, and 2.8% demonstrated pseudoneutralization ability [bib_ref] Serologic markers for ebolavirus among healthcare workers in the Democratic Republic of..., Hoff [/bib_ref]. Outside of the infection, it is possible that seroreactivity was generated through low viral inocula, inactivated virus, isolated viral antigens in the workplace, or while gathering or consuming bushmeat or other foods [bib_ref] Human exposure to wild animals in the Sankuru Province of the Democratic..., Rimoin [/bib_ref].
It is impossible to determine if these HCWs were asymptomatic or paucisymtomatic. The initial symptoms associated with EVD are nonspecific and mirror common diseases in DRC, such as typhoid and malaria, thus may not initially be recognizable as EVD. It is possible that EBOV could be transmissible to others from individuals with mild symptoms, although this may be rare [bib_ref] Transmission of Ebola hemorrhagic fever: a study of risk factors in family..., Dowell [/bib_ref] [bib_ref] Transmission of Ebola viruses: what we know and what we do not..., Osterholm [/bib_ref]. Our findings do not indicate if exposure is associated with immunity. This analysis, along with further research on the association between seroreactivity and immunity, could identify HCWs who are naturally immune to EVD. During future outbreaks, HCWs could act as caregivers in Ebola treatment centers and may reduce the stigma associated with utilizing survivors. Additionally, a safe and effective Ebola vaccine is now licensed and will likely be considered for preventative use in high-risk populations such as HCWs [bib_ref] Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease:..., Henao-Restrepo [/bib_ref]. As with other vaccine-preventable diseases, understanding preexisting immunity and the proportion of the population at risk will be critical to determine what vaccine coverage rates are needed for effective outbreak prevention and control.
While the HCWs in our study, as a whole, showed high exposure to EBOV, specific subgroups were associated with higher odds of EBOV seroreactivity. Being a traditional healer or pastor was associated with increased odds of seroreactivity compared to nurses. Traditional medicine is common throughout sub-Saharan African and in the DRC, with the number of traditional healers surpassing doctors and nurses in many rural areas [bib_ref] Integrating traditional healers into the health care system: challenges and opportunities in..., Krah [/bib_ref] [bib_ref] Trends and challenges of traditional medicine in Africa, Abdullahi [/bib_ref] [bib_ref] Who are the real community health workers in Tshopo Province, Democratic Republic..., Dalglish [/bib_ref]. Thus, in many settings, traditional healers are likely to be the first source of treatment when someone falls ill, but often do not have PPE or other medical resources to treat EVD patients safely, leading to exposure [bib_ref] The impact of traditional and religious practices on the spread of Ebola..., Manguvo [/bib_ref]. Additionally, those in administrative roles are also associated with increased odds of seroreactivity compared to nurses. We hypothesize that these individuals might come in contact with EVD patients unknowingly, as patients at health facilities seeking care, without wearing proper PPE or following consistent hand washing practices. Alternatively, this increase could be explained by a community-level exposure independent of their profession. Not surprisingly, those who used any PPE showed significantly reduced odds of seroreactivity in the multivariable model. Furthermore, both use of a facemask and gloves were independently associated with lower odds of seroreactivity in the multivariable model.
Demographic risk factors for EBOV infection and EVD, such as age, sex, and ethnicity, are not well characterized [bib_ref] Ebola virus disease, Jacob [/bib_ref]. While age was not a significant predictor of seroreactivity, being female was associated with lower odds compared to being male, a pattern that has been documented previously [bib_ref] Serologic evidence of ebolavirus infection in a population with no history of..., Mulangu [/bib_ref]. This might be explained by a male dominated workforce and therefore an increased risk of exposure to EBOV in this nontraditional population.
Our study is subject to a number of limitations. This is a cross-sectional study targeting a specific population and therefore the results may not be generalizable to the general population. We attempted to enroll all HCWs in Boende health zone; however, without a comprehensive list of HCWs, it was impossible to determine exact participation rates in our study. Additionally, we interviewed study participants a year after the outbreak, and so there is a possibility of misclassification due to recall inaccuracies, particularly for questions on exposure during the outbreak. However, we would expect misclassification to be nondifferential and unrelated to serologic results. We note that a small number of participants suspected that they had become infected with EVD, although none were tested, and only 7 of these 28 were seroreactive. Exclusion of these individuals in sensitivity analyses had no effect on our results.
To date, there is no gold standard serologic test for EBOV and no assay has been approved by the Food and Drug Administration. Previous work has shown that seroreactivity is difficult to define and serologic tests may often overestimate seroreactivity, thus we must be cautious of the interpretations [bib_ref] Serologic prevalence of Ebola virus in equatorial Africa, Steffen [/bib_ref] [bib_ref] Serologic markers for ebolavirus among healthcare workers in the Democratic Republic of..., Hoff [/bib_ref]. We used a commercially available test and considered various cutoffs for seroreactivity. While we choose to use a higher cutoff than the manufacturer's suggestions (1.0 units/ mL), other studies suggest a more conservative cutoff may be warranted due to the potential for cross-reactivity and high background [bib_ref] Minimally symptomatic infection in an Ebola 'hotspot': a cross-sectional serosurvey, Richardson [/bib_ref] [bib_ref] Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immunoassay requires..., Logue [/bib_ref]. We conducted sensitivity analyses with a higher cutoff to account for these concerns and overall seroprevalence in this population was reduced to 6%. Similar associations were observed and are presented in [fig_ref] Table 1: Sample Characteristics of 582 Health Care Workers from Boende Health Zone in... [/fig_ref] Our results provide additional evidence for asymptomatic or paucisymptomatic EVD in the DRC and sub-Saharan Africa. Further studies to distinguish between asymptomatic or paucisymptomatic infections should be conducted along with studies to determine whether these individuals are infectious and if seroreactivity is associated with immunity. Additionally, more detailed analyses looking at additional risk factors should be considered. Regardless, high EBOV seroreactivity in HCWs underscores the importance of appropriate infection prevention control practices and education in health care settings to prevent nosocomial disease transmission of Ebola and other communicable diseases.
[fig] Figure 1: Location of the serosurvey in Boende town, Tshuapa province, Democratic Republic of the Congo. [/fig]
[table] Table 1: Sample Characteristics of 582 Health Care Workers from Boende Health Zone in the Democratic Republic of the Congo, November 2015 The other category comprised physicians, epidemiologists, communication specialists, technicians, students, and maintenance workers.Fifty-two participants had not been present for an Ebola outbreak and were not eligible for the question. [/table]
[table] Table 3: Adjusted Odds Ratios of Seroreactivity (GP > 2.5) to Ebola GP by Possible Community Exposures to Ebolavirus Among 530 Health Care Workers in Boende Health Zone in the Democratic Republic of the Congo Who Have Been Present at an Ebola Outbreak, November 2015 Adjusted for age and sex. b n = 90, the number of HCW who had contact with a confirmed, suspected, or probable EVD case. [/table]
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Improving object detection quality with structural constraints
Recent researches revealed object detection networks using the simple "classification loss + localization loss" training objective are not effectively optimized in many cases, while providing additional constraints on network features could effectively improve object detection quality. Specifically, some works used constraints on training sample relations to successfully learn discriminative network features. Based on these observations, we propose Structural Constraint for improving object detection quality. Structural constraint supervises feature learning in classification and localization network branches with Fisher Loss and Equi-proportion Loss respectively, by requiring feature similarities of training sample pairs to be consistent with corresponding ground truth label similarities. Structural constraint could be applied to all object detection network architectures with the assist of our Proxy Feature design. Our experiment results showed that structural constraint mechanism is able to optimize object class instances' distribution in network feature space, and consequently detection results. Evaluations on MSCOCO2017 and KITTI datasets showed that our structural constraint mechanism is able to assist baseline networks to outperform modern counterpart detectors in terms of object detection quality.OPEN ACCESSCitation: Rong Z, Wang S, Kong D, Yin B (2022) Improving object detection quality with structural constraints. PLoS ONE 17(5): e0267863. https://
# Introduction
Object detection is a fundamental computer vision technology with a broad range of application scenarios, such as autonomous driving. It's a compound task of object classification and localization. Modern object detectors are trained by matching their detection results with ground truth labels, and then minimizing the loss measuring the differences of these label-prediction matches. Each match's loss is constituted with two terms, measuring classification error and localization error respectively. The complete loss is the sum of the two terms of all matches. In such a loss, each detection result is evaluated independently and only required to fit to the matched ground truth label. Though this loss form is simple, recent researches revealed that object detection networks could not be effectively trained by directly minimizing such a loss in many cases [bib_ref] Dollá r P. Focal Loss for Dense Object Detection, Lin [/bib_ref] , while some researches showed that object detection quality could be effectively improved with additional constraints on intermediate network features. Specifically, recent researches on network-based clustering [bib_ref] Deep Self-Evolution Clustering, Chang [/bib_ref] showed that feature learning could be effectively guided for the benefit of the main task, under constraints on mutual relations between training samples. This indicates it's possible to optimize object class distributions in network feature space for the benefit of object recognition. Thus, it's reasonable to expect object detection quality improvement by complementing the basic loss form of modern detectors with additional constraints on training sample relations in intermediate feature space. This work presents a training-sample-relation-based constraint on object detection network training for improving detection quality. We name these Structural Constraints, because these constraints exert influence on the structure of training sample distribution in object detection network feature space (as is shown later in . Structural constraints append two terms to the basic loss, Fisher Loss and Equi-proportion Loss, for constraining the relations of training samples in classification branch space and localization branch space respectively. For an arbitrary pair of training samples, Fisher loss measures the difference between pairwise sample feature similarity and pairwise classification target similarity, while equi-proportion loss measures the difference between pairwise sample feature similarity and pairwise localization target similarity. Thus, under the constraint of these two terms, training sample feature distributions in classification branch and localization branch more resemble ground truth label distributions. As a result, features of these network branches could be more easily transformed to accurate detection results. Structural constraints could be applied to object detection networks of various architectures, like single-stage, two-stage and multi-stage networks, without changing the original network structures or influencing detection rates. In our experiments, we evaluated structural constraints' effect on representative object detection networks of various architectures on different image datasets. These experiment results demonstrated that structural constraints could improve object detection quality noticeably on a broad range of detectors.
To summarize, novel contributions of this work are:
- proposing Fisher loss function as part of structural constraints to constrain training sample feature relations for improving classification performance of object detection networks;
- proposing equi-proportion loss function as part of structural constraints to constrain training sample feature relations for improving localization performance;
- a mechanism for applying structural constraints to various object detection network architectures.
The rest of this paper is organized as follows: Section 2 reviews related works, Section 3 describes in detail structural constraint and the mechanism of applying it to networks, Section 4 presents our experiment results and analysis, and finally Section 5 concludes this work.
# Related works
In this section, we review some previous works closely related to structural constraints proposed in this work, and we confine the scope to works based on neural networks. At first, we review some deep learning models for image recognition with feature learning constraints; then, we review representative object detection networks of various architectures.
## Feature learning constraints
Feature learning constraints are widely adopted in deep-learning-based image recognition domain. Some works on object detection use feature learning constraints to improve object detection quality. RIFD-CNNused two types of constraints on its network's intermediate layer features, one for rotation invariance and one for Fisher discrimination. Its rotation invariance constraint requires the intermediate feature representation of each training sample image to be similar to the average intermediate feature representation of the rotated versions of the image, so the subsequent classification based on this type of features will be robust against influence of object rotation. Its Fisher discrimination constraint requires each class's training sample intermediate features to lie close to the mean of the class, and each class's mean feature to lie distant from the global mean of all classes, so the subsequent classification layer could easily and accurately separate the classes from each other. Using these two constraints, RIFD-CNN achieved significant object detection accuracy improvement.
DETRis another object detection network using feature learning constraints. DETR uses transformer to process feature maps from its backbone into detection results. Its transformer's encoder consists of multiple layers of attention mechanism, and the detection results are produced by the last attention mechanism layer. However, other attention mechanism layers' intermediate features are also required to be transformed into accurate detection results through the same detection head shared with the last layer. This deep supervision is in essence a type of feature learning constraint: the supervision on the intermediate attention mechanism layers constrains their output features to facilitate the subsequent inference for better detection accuracy.
Feature learning constraints have also been used to solve image clustering problems. Deep Self-evolution Clustering (DSEC) [bib_ref] Deep Self-Evolution Clustering, Chang [/bib_ref] network and Deep Adaptive Clustering (DAC) [bib_ref] Deep Adaptive Image Clustering, Chang [/bib_ref] network constrain their output features' pairwise relationships to make these features directly express cluster identities. These clustering networks' constraints require the dot products of aribitrary pairs of output features to be close to corresponding pseudo labels. These pseudo labels reflect cosine similarities of the feature pairs. As a result, the training of these networks under this type of constraints gradually makes the output features to be one-hot vectors which express cluster identities directly. Factually, this type of constraints on pairwise feature relationships are the only content in these two clustering networks' training objective functions.
Compared with the feature learning constraints in the works described above, structural constraints in this work exhibit both similarity and difference. Like RIFD-CNN, structural constraints are applied over intermediate layer features of object detection networks; like DSEC and DAC, structural constraints are based on pairwise training sample feature relations. However, the combination of these two characteristics is absent in all these works. Besides, as constraints for object detection networks, RIFD-CNN's constraints are applied for classification merely, while structural constraints are applied for both classification and localization. Furthermore, RIFD-CNN did not constrain inter-class training sample relations, while our structural constraints' Fisher loss constrains both inter-and intra-class relations over all training sample pairs.
## Object detection network architectures
Until now, object detection networks exhibited two types of architectures: networks generating detections in a single stage, and networks generating detections through several stages of refinements. We review these architectures below.
2.2.1 Single-stage object detection networks. Single-stage object detection networks transform input images' backbone feature maps into detection results directly, through a single detection head. SSD [bib_ref] Single Shot MultiBox Detector, Liu [/bib_ref] is the forerunner of this architecture. It scatters boxes of various sizes and aspect ratios over input images' feature maps and infers classes and adjustments for these boxes to form detection results. Detection results across feature map pyramid levels are synthesized to infer final detection results. The boxes initially scattered are then known as anchors.
YOLO [bib_ref] You Only Look Once: Unified, Real-Time Object Detection, Redmon [/bib_ref] is another single-stage detection network that is fast at inference. It additionally infers a confidence value for each bounding box, which represents probability of existence of objects within the bounding box, and these confidence values participate in the decision of final detection results. However, YOLO's detection quality is not satisfying.
RetinaNet [bib_ref] Dollá r P. Focal Loss for Dense Object Detection, Lin [/bib_ref] is a high-detection-quality single-stage object detection network. It focuses on dealing with imbalance between foreground and background training samples, which is a crucial cause of poor detection quality of many other single-stage networks. It proposes Focal Loss to replace the widely adopted cross entropy loss for classification task. By using focal loss, RetinaNet is able to allocate more weights on poorly classified hard samples during training, and make the trained network better generalize to test data.
2.2.2 Object detection networks with several stages. Another kind of object detection networks are constituted with more than one stage. These networks could be further divided into two groups according to number of network stages: two-stage networks and multi-stage (more than two) networks. The first network stage of all these object detection networks are responsible for generating region proposals, also known as RoIs (regions of interest). Twostage networks then refine the region proposals with a detection head to produce final detection results, while multi-stage networks refine the region proposals with several detection heads in sequence. We review representatives of these architectures below.
Two-stage object detection networks. Two-stage object detection networks appeared early among all architectures, and usually produce better detection quality than single-stage networks. Faster RCNN [bib_ref] Towards Real-Time Object Detection with Region Proposal Networks, Ren [/bib_ref] is the forerunner of this architecture. Faster RCNN introduced RPN (Region Proposal Network) upon the basis of Fast RCNN. RPN takes backbone feature maps as input and inferences RoIs and corresponding confidence values. These RoIs are then used to extract features from backbone feature maps through RoI pooling operation, and these features are passed into a fully connected detection head to inference detection results.
R-FCN [bib_ref] Object Detection via Region-based Fully Convolutional Networks, Dai [/bib_ref] focuses on accelerating inference rate of Faster RCNN by reducing redundant computation of detection head. R-FCN's detection head is constituted with convolutional layers, and is able to generate a special feature map of which different channels are sensitive to different parts of target objects. Then, RoI pooling over this feature map could easily decide whether an RoI accurately localizes an object and corresponding class, by filling RoI parts with features from corresponding channels. Since most necessary computation is done by the convolutional detection head and the remaining RoI pooling operations cost only subtle computation, R-FCN's inference is time-efficient.
Double-head RCNN [bib_ref] Rethinking Classification and Localization in R-CNN, Wu [/bib_ref] is another two-stage network whose second stage is composed of two detection heads in parallel, one fully connected head and one convolutional head. This design is based on the observation that fully connected layers are sensitive to spatial completeness of objects, while convolutional layers are robust against occlusion and deformation. Thus Double-head RCNN uses its fully connected head to infer classification scores which should reflect localization quality, and uses its convolutional head to infer bounding boxes to better generalize to various object appearances and influencing contents.
Multi-stage object detection networks. Multi-stage object detection networks extend twostage architecture by appending additional detection heads, refining RoIs with more stages of inferences. Cascade RCNN [bib_ref] IEEE Conference on Computer Vision and Pattern Recognition, Cai [/bib_ref] is a typical multi-stage object detection network. During Cascade RCNN training, each stage's detection head is trained from detection results of its previous stage. At inference, each stage's detection head takes features from RoI pooling based on its previous stage's detection boxes, and generates new detection results. The final detection results take the last stage's detection head's output boxes as localization results, and take the averages of all detection heads' class scores as classification results. The increased network stages improved detection quality noticeably, making Cascade RCNN one of the most accurate object detectors by then.
Hybrid Task Cascade [bib_ref] Hybrid Task Cascade for Instance Segmentation, Chen [/bib_ref] is a multi-stage network capable of both object detection and instance segmentation. Hybrid Task Cascade inherited the network structure of Cascade RCNN, and introduced additional components and links. It introduced a semantic segmentation convolutional branch to provide helpful inputs to its detection heads and mask heads. The detection quality of Hybrid Task Cascade is outstanding in multi-stage group, but the whole of its network is cumbersome.
All representative object detection networks mentioned above and many others lack constraints on relationships between training samples in feature spaces, so structural constraints proposed in this work are able to complement them in this respect. We will show that structural constraints are applicable to all these architectures through a unified mechanism in next section.
## Structural constraint mechanism
In this section, we describe sturctural constraint mechanism for object detection in detail. Firstly, we explain the motivation of structural constraints. Then, we present the definition of structural constraints. After these, we describe the mechanism of combining structural constraints with object detection networks.
## Motivation
The reason of we proposing structural constraints is based on two observations: first, the lack of constraints on training sample relationships in modern object detection networks; second, the importance of feature learning exhibited in many other image recognition tasks. As described in Section 1, it could be observed that most modern object detection networks' loss functions usually have a form like this:
[formula] X i L cls ðp i ; p gt i Þ þ L loc ðb i ; b gt i Þð1Þ [/formula]
where L cls and L loc are two loss terms for measuring classification error and localization error respectively. For each match, the difference between the estimated class probability vector p i and the corresponding ground truth vector p gt i is calculated by L cls , and the difference between the estimated bounding box b i and the corresponding ground truth box b gt i is measured by L loc . Loss functions like this only force each detection result to fit to its matched ground truth. They are simple in form, but could not be effectively minimized in many cases, since the supervision on object classification could be severely influenced by large amount of background training samples [bib_ref] Dollá r P. Focal Loss for Dense Object Detection, Lin [/bib_ref]. We observed that additional supervision on one training sample could come from the other training samples, since one training sample could be represented by its relative differences from the others. This could be understood by looking at some works on image clustering, such as DSEC [bib_ref] Deep Self-Evolution Clustering, Chang [/bib_ref] , where the clustering network was effectively trained under the supervision on similarity of each pair of training samples. Thus, structural constraints are designed to supervise the differences between each pair of sample detections. Because of that object detection consists of classification and localization, structural constraints use two types of loss functions to measure sample pairs' classification differences and localization differences, namely Fisher loss and equi-proportion loss.
We also observed that proper supervision on object detection networks' intermediate features could effectively improve detection quality. Examples are RIFD-CNN's rotation invariance constraint and Fisher discrimination constraint on its backbone's intermediate layers, and DETR's auxiliary supervisions on multiple levels of transformer decoders. Apart from this, we try to avoid disrupting optimization of the main objective in Eq (1). Thus, instead of being applied over object detection networks' final outputs, structural constraints are applied over the networks' intermediate features to guide the feature learning.
## Definition
Structural constraints take training samples' intermediate features as input. To supervise training samples' relations during classification and localization, structural constraints use Fisher loss and equi-proportion loss to constrain pairwise feature differences respectively. These losses in structural constraints and the basic object detection objective in Eq (1) altogether form the new training objective.
Fisher loss in structural constraints calculates the similarity between an arbitrary pair of intermediate features of training samples, and supervises this with the corresponding pair of class labels' similarity. It's expressed as:
[formula] L Fisher ðf i ; f j ; p gt i ; p gt j Þ ¼ ½sðf i Þ � sðf j Þ À p gt i � p gt j � 2ð2Þ [/formula]
where σ(�) is sigmoid function, f i is a transformed intermediate feature vector of training sample i, and p gt i 2 ½0; 1� C is the corresponding one-hot class label, with C being the number of object classes. Fisher loss L Fisher calculates the similarity between f i and f j , and the similarity between p gt i and p gt j , both in terms of dot production. The squared difference between these two similarities is used as the loss value. To make the comparison between these similarities fair, f i is obtained by linearly transforming the intermediate feature into the same dimensionality as p gt
i . Since f i acts as a proxy of the intermediate feature, we name it Proxy Feature. Before calculating the similarity, the proxy feature vectors' elements are transformed by σ(�) into the same range [0, 1] as p gt i . By supervising the similarity between proxy feature vectors, Fisher loss drives the similarity between the underlying intermediate features to be consistent with the similarity of the corresponding class labels. As a result, Fisher loss produces the effect of reducing intra-class variance and increasing inter-class separation of training sample distribution, which benefits object classification.
Equi-proportion loss is another loss term in structural constraints. It also measures the similarity between an arbitrary pair of intermediate features, but supervises this with the corresponding pair of localization labels. It's expressed as:
[formula] L equip ðf 0 i ; f 0 j ; b gt i ; b gt j Þ ¼ ksð sðf 0 i Þ sðf 0 j Þ Þ À sð sðb gt i Þ sðb gt j Þ Þk 2ð3Þ [/formula]
where f 0 i is proxy feature of training sample i, and b gt i 2 R 4 is the corresponding localization label. f 0 i is linearly transformed from the intermediate feature into same dimensionality as b gt i , to facilitate the comparison between training sample difference and localization label difference. Since b gt i ; b gt j are not bounded, we measure their relative difference in terms of elementwise ratios, and so is the difference between f 0 i and f 0 j measured. The squared magnitude of the difference between these two sets of ratios is used as the value of L equip . Under the guidance of equi-proportion loss, the intermediate features of training samples tend to be sensitive enough to reflect the differences between their localization labels, and benefit bounding box regression.
After applying structural constraint constituted with Fisher loss and equi-proportion loss, the object detection network training objective is rewritten as:
[formula] X i ½L cls ðp i ; p gt i Þ þ L loc ðb i ; b gt i Þ� þ X i;j ½L Fisher ðf i ; f j ; p gt i ; p gt j Þ þ L equip ðf 0 i ; f 0 j ; b gt i ; b gt j Þ�ð4Þ [/formula]
where Fisher loss and equi-proportion loss are evaluated for all pairs of training samples. This sum of original loss and structural constraint terms is used to calculate back-propagations during end-to-end object detection network training processes. Thus, training with this new objective not only optimizes the main objective of object detection, but also optimizes the structure of training sample distribution in intermediate feature space which benefits the main objective in return.
## Combination with various object detection architectures
Structural constraints supervise intermediate features of object detection networks, that is, applied over intermediate network layers, so how they are combined with networks depends on the forms of these layers, which differ among object detection architectures. We describe how structural constraints are combined with single-stage, two-stage and multi-stage object detection networks respectively below. Single-stage case. Single-stage object detection networks' detection heads transform backbone feature maps with two-dimensional convolution (Conv2D) to generate classification outputs and localization outputs. Because that the dimensionality of proxy features used in Fisher loss and equi-proportion loss calculation must be unified with the dimensionality of classification outputs and localization outputs respectively, structural constraints in singlestage networks use Conv2D layers to transform intermediate features of training samples into the needed proxy features. This could be expressed as:
[formula] ff i g i ¼ Conv2D Fisher ðFÞ ff 0 i g i ¼ Conv2D equip ðFÞð5Þ [/formula]
where Conv2D Fisher and Conv2D equip are convolution layers generating proxy features for Fisher loss and equi-proportion loss respectively, and F is intermediate feature collection. Con-v2D Fisher and Conv2D equip take F as input and generate proxy feature collections ff i g i ; ff 0 i g i . It should be noticed that F, {f i } i and ff 0 i g i take the form of feature tensors in this case. With proxy features obtained, the rest of structural constraint evaluation is exactly same as the description in Section 3.2. The complete mechanism in single-stage case is illustrated in [fig_ref] Fig 1: Illustration of structural constraint mechanisms in object detection networks of various architectures [/fig_ref] Two-stage case. Two-stage object detection networks firstly generate RoIs with their RPNs, and then their detection heads infer detection results from these RoIs. Their detection heads usually consist of fully-connected (FC) layers. Thus, for the same reason as in singlestage case, we set up special FC layers for transforming intermediate features into proxy features whose dimensionality is unified with detection head outputs. This could be expressed as:
[formula] ff i g i ¼ FC Fisher ðFÞ ff 0 i g i ¼ FC equip ðFÞð6Þ [/formula]
where FC Fisher and FC equip are the FC layers that generate proxy features for Fisher loss and equi-proportion loss respectively. In this case, the intermediate feature collection F comes from RoI pooling. The rest of structural constraint evaluation is still same as the description in Section 3.2. Apart from the detection heads, structural constraints could also be applied to RPNs of two-stage networks, because these RPNs are identical to single-stage networks' detection heads. This means the aforementioned mechanism for single-stage case could be directly applied to these RPNs. The complete structural constraint mechanism for two-stage case is illustrated in [fig_ref] Fig 1: Illustration of structural constraint mechanisms in object detection networks of various architectures [/fig_ref] Multi-stage case. Multi-stage object detection networks extend two-stage architecture by using multiple detection heads to refine detection results sequentially. Thus, compared with two-stage networks, the constituting modules of multi-stage networks remain unchanged. This means how structural constraints are applied to detection heads and RPNs in multi-stage networks is exactly same as the two-stage case. For structural constraints on detection heads, the proxy features are generated in the same manner as Eq (6); on RPNs, they are generated in the same manner as Eq [bib_ref] Deep Adaptive Image Clustering, Chang [/bib_ref]. All the rest of structural constraint evaluation still obey Section 3.2. The complete mechanism for multi-stage case is illustrated in [fig_ref] Fig 1: Illustration of structural constraint mechanisms in object detection networks of various architectures [/fig_ref] In all cases above, structural constraint mechanisms exist during the training period of these object detection networks, and guide the intermediate feature learning by handling proxy features. At inference time, all calculations related to structural constraints are absent, as well as all exclusive network layers (Conv2D Fisher/equip , FC Fisher/equip ), so detection rates and deployment sizes of these networks are not influenced.
## Plos one
## Experiments
To verify the effectiveness of structural constraints, we experimented with multiple object detection networks over several image datasets, and examined the training processes and network behaviors. In this section, we present these experiment results.
## Experiment settings
We describe settings of the experiments firstly. These include settings of networks, training and testing. All hyper-parameters listed below are set to default values of MMDetectionconfiguration files.
Networks. The default settings of object detection networks used in the experiments are: ResNet-101 [bib_ref] Deep Residual Learning for Image Recognition, He [/bib_ref] as backbone, FPN [bib_ref] Feature Pyramid Networks for Object Detection, Lin [/bib_ref] as neck, and Greedy NMS [bib_ref] Object Detection with Discriminatively Trained Part-Based Models, Felzenszwalb [/bib_ref] for post-processing. All multi-stage networks use 3 stages of detection heads. All object detection networks are implemented with MMDetection toolboxand PyTorch deep learning library [bib_ref] PyTorch: An Imperative Style, High-Performance Deep Learning Library, Paszke [/bib_ref].
# Experiment results
We present experiment results on structural constraint mechanism in this subsection. Firstly, we present ablation evaluation results to show influences of different factors in the mechanism. Then, we compare object detection quality of our structural-constraint-applied networks with other modern detectors. Finally, we analyze behaviors of structural constraint mechanism through visualization.
## Ablation evaluation.
We performed ablation evaluations on structural constraint mechanism to investigate different factors' influences on object detection quality, including the constituting loss terms L Fisher and L equip as well as different combination manners. We report our evaluation results on two widely used image datasets, MSCOCO2017 [bib_ref] Microsoft COCO: Common Objects in Context, Lin [/bib_ref] and KITTI [bib_ref] Are we ready for Autonomous Driving? The KITTI Vision Benchmark Suite, Geiger [/bib_ref] , respectively.
Evaluations on MSCOCO2017. For ablation on MSCOCO2017, all object detection networks are trained over the train2017 subset, and tested over the val2017 subset. We choose RetinaNet as the evaluation subject for single-stage architecture, Faster RCNN for twostage, and Cascade RCNN for multi-stage. The ablation evaluation results are shown in [fig_ref] Table 1: Ablation evaluations of structural constraint mechanism on MSCOCO2017 [/fig_ref]. The network names containing "+L Fisher/equip " indicate that Fisher loss or equi-proportion loss is applied to the detection heads of those networks, and names with "þL Fisher =equip 2 " indicate Fisher loss or equi-proportion loss is applied to both the detection heads and RPNs of those networks (in two-or multi-stage case). It could be observed that structural constraint mechanism is able to improve object detection qualities of all network subjects on this general object detection task. Specifically, the complete structural constraint mechanism that includes both Fisher loss and equi-proportion loss produced the most obvious improvement in some cases, like Faster RCNN þ L 2 Fisher þ L 2 equip . We also evaluated the influence of batch size, and the networks marked with " � " are trained with smaller batch sizes (half). It could be observed that structural constraint mechanism is robust against batch size changes.
Evaluations on KITTI. We use the 2D object detection subset in KITTI to perform ablation evaluations, which contains 7481 labeled driver-view images. For all evaluated network subjects, the first 6000 images are used for training and the rest 1481 images for testing. We adopted Pascal-VOC-styled metrics which evaluate class-wise average precisions and the global mean average precision (MAP). We choose RetinaNet and SSD as evaluation subjects for single-stage architecture, Faster RCNN for two-stage, and Cascade RCNN for multi-stage. The evaluation results are shown in [fig_ref] Table 2: Ablation evaluations of structural constraint mechanism on KITTI [/fig_ref]. It could be observed that structural constraint mechanism is able to produce object detection quality improvement for all these network architectures. It's also observable that the improvement happened on multiple classes simultaneously, such as the case of Faster RCNN þ L 2
Fisher . Besides, structural constraint mechanism still exhibits robustness against batch size settings, which could be observed from the evaluations on Cascade RCNN.
## Comparison with other object detectors.
We present object detection quality comparisons between modern object detectors and our networks with structural constraints in this subsection. These comparisons were carried out over MSCOCO2017 and KITTI. We give descriptions respectively in the following.
Comparison on MSCOCO2017. The training set and testing set for this comparison are same as the settings in last subsection. The evaluation results are presented in . SCM-Two and SCM-Multi are our two-stage and multi-stage object detection networks with structural constraint mechanisms. SCM-Two is configured as Faster RCNN þ L 2 Fisher þ L 2 equip , and SCM-Multi as Cascade RCNN þ L 2
Fisher . SSD300 and SSD512 are SSD networks with input image sizes as 300 × 300 and 512 × 512 respectively. It could be observed that our SCM-Two [fig_ref] Table 4: Object detection quality comparison of our structural-constraint-applied networks and other detectors on... [/fig_ref]. Since KITTI's leaderboard publishes detection precisions on car, pedestrian and cyclist, we compare performances on these three classes and the global mean average precisions (MAP). It could be observed that our SCM-Multi network achieved top values on all these metrics. According to these ablation evaluations and comparisons with other modern detectors on different datasets, it's shown that structural constraint mechanism is able to improve object detection quality on various network architectures, and is able to assist some prototype networks to achieve advanced performances.
# Visualization analysis
We analyze behaviors of structural constraint mechanism during training and testing in this subsection. For this purpose, we visualized changing of the loss terms in structural constraint, their influences on feature space and some final detection results.
Changing of loss values. We plotted curves of Fisher loss and equi-proportion loss during training of object detection networks of different architectures. The observation subjects include RetinaNet, SSD, Faster RCNN and Cascade RCNN, all with structural constraints applied. These loss curves are shown in [fig_ref] Fig 2: The curves of Fisher and equi-proportion losses [/fig_ref] Both losses were obviously dropping during all these training processes. This observation indicates that the loss terms in structural constraints are effectively minimized, so they are indeed guiding networks' training.
Influence on network feature space. To observe the influences of structural constraint mechanism on object detection networks' feature spaces, we adopted t-SNE [bib_ref] Visualizing data using t-SNE, Der Maaten [/bib_ref] to project high-dimensional backbone features to 2D space for visualization. These backbone features were obtained by feeding the networks with images of object classes. These images are sampled from KITTI according to its bounding box labels and are of class Car or Pedestrian (Ped). The extracted backbone features are then resized to a uniform size for the convenience of t-SNE transform. The visualization results are shown in The network subjects are Faster RCNN and Cascade RCNN. It could be observed that with greater extent of structural constraint application, the distributions of Car and Ped are less mixed and easier to separate. This is a beneficial behavior to object classification, and is consistent with the intention of structural constraints. Detection result visualization. In [fig_ref] Fig 4: Detection results of Faster RCNN [/fig_ref] we visualized some detection results on MSCOCO2017 images (val2017). We compared detection results of Faster RCNNs with and without structural constraints applied. It could be observed that the application of structural constraints made the detector more accurate at localization and give less false positives.
# Conclusion
In this work, we introduced our structural constraint mechanism for improving object detection quality. Structural constraint mechanism supervises object detection networks' intermediate feature spaces, and guides the training processes to optimize object class instances' distributions within the spaces. It constrains feature similarities of training sample pairs to be consistent with corresponding ground truth label similarities. With the aid of proxy feature design, structural constraint could be applied to all types of object detection network architectures. Experiment results indicate our structural constraint mechanism is able to optimize networks' intermediate features and consequently final detection results. It should be pointed out that calculation of structural constraint is done for all possible pairs of training samples, which has high GPU memory demand. We will address this issue in our future work.
# Author contributions
Conceptualization: Zihao Rong.
Data curation: Zihao Rong.
[fig] Fig 1: Illustration of structural constraint mechanisms in object detection networks of various architectures. (a) single-stage, (b) two-stage, and (c) multi-stage. https://doi.org/10.1371/journal.pone.0267863.g001 [/fig]
[fig] Fig 2: The curves of Fisher and equi-proportion losses (L Fisher and L equip ) during the training of object detection networks of different architectures. Upper row: Fisher losses; lower row: equi-proportion losses. "s#" in legends indicates the loss corresponds to stage # in the case of multi-stage networks. https://doi.org/10.1371/journal.pone.0267863.g002 [/fig]
[fig] Fig: 3. t-SNE visualization of Car and Pedestrian (Ped) instance distributions in feature spaces of object detection networks with and without structural constraints applied. [/fig]
[fig] Funding acquisition: Dehui Kong. Methodology: Shaofan Wang. Project administration: Dehui Kong. Resources: Dehui Kong, Baocai Yin. Software: Zihao Rong. Supervision: Shaofan Wang, Dehui Kong. Validation: Shaofan Wang. Visualization: Zihao Rong. Writing -original draft: Zihao Rong. Writing -review & editing: Shaofan Wang. [/fig]
[table] PLOS ONE: | https://doi.org/10.1371/journal.pone. [/table]
[table] Table 1: Ablation evaluations of structural constraint mechanism on MSCOCO2017. Note: the values where improvements happen are in bold face. " � " indicates that the network is trained using smaller batch size. https://doi.org/10.1371/journal.pone.0267863.t001 network produced identical object detection quality with many other detectors, and our SCM-Multi network achieved top values under most metrics.Comparison on KITTI. In this comparison, the training setting of our network SCM-Multi is same as the last subsection, and it's configured as Cascade RCNN þ L 2 Fisher þ L 2 equip . Other detectors' evaluation results are obtained from KITTI's official website. The comparison is shown in [/table]
[table] Table 2: Ablation evaluations of structural constraint mechanism on KITTI.Table 3. Object detection quality comparison between structural-constraint-applied networks and other detectors on MSCOCO2017.Note: the top value under each metric is in bold face.https://doi.org/10.1371/journal.pone.0267863.t003 [/table]
[table] Table 4: Object detection quality comparison of our structural-constraint-applied networks and other detectors on KITTI.Note: the top value under each metric is in bold face.https://doi.org/10.1371/journal.pone.0267863.t004 [/table]
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Pulmonary benign metastasizing leiomyoma: A case report
A B S T R A C TUterine leiomyoma is the most common benign gynecological tumor. Rarely, it has benign extra-uterine growth patterns, including benign metastasizing leiomyoma (BML), with lungs being the most common metastatic site.We present a case of a 47-year-old female who, 3 years prior to presentation, underwent abdominal supracervical hysterectomy for benign leiomyoma. Approximately 6 months prior to presentation, she was seen for shortness of breath and chest pain. A CT of the chest revealed multiple new non-calcified pulmonary nodules bilaterally. PET/CT demonstrated mild FDG uptake in multiple lung nodules, with no significant extra-thoracic sites of abnormal FDG uptake. A CT guided lung biopsy showed a low grade, smooth muscle tumor. Immunohistochemical staining was positive for smooth-muscle actin and desmin, estrogen and progesterone receptor and was negative for CD117, HMB-45, CD34, pan cytokeratin and EMA. She underwent wedge resection of one of the nodules which confirmed the above findings. A cytogenetic analysis was also performed, which was consistent with pulmonary BML. She ultimately underwent left lower lobe resection and was started on a daily aromatase inhibitor.BML is a rare disease usually seen in women of reproductive age. The pathogenesis and treatment remain controversial. BML mostly tends to have an indolent course and a favorable outcome.
# Introduction
Uterine leiomyoma is one of the most common benign gynecological tumors [bib_ref] Pathology and pathophysiology of uterine smooth-muscle tumors, Robboy [/bib_ref] [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report, Fan [/bib_ref] [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report and literature review, Chen [/bib_ref]. Rarely, it has been shown to have benign extra-uterine growth patterns, including benign metastasizing leiomyoma (BML) [bib_ref] Pathology and pathophysiology of uterine smooth-muscle tumors, Robboy [/bib_ref] [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report, Fan [/bib_ref] [bib_ref] Uterine smooth-muscle tumors with unusual growth patterns, Vaquero [/bib_ref]. Lungs are the most common metastatic site amongst others such as lymph nodes, deep soft tissues, central nervous system, mesentery, bones and heart [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report, Fan [/bib_ref] [bib_ref] Benign metastasizing leiomyoma: a rare type of lung metastases-two case reports and..., Taftaf [/bib_ref] [bib_ref] Pulmonary benign metastasizing leiomyoma: report of three cases, Jeon [/bib_ref] [bib_ref] Benign pulmonary metastasizing leiomyomatosis: case report, Cobellis [/bib_ref] [bib_ref] Hormonal manipulation of benign metastasizing leiomyomas: report of two cases and review..., Rivera [/bib_ref] [bib_ref] Benign metastasizing leiomyoma: a rare cause of multiple pulmonary nodules, Rege [/bib_ref]. Most pulmonary BMLs (PBML) are discovered incidentally in asymptomatic patients with scattered, wellcircumscribed, multiple, non-calcified bilateral nodules on imaging studies [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report, Fan [/bib_ref] [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report and literature review, Chen [/bib_ref] [bib_ref] Benign pulmonary metastasizing leiomyoma of the uterus: a case report, Ma [/bib_ref].
## Case description
We present a case of a 47-year-old, morbidly obese African American female, G6 P4-0-2-4, with a 22 pack per year smoking history who had quit 5 years prior to presenting. She had an abdominal supracervical hysterectomy 3 years prior to presentation for uterine fibroids.
Surgical pathology had confirmed benign leiomyomata with no malignant findings. A CT of the abdomen/pelvis was performed a year later for abdominal pain which showed a 42 × 47 mm right ovarian cyst and multiple ventral hernias. She did not follow up for the ovarian cyst.
Six months prior to presentation the patient was seen in the hospital for shortness of breath and chest pain. She underwent a CT of the chest with pulmonary embolus (PE) protocol, which showed multiple, noncalcified pulmonary nodules bilaterally, with the largest being 14 mm in the left lower lobe (LLL), accompanied by a 10 mm nodule in the right middle lobe amongst others, concerning for metastatic disease. No pulmonary embolism was seen [fig_ref] Figure 1: A [/fig_ref]. As part of the initial workup, given her history of ovarian cyst, she underwent a CT of the abdomen and pelvis again which showed bilateral cystic masses in the adnexa, likely ovarian in nature. She subsequently had a pelvic ultrasound confirming right and left ovarian cysts which were 13 × 24 × 20 and 33 × 23 × 27 mm, respectively. No other masses were seen.
This prompted a repeat visit to her gynecologic oncologist. Her tumor markers, including CA-125 and CEA, were normal. FSH was normal as well. Based on the nature of the cysts and negative tumor markers, metastatic ovarian cancer to the lungs was deemed highly unlikely and a referral was made to the lung cancer center. PET/CT was ordered to further assess the nodules and assess for any extra-pulmonary areas of abnormal uptake. It again showed the largest nodule measuring 13 × 14 mm in the LLL, and a middle lobe nodule measuring 10 × 10 mm, both demonstrating mild FDG uptake [fig_ref] Figure 2: A [/fig_ref] and B).
When compared with a CT performed four years prior, the LLL nodule was new and the middle lobe nodule had increased in size since that time [fig_ref] Figure 3: CT chests with PE protocol comparing Right Middle Lobe nodule over 4... [/fig_ref]. Also, at least 7 other smaller nodules, which were beneath the threshold of reliable characterization with PET FDG imaging, were seen with no significant extra-thoracic sites of abnormal FDG uptake.
Based on these findings she underwent a CT guided biopsy of the LLL nodule. Pathology identified a low grade (2/8 HPF mitosis), benign appearing, smooth muscle tumor. Immunohistochemical staining showed smooth-muscle actin and desmin, but was negative for CD117, HMB-45, CD34, pan cytokeratin and EMA. An estrogen receptor was present with patchy positivity and a progesterone receptor was strongly and diffusely positive. Given her history, these findings were suggestive of benign metastasizing uterine leiomyoma.
Due to concerns about a possible sampling error missing features of a more aggressive leiomyosarcoma, as well as to get fresh tissue for cytogenetic analysis, she underwent a video-assisted thoracoscopic surgery (VATS) wedge resection of the LLL nodule. Pathology again confirmed cytologically bland-appearing spindle cell neoplasm, with entrapment of bronchiolar epithelium and no significant nuclear atypia or necrosis. Mitotic activity was low (up to 2 mitoses per 10 high power fields), with no margin involvement identified. Properly controlled immunostaining showed the neoplastic cells to lack expression of HMB-45 and CD117 (c-kit) (no support for lymphangioleiomyomatosis or gastrointestinal stromal tumor, respectively) as before. A properly controlled DOG-1 (discovered on GIST) immunostain was positive, a result typically associated with gastrointestinal stromal tumor. However, this result has also been reported for uterine type retroperitoneal leiomyomas and peritoneal leiomyomatosis [bib_ref] DOG1 antibody in the differential diagnosis of gastrointestinal stromal tumors: a study..., Miettinen [/bib_ref] [fig_ref] Figure 4: A [/fig_ref]. Cytogenetic analysis was also performed. Karyotyping showed an abnormal result, including loss of chromosomes 19 and 22 and deletion of 1p, in addition to other abnormalities . This was consistent with the diagnosis of pulmonary benign metastasizing leiomyoma.
The patient was started on the daily aromatase inhibitor, Anastrozole. She tolerated the medication without any side effects. Three-month follow up CT scans showed a decrease in the size of pulmonary nodules. Six and 12-month CT scans have shown stable disease.
# Discussion
BML is a rare disease, first described in 1939 by Steiner [bib_ref] Metastasizing fibroleiomyoma of the uterus: report of a case and review of..., Steiner [/bib_ref] , with about 100 cases described in the literature to date [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report, Fan [/bib_ref]. The pathogenesis and etiology have remained controversial with hematogenous spread of benign uterine tumor, local smooth muscle tissue proliferation or metastatic low grade leiomyosarcoma as proposed etiologies [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report, Fan [/bib_ref]. It is usually seen in women of reproductive age with a history of uterine leiomyoma who underwent hysterectomy, favoring the hematogenous/ iatrogenic spread of the tumor, yet in some cases lung nodules existed even before hysterectomy, as in our case [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report and literature review, Chen [/bib_ref] [bib_ref] Hormonal manipulation of benign metastasizing leiomyomas: report of two cases and review..., Rivera [/bib_ref]. However, the cytogenetic studies demonstrating a monoclonal origin of both uterine and lung tumors, along with positive hormone receptors and response to hormonal therapy, support the hypothesis [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report, Fan [/bib_ref] [bib_ref] Benign metastasizing leiomyoma: a cytogenetically balanced but clonal disease, Tietze [/bib_ref] [bib_ref] Pulmonary benign metastasizing leiomyoma associated with intravenous leiomyomatosis of the uterus: clinical..., Lee [/bib_ref]. Cytogenetic studies have also shown that BML is a genetically distinct and definable entity with a 19q and 22q deletions, amongst others. Such changes are also found in a small genetically distinctive subset of uterine leiomyomata, supporting a common origin [bib_ref] Distinctive cytogenetic profile in benign metastasizing leiomyoma: pathogenetic implications, Nucci [/bib_ref].
The average age of patients diagnosed with BML is 48 years with about a 3 month to 20 year span between hysterectomy and lung findings [bib_ref] Benign metastasizing leiomyoma: clinical, imaging, and pathologic correlation, Abramson [/bib_ref] [bib_ref] Immunohistological detection of estrogen and progesterone receptors in multiple and well differentiated..., Jautzke [/bib_ref]. Open/thoracoscopic lung biopsy is the standard diagnostic modality [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report and literature review, Chen [/bib_ref].
Pulmonary smooth muscle proliferations can either be primary, including hamartomas, lymphangioleiomyomatosis, leiomyoma and leiomyosarcoma, or metastatic, including metastatic leiomyosarcoma and BML. The low mitotic index (< 5 mitoses per 10 high power fields), lack of nuclear pleomorphism, lack of local invasion and distinctive karyotypic profile helps differentiate BML from other possible diagnoses [bib_ref] Uterine smooth-muscle tumors with unusual growth patterns, Vaquero [/bib_ref] [bib_ref] Distinctive cytogenetic profile in benign metastasizing leiomyoma: pathogenetic implications, Nucci [/bib_ref].
Due to the rarity of the disease, currently there are no treatment guidelines for BML. Multiple options have been reported in the literature, including close observation, surgical resection or antiestrogen therapy (e.g., selective estrogen receptor modulator, progesterone, aromatase inhibitors, oophorectomy and gonadotropin-releasing hormone analogues) [bib_ref] Benign metastasizing leiomyoma: a rare type of lung metastases-two case reports and..., Taftaf [/bib_ref] [bib_ref] Pulmonary benign metastasizing leiomyoma: a case report and review of the literature, Fu [/bib_ref]. The preferred treatment is surgical resection if possible with hormonal therapy as an alternate. BML tends to typically have an indolent course and a favorable outcome, although pulmonary lesions may continue to progress, resulting in pulmonary insufficiency and even death [bib_ref] Distinctive cytogenetic profile in benign metastasizing leiomyoma: pathogenetic implications, Nucci [/bib_ref].
# Conclusion
Despite BML being a rare condition, it should be considered in the differential diagnosis in women of reproductive age with a history of uterine leiomyoma presenting with pulmonary nodules, solitary or multiple. Accurate histopathological analysis along with immunohistochemical staining and cytogenetic analysis are necessary to exclude other spindle cell neoplasms.
## Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. with entrapped benign bronchiolar epithelium (epithelial entrapment rather than obliteration being a reflection of indolent rather than aggressive growth of the neoplasm). C: High power view of the lung wedge resection specimen showing the cytologically bland appearance of the spindle cells (lack of any significant nuclear atypia); also, inconspicuous mitoses (no significant mitotic activity), and no necrosis. D: Core biopsy, desmin immunostain (marker of smooth muscle and skeletal muscle differentiation), showing strong and diffuse expression in the neoplastic cells. The appearance of the smooth muscle actin (SMA) immunostain, which was also done on the core, is identical. E: Core biopsy, progesterone receptor immunostain, showing strong and diffuse nuclear expression in the neoplastic cells. The estrogen receptor immunostain showed similar results. F: Wedge resection specimen, DOG-1 immunostain, showing unexpected diffuse expression in the neoplastic cells. The CD117 (c-kit) immunostain was negative. The DOG-1 is typically positive for GIST, but has also been reported to show expression in some uterine type retroperitoneal leiomyomas and peritoneal leiomyomatosis.
# Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
[fig] Figure 1: A: CT chest with PE protocol showing a non-calcified 14 mm pulmonary nodule in left lower lobe. B: CT chest with PE protocol showing a non-calcified 10mm pulmonary nodule in Right Middle Lobe. C: CT chest with PE protocol showing small non-calcified pulmonary nodules in Right and Left Upper Lobes. [/fig]
[fig] Figure 2: A: PET/CT scan showing mild FDG uptake in the Left Lower Lobe Nodule. B: PET/CT scan showing mild FDG uptake in the Right Middle Lobe Nodule. [/fig]
[fig] Figure 3: CT chests with PE protocol comparing Right Middle Lobe nodule over 4 years. [/fig]
[fig] Figure 4: A: Low power view of the lung wedge resection specimen showing a well-circumscribed nodule composed of cytologically bland appearing spindle cells arranged in a fascicular pattern. B: Medium power view of the lung wedge resection specimen showing the fascicular pattern of the nodule [/fig]
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## Figure s1. mitochondrial localization of trap1 and inhibition of its expression in several
Tumor Cell Types, Related to (A) Immuno-electron microscopy inspection shows most of TRAP1 along the inner mitochondrial membranes (arrows) of HCT116 colorectal carcinoma cells. (B) Trypsin treatment of isolated mitochondria from osteosarcoma SAOS-2 cells shows that TRAP1 is partially cleaved at the highest trypsin concentration, but it displays a pattern similar to that of the matrix protein cyclophilin D (CyP-D), thus indicating that most of TRAP1 is found in the internal mitochondrial compartments.
Mitochondria (70 μg per point) were treated for 1 h with the reported quantities of trypsin (in micrograms) and analyzed by Western immunoblot. Where indicated, 0.1% SDS was added before trypsin to achieve complete digestion. Blots were probed for Bcl-X as a marker of the outer mitochondrial membrane and for CyP-D as a matrix marker. (A, B), analysis of the succinate:coenzyme Q reductase (SQR) enzymatic activity of complex II. In (A), representative spectrophotometric traces performed on SAOS-2 mitochondria are reported. In (B), analysis of the succinate:coenzyme Q reductase (SQR) enzymatic activity of complex II on HCT116 mitochondria. In the left part, representative spectrophotometric traces are reported; in the right part, analysis was performed as in . The TRAP1 inhibitor 17-allylamino-17demethoxygeldanamycin (17-AAG) was added 5 min before starting recordings. In (C), spectrophotometric analysis of the COX enzymatic activity was performed on SAOS-2 cells. Mock indicates SAOS-2 cells transfected with a scrambled shRNA; shTRAP1 indicates SAOS-2 cells transfected with a TRAP1 shRNA. (D) Western immunoblot showing that no changes in SDHB protein expression occur with or without TRAP1 and during the focus-forming assay (FF samples were obtained at the 15 th experimental day). GAPDH was used as a loading control. (E) N-acrydine 4 orange (NAO) cytofluorimetric analyses indicate that no change in mitochondrial mass occur between SAOS-2 mock and shTRAP1 cells. (F) Western immunoblot comparing Hsp90 and TRAP1 expression level in mock and shTRAP1 SAOS-2 cells. As Hsp90 protein levels were identical in mock and shTRAP1 cells, the selective effect of 17-AAG on mock cells can be reasonably ascribed to its TRAP1 inhibition. Blots were probed with an anti-Bcl-X antibody to check for mitochondrial protein load. All along the In order to re-express TRAP1 in interfered cells in a shRNA-insensitive way, shTRAP1 cells obtained with sequence b), which is highly divergent between human and mouse genes, were transfected with a mouse TRAP1 cDNA (Origene). HIF1 stable interference was achieved with the following HIF1 shRNAs from Sigma:
CCGGCCAGTTATGATTGTGAAGTTACTCGAGTAACTTCACAATCATAACTGGTTTTT;
CCGGTGCTCTTTGTGGTTGGATCTACTCGAGTAGATCCAACCACAAAGAGCATTTTT;
HIF1 interference was achieved with the following shRNAs from Sigma:
[formula] CCGGGCCTACACTCTCCAACACAATCTCGAGATTGTGTTGGAGAGTGTAGGCTTTTT; 8 CCGGCCTTTGTCTTTCTGTGTACTTCTCGAGAAGTACACAGAAAGACAAAGGTTTTT; [/formula]
CCGGCATTGTCCAGAGGGCTATTAACTCGAGTTAATAGCCCTCTGGACAATGTTTTT;
scrambled shRNAs were always used as negative controls. Stable interfered cells were selected in 0.8 g/ml puromycin (Sigma). TRAP1 mutant lacking the mitochondrial import sequence (N TRAP1) was a Δ1-59-Myc construct generated as in . MEF cells were transfected with Lipofectamine 2000 (Invitrogen) and selected in G418 (0.5 mg/ml; Sigma) for the expression of TRAP1 cDNA (cloned in a pCMV6 vector, Origene). The rate of cell growth was measured with a Scepter TM cell counter (Millipore).
## Tissue samples
Specimens from both tumor and normal, non-infiltrated peritumoral mucosa were obtained from patients with colorectal carcinoma during surgical cancer removal, after an expressed written informed consent to use biological specimens for investigational procedures was obtained from all patients. Samples were cut into 125 mm 3 pieces and one specimen was fixed in formalin to confirm the histopathological diagnosis, while the others were frozen in liquid nitrogen for further analyses.
TRAP1 expression was evaluated by Western immunoblot followed by densitometric analysis, and its level, which was considered as induced when the ratio between tumor and non-infiltrated surrounding mucosa was ≥3 [bib_ref] TRAP1, a novel mitochondrial chaperone responsible for multi-drug resistance and protection from..., Costantino [/bib_ref] , increased in all samples from metastatic neoplasias and in the majority of initial, non-metastatic tumors . For ETC complex II evaluations, at least three samples with increased TRAP1 expression and three samples without any TRAP1 expression changes were analyzed.
## In vitro tumorigenesis assays
For the focus-forming assay, 10 6 cells were plated in 10 cm Petri dishes (BD Falcon) in Dulbecco's modified Eagle's (DMEM) medium supplemented with 10% fetal bovine serum (Gibco). When cells reached sub-confluence, serum concentration was decreased to values ranging between 0.5% 9 and 2%, which did not induce cell death per se (data not shown), and changed every 4 th day.
Mitochondria utilized for the determination of ETC complex enzymatic activities or cell lysates used for Western immunoblot assays were obtained at the 15 th day after serum decrease, i.e. 1-2 days before cells that did not form foci started a massive death process (see below for sample preparation). 25 days after serum decrease foci appeared as thick masses and cords of cells. Plates were washed in PBS, fixed in methanol for 30 min and foci colored with GIEMSA solution for 1 h.
After washing in deionized water, size and number of foci were analyzed with an Image Analyzer custom software . For the soft agar assay, cells were grown in 6 cm Petri dishes Dishes were then maintained in a humidified atmosphere of 5% CO 2 -95% air at 37°C for three weeks, adding medium (DMEM 0.5% serum) on the top of the two layers every 7 th day. At the 25 th day, dishes were washed in PBS and colonies were stained with Crystal Violet 0.005% and analyzed with Image Analyzer software.
## In vivo tumorigenesis assays
Experiments were performed in 5-week-old female CD1 nude mice (Charles River Laboratories) treated in accordance with the European Community guidelines. Twelve mice were injected subcutaneously bilaterally in the flanks with 1.5x10 7 SAOS-2 mock or shTRAP1 cells in 200 l of serum-free sterile PBS. In a subset of animals, tumor growth was favored by injecting cells in PBS mixed with Matrigel in a 1:1 ratio. Tumor growth was evaluated on alternate days by calliper measuring. After three weeks, mice were sacrificed and tumors stored at -80°C or fixed in formaldehyde and maintained in 70% ethanol for immunohistochemical analyses.
## 10
## Cytofluorimetric analyses
Flow cytometry recordings were performed to determine cell death, as described [bib_ref] Hepatocyte growth factor installs a survival platform for colorectal cancer cell invasive..., Fassetta [/bib_ref] , and mitochondrial mass. Briefly, cells were incubated at 37°C in 135 mM NaCl, 10 mM HEPES, 5 mM CaCl 2 with FITC-conjugated Annexin-V (Boehringer Mannheim) and propidium iodide (PI, 1 g/ml; Sigma), to detect phosphatidyl-serine exposure on the cell surface (increased FITC-conjugated Annexin-V staining) and loss of plasma membrane integrity (PI permeability and staining). N-acrydine orange (NAO 200 nM, Invitrogen), which binds to cardiolipin in mitochondrial membranes, was utilized to evaluate mitochondrial mass. Samples were analyzed on a FACS Canto II flow cytometer (Becton Dickinson). Data acquisition and analysis were performed using FACSDiva software.
## Mitochondria purification and quantification
Mitochondria were isolated after cell disruption with a glass-Teflon or electrical potter (Sigma) in a buffer composed by 250 mM sucrose, 10 mM Tris-HCl, 0.1 mM EGTA-Tris, pH 7.4. Nuclei and plasma membrane fractions were separated by a first mild centrifugation (700g, 10 min); mitochondria were then spinned down at 7000g, 10 min, and washed twice (7000g, 10 min each).
All procedures were carried out at 4°C. In order to define submitochondrial protein localization, isolated mitochondria were digested with trypsin at different concentrations at 4°C for 1h. Where indicated, 0.1% SDS was added before trypsin. Trypsin was then inactivated with a protease inhibitor cocktail (Sigma).
## Western immunoblots, immunoprecipitations and crosslinkings
For Western immunoblots analyses, cells kept in standard culture conditions or in focus-forming assays were lysed at 4°C in a buffer composed by 140 mM NaCl, 20 mM Tris-HCl pH 7.4, 5 mM EDTA, 10% glycerol, 1% Triton X-100, in the presence of phosphatase and protease inhibitors (Sigma). Lysates were then cleared with a centrifugation at 13000g for 30 min at 4°C, and proteins were quantified using a BCA Protein Assay Kit (Thermo Scientific-Pierce).
Protein immunoprecipitations were carried out on 3 mg of total cellular extracts. Lysates were pre-cleared with an incubation with protein A-Sepharose (Sigma) for 1 h at 4°C and then incubated in agitation for 18 h at 4°C with the antibody conjugated to fresh protein A-Sepharose beads. Where indicated, an anti mouse IgG was added as a negative isotype control. Beads were then washed several times in lysis buffer.
For the crosslinking assays, isolated mitochondria were suspended in PBS buffer and incubated with dimethyl 3,3-dithiobis-propionimidate (DTBP, Sigma, 1 mM), a membranepermeable, homo-bifunctional reagent that reacts with the primary amines of two interacting proteins at an average distance of about 8 Å , for 15 minutes at room temperature and then spinned at 7000 rcf for 5 minutes. Pellet was then lysed as above, and lysates were ultracentrifuged at 100000 rcf for 25 minutes at 4°C prior to TRAP1 immunoprecipitation.
Proteins extracted from total cell lysates or from immunoprecipitations were then boiled for
## Etc complex ii and iv activity assays
To measure the enzymatic activity of respiratory chain complex II, cells or biopsies were homogenized with an electric potter (Sigma) in a buffer composed by 250 mM sucrose, 10 mM To measure the enzymatic activity of respiratory chain Complex IV, cells were subjected to three cycles of freezing and thawing in liquid nitrogen. These cellular homogenates were used for spectrophotometric recordings (550 nm, 37°C) of the oxidation of reduced cytochrome c, (ε=18.5 mM−1 cm−1) in a buffer composed by potassium phosphate 100 mM pH 7, water and laurylmaltoside 10% p/v. The reaction started by the addition of cellular homogenates and the decrease in absorbance was followed for 3 min. Reduced cytochrome c was prepared immediately before use by adding a few grains of sodium dithionite. Each measurement of ETC complex IV activity was normalized for protein amount and for CS activity, as above.
## Oxygen consumption rate experiments
The rate of oxygen consumption was assessed in real-time with the XF24 Extracellular Flux Analyzer (Seahorse Biosciences), which allows to measure OCR changes after up to four sequential additions of compounds. Cells (5x10 4 /well) were plated the day before the experiment in a DMEM/10% serum medium; experiments were carried out on confluent monolayers. Before starting measurements, cells were placed in a running DMEM medium (supplemented with 25 mM
## Determination of intracellular succinate level
To determine the intracellular succinate level, cells were first washed three times in PBS at room temperature and then quickly scraped in a buffer composed by 30% acetonitrile, 50% methanol and 20% water on ice. Lysates were collected and frozen in a cold solution with methanol and dry ice for 5 min in order to favor the metabolic quenching needed for metabolites extraction. The insoluble material was immediately spun down in a cooled centrifuge at 16000 g for 15 min at 0°C and the supernatant was collected for subsequent analysis. Metabolites were separated using a liquid chromatography (LC) system and analyzed by mass spectrometry (MS). A ZIC-HILIC column (4.6 mm×150 mm, guard column 4.6 mm×10 mm, Merck, Germany) was used for LC separation using formic acid, water acetonitrile as component of the mobile phase. Results were normalized to protein concentration, measured from parallel cell culture using BCA kit as described above.
## Intracellular atp determination
Intracellular ATP was quantified by the luciferin/luciferase method using the ATP determination kit by Invitrogen/Molecular Probes following manufacturer's instructions. Cells were kept for two hours in the different experimental conditions, washed in PBS, lysed in boiling water to avoid ATPase activity. 2.5 g of proteins were analyzed in a 100 l final volume. Each experiment was performed in triplicate.
[fig] 3, Figure S2: (C-E) Western immunoblot showing TRAP1 expression level in human osteosarcoma SAOS-2 cells (C), in human colorectal carcinoma HCT116 cells (D), and in human cervix carcinoma HeLa cells (E) stably transfected with a scrambled shRNA (mock) or with different TRAP1 shRNAs dubbed shTRAP1a, shTRAP1b and shTRAP1c. GAPDH is shown as a loading control. TRAP1 Expression Inhibits ETC Complex II without Affecting ETC Complex IV Activity, SDH Protein Expression Level, or Mitochondrial Mass, Related to Figure 4 [/fig]
[fig] Figure S3: TRAP1 Decreases Cell Oxygen Consumption Rate in Cancer Cell Models, Related to Figure 5 [/fig]
[fig] 6, Figure S4: A, B) Representative traces of oxygen consumption rate (OCR) experiments performed on monolayers of living human cervix carcinoma HeLa cells (A), or of human epithelial prostate RWPE-2 cells transformed by v-Ki-Ras expression (B). Subsequent additions of the ATP synthase inhibitor oligomycin, of the uncoupler FCCP, of the ETC complex I inhibitor rotenone and of the ETC complex III inhibitor antimycin A were carried out. Mock indicates cells stably transfected with a scrambled shRNA; shTRAP1 indicates cells stably transfected with a TRAP1 shRNA. 5 The OCR Is Increased by TRAP1 Inhibition in Mock Cells and Downregulated by SDH Inhibition in shTRAP1 Cells, Related to Figure 5 (A) Effect of the TRAP1 inhibitor 17-AAG on the OCR of SAOS-2 cells before oligomycin addition; data are shown as fold increase with respect to untreated mock cells. 17-AAG was preincubated within cell monolayers for 30 minutes. (B, C) Representative traces of OCR experiments. Subsequent additions of the ATP synthase inhibitor oligomycin, of the uncoupler FCCP, of the ETC complex I inhibitor rotenone and of the ETC complex III inhibitor antimycin A were carried out. The SDH inhibitors 3-nitro propionic acid (3-NP; B) and thenoyltrifluoroacetone (TTFA; C) were added to cell monolayers immediately before starting the experiments. 7SUPPLEMENTAL EXPERIMENTAL PROCEDURES Cell Culture and TransfectionsHuman SAOS-2 osteosarcoma cells, human HCT-116 colorectal carcinoma cells, human HeLa cervix carcinoma cells, and MEF cells obtained from C57BL/6J mice through SV40immortalization were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2%-10% fetal bovine serum (Invitrogen); RWPE prostate epithelial cells were grown in keratinocyte medium (Gibco) supplemented with EGF and bovine pituitary extract; 100 units/ml penicillin and 100 g/ml streptomycin were added to all media, and cells were kept in a humidified atmosphere of 5% CO 2 /95% air at 37°C. Hypoxic treatments were achieved by incubating cells in an InVivo2 300 hypoxic chamber (Ruskinn Technology). TRAP1 stable interference of human cells was achieved by transfecting cells with a panel of TRAP1 shRNAs from Sigma: [/fig]
[fig] 5: min in Laemmli sample buffer, separated in reducing conditions on SDS-polyacrylamide gels and transferred onto Hybond-C Extra membranes (Amersham) following standard methods. Primary antibodies were incubated 16 h at 4°C, and horseradish peroxidase-conjugated secondary antibodies were added for 1 h. Proteins were visualized by enhanced chemiluminescence (Millipore). Anti human TRAP1 (sc-13557), anti SDHA (sc-166947), anti SDHB (sc-59688) mouse monoclonal antibodies and anti actin (sc-1615) goat polyclonal antibody were all from Santa Cruz; anti HIF1 (610959) and anti human and mouse TRAP1 (612344) mouse monoclonal antibodies were from Becton Dickinson; anti GAPDH (MAB374) mouse monoclonal antibody was from Chemicon; anti caspase-3 (8G10), rabbit monoclonal antibody, anti Bcl-X (54H6) and anti Hydroxy-HIF1(Pro564; D43B5) rabbit polyclonal antibodies were from Cell Signaling; anti Hydroxy-HIF1(Pro402; 07-1585) rabbit polyclonal antibody was from Millipore; anti Cyp-D (AP1035) [/fig]
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Determination of the Volatile Composition in Brown Millet, Milled Millet and Millet Bran by Gas Chromatography/Mass Spectrometry
The volatile compounds from brown millet (BM), milled millet (MM) and millet bran (MB) were extracted using simultaneous distillation/extraction with a Likens-Nickerson apparatus. The extracts were analysed using gas chromatography coupled with mass spectrometry (GC-MS). A total of 65 volatile compounds were identified in all of the samples. Among these compounds, 51, 51 and 49 belonged to BM, MM and MB, respectively. Aldehydes and benzene derivatives were the most numerous among all of the compounds. Three compounds (hexanal, hexadecanoic acid and 2-methylnaphthalene) were dominant in the BM and MM materials. Eight compounds (hexanal, nonanal, (E)-2-nonenal, naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, hexadecanoic acid and 2-pentylfuran) were dominant in the MB materials. Apart from the aromatic molecules, which were present in all fractions, compounds present only in BM, MM or MB were also identified.
# Introduction
Foxtail millet (Setaria italica Beauv) is one of the oldest cultivated cereals, which has its origins in China and subsequently was extended into India and over most of Africa and parts of the southern United States [bib_ref] Chemical characteristics and fatty acid profile of foxtail millet bran oil, Liang [/bib_ref]. Foxtail millet plays a very important role in the agriculture and food industry of many developing countries because of its capacity to grow under adverse heat and limited rainfall conditions. This millet is rich in carbohydrates [bib_ref] Free sugar and non-starchy polysaccharides of finger millet (Eleusine coracana), pearl millet..., Malleshi [/bib_ref] [bib_ref] Carbohydrate composition of finger millet (Eleusine coracana) and foxtail millet (Setaria italica), Wankhede [/bib_ref] [bib_ref] Functional characteristics of starches from proso and foxtail millets, Lorenz [/bib_ref] and protein [bib_ref] Seed protein of millet: Amino acid composition, proteinase inhibitors and in-vitro protein..., Ravindran [/bib_ref] [bib_ref] Nutrient composition and protein quality of minor millets, Geervani [/bib_ref] and also contains oils [bib_ref] Chemical characteristics and fatty acid profile of foxtail millet bran oil, Liang [/bib_ref] and vitamins [bib_ref] Study on determination of vitamin E content in millets by high performance..., Xiong [/bib_ref]. It has been recorded that foxtail millet has many nutritional and medical functions. It has been used in China as a Traditional Medicine to invigorate the stomach, strengthen the spleen, quench thirst and promote urination. In some cases, this millet can also be used in the treatment of diabetes and cardiovascular diseases [bib_ref] Effects of dietary Korean protein of foxtail-millet on plasma adiponectin, HDL-cholesterol, and..., Choi [/bib_ref].
In recent years, increasingly more foxtail millet products have entered into the daily lives of people, including millet porridge, millet wine, and millet nutrition powder. In addition to its nutritional benefits, the unique odour of the millet is one of the main reasons why consumers prefer it. Odour is considered a critical quality trait in cereals; it affects consumer preference because it travels to the nose during consumption and stimulates the olfactory receptors in the nasal cavity [bib_ref] Measurement of volatile release in the mouth, Linforth [/bib_ref]. The aromatic characteristics of various cereals, such as rice [bib_ref] Rice product volatiles: A review, Maga [/bib_ref] [bib_ref] Rice aroma and flavor: A literature review, Champagne [/bib_ref] , corn [bib_ref] Importance of 2-aminoacetophenone to the flavor of masa corn flour products, Buttery [/bib_ref] [bib_ref] Studies on flavor volatiles of some sweet corn products, Buttery [/bib_ref] [bib_ref] Volatile flavor components of corn tortillas and related products, Buttery [/bib_ref] , wheat [bib_ref] Flavour of sourdough wheat bread crumb, Hansen [/bib_ref] [bib_ref] Identification and quantification of potent odorants formed by toasting of wheat bread, Rycchlik [/bib_ref] [bib_ref] Potent odorants of the wheat bread crumb: Differences to the crust and..., Schieberle [/bib_ref] and buckwheat [bib_ref] Aroma compounds in buckwheat (Fagopyrum esculentum Moench) groats, flour, bran, and husk, Janeš [/bib_ref] [bib_ref] Comparison of isolation methods for the determination of buckwheat volatile compounds, Prosen [/bib_ref] [bib_ref] Identification of buckwheat (Fagopyrum esculentum Moench) aroma compounds with GC-MS, Janeš [/bib_ref] [bib_ref] Salicylaldehyde is a characteristic aroma component of buckwheat groats, Janeš [/bib_ref] , have been investigated from the perspective of the volatile compounds they contain. Aldehydes, alcohols, ketones, benzene derivatives, hydrocarbons, acids, esters, heterocycles and sulphur-containing compounds are the main volatile compounds of these cereals. Among them, odour active compounds of rice were analysed in detail, and 2-acetyl-1-pyrroline was reported as the most important odour active compounds contributing to the odour of popcorn [bib_ref] Rice product volatiles: A review, Maga [/bib_ref] [bib_ref] Rice aroma and flavor: A literature review, Champagne [/bib_ref]. Although the volatile constituents of the grain are believed to play a major role in cereal quality, relatively little information is available concerning these components in foxtail millet.
Dehusking and polishing are important steps in grain processing. These steps not only improve the sensory and edible quality of grain but also yield the main product and by-products, including brown grain, milled grain and bran. Compared with milled grain, brown grain is a nutritionally valuable food. The bran layers of brown grain kernels are rich in dietary fibre, minerals, oil, protein and vitamins (particularly B vitamins) [bib_ref] Maltooligosaccharides extracted from outer-layer of rice grain, Tajima [/bib_ref] [bib_ref] Quality characteristics of long-grain rice milled in two commercial systems, Chen [/bib_ref] [bib_ref] Bio-functional components in the processed pre-germinated brown rice by a twin-screw extruder, Ohtsubo [/bib_ref] [bib_ref] Effect of milling on colour and nutritional properties of rice, Lamberts [/bib_ref]. When added to the diet, bran layers are effective in reducing cholesterol levels in humans [bib_ref] Stabilized rice bran and oat bran lower cholesterol in humans, Hegsted [/bib_ref] [bib_ref] Influence of rice bran, oat bran and wheat bran on cholesterol and..., Kahlon [/bib_ref] [bib_ref] Cholesterol lowering in hamsters fed rice bran at various levels, defatted rice..., Kahlon [/bib_ref]. The utilisation of brown grain and its bran has gradually become one of the main directions of development in diet adjustments. However, most of these studies have focused on nutrition but have ignored differences in the volatile compounds.
To conduct a thorough investigation into the volatile flavours of foxtail millet and its foment its increased utilisation, an evaluation of the differences in volatile compounds among brown millet (BM), milled millet (MM) and millet bran (MB) is essential. Therefore, the main objective of this research was to identify and compare the volatile components of BM, MM and MB using simultaneous distillation extraction (SDE) and gas chromatography-mass spectrometry (GC-MS).
# Results and discussion
In this study, the volatile compounds from brown millet, milled millet and millet bran were extracted using the simultaneous distillation method and detected using GC and GC-MS. The experimental results are provided in [fig_ref] Table 1: Volatile components identified in BM, MM and MB [/fig_ref]. A total of 65 volatile compounds were tentatively identified. These compounds included aldehydes (17 compounds), alcohols (five compounds), ketones (nine compounds), hydrocarbons (seven compounds), acids (three compounds), benzene derivatives (12 compounds), esters (four compounds), furans (five compounds) and S-containing compounds (three compounds). In total, 52 compounds were identified: 51 in BM, 51 in MM, and 49 in MB. Aldehydes and benzene-containing compounds were the most numerous among all the compounds. The major volatile components present in BM, MM and MB were hexanal (12.82 ± 0.42%), hexadecanoic acid (16.43 ± 1.28%) and 2-methylnaphthalene (8.87 ± 0.54%); hexanal (7.49 ± 0.42%), hexadecanoic acid (35.77 ± 1.62%), 2-methylnaphthalene (5.36 ± 0.21%) and hexanal (7.69 ± 0.27%); and nonanal (5.89 ± 0.17%), (E)-2-nonenal (5.62 ± 0.18%), naphthalene (6.75 ± 0.34%), 2-methylnaphthalene (14.18 ± 0.26%), 1-methylnaphthalene (5.61 ± 0.39%), hexadecanoic acid (8.98 ± 0.29%) and 2-pentylfuran (5.11 ± 0.18%), respectively.
## Aldehydes
Aldehydes represented the largest group in the number of volatile components identified in all of the samples, comprising 21.81-34.21% of the total volatile compounds detected. The composition of aldehydes differed both qualitatively and quantitatively among the three samples. The volatile fraction was mainly composed of hexanal, nonanal and (E)-2-nonenal; the three aldehydes had concentrations of >5% (and existed in at least one of the samples), and hexanal was the most abundant aldehyde (>7%). BM, MM and MB contained 16, 15 and 13 aldehydes, respectively. (E,E)-2,6-Nonadienal, tridecanal and (E)-heptenal were absent in BM, MM and MB, respectively. Tetradecanal was unique to BM, whereas dodecanal was unique to MM. Aldehydes usually are derived from the autoxidation and enzymolysis oxidation of the double carbon-carbon bond of unsaturated fatty acids present in cereals [bib_ref] Contribution of volatiles to rice aroma, Buttery [/bib_ref] [bib_ref] Quantitative and sensory studies on tomato paste volatiles, Buttery [/bib_ref] [bib_ref] Volatile aldehydes in smoked fish: Analysis methods, occurence and mechanisms of formation, Varlet [/bib_ref]. In general, aldehydes have a great impact on the aroma of a cereal product because of their low odour threshold values. Aldehydes detected in the three samples belonged to the n-alkanal, 2-alkenal, and 2,4-alkadienal classes, respectively. The n-alkanals from hexanal to decanal are aromatics and provide grassy, green and fatty (hexanal), fatty and citrus (heptanal), fatty, soapy and green (octanal), fatty, citrus and green (nonanal) and fatty and grass (decanal) characteristics [bib_ref] Contribution of volatiles to rice aroma, Buttery [/bib_ref] [bib_ref] Quantitative and sensory studies on tomato paste volatiles, Buttery [/bib_ref] [bib_ref] Volatile aldehydes in smoked fish: Analysis methods, occurence and mechanisms of formation, Varlet [/bib_ref].
In addition, C 13 -C 18 aldehydes of low concentrations were expressed in the samples; these long-chain aldehydes typically have high thresholds [bib_ref] Volatile aldehydes in smoked fish: Analysis methods, occurence and mechanisms of formation, Varlet [/bib_ref] ; therefore, they may contribute little to the flavours of the samples. The 2-alkenals and 2,4-alkadienals have very low odour thresholds, which is the reason they are prevalent in cereal in small quantities. (E)-2-Nonenal, (E)-2-octenal, (E)-2-heptenal, (E)-2-hexenal belong to the 2-alkenals; with increasing C-chain length, the odour becomes less citrusy and fruity and more fat-like, and the odour threshold decreases [bib_ref] Aroma chemicals for savory flavours, Rowe [/bib_ref]. In terms of the 2,4-alkadienals, (E,E)-2,4-nonadienal and (E,E)-2,4-decadienal have been reported as the key odorants in rice, responsible for the "nutty and fatty" and "fatty" characteristics [bib_ref] Characterization of volatile aroma compounds in cooked black rice, Yang [/bib_ref] [bib_ref] Comparison of odor active compounds from six distinctly different rice flavor types, Yang [/bib_ref]. Abundant aldehydes detected in all of the samples are very important in the odour of the millet.
## Benzene derivatives
Benzene derivatives were the second largest group identified in the samples. In BM, MM and MB, nine, nine and 11 benzene derivatives were detected, respectively. From highest to lowest, the benzene derivative contents were: 32.94% in MB, 21.67% in BM and 15.90% in MM. Naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, and 1-ethylnaphthalene were present in concentrations of >5% (and existed in at least one of the samples). Ethylbenzene and 1,6-dimethylnaphthalene were only present in MM, and 1,2,4,5-tetramethylbenzene was only detected in MB. The compounds 1,3-dimethylbenzene and 1-ethylnaphthalene were missing in MB. Among benzene derivatives, naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, biphenyl, 1-ethylnaphthalene, 1,6-dimethyl-naphthalene, fluorene and phenanthrene, which belong to polycyclic aromatic hydrocarbon (PAH), should be noted. Recent epidemiological studies have revealed that dietary exposure to PAHs is associated with an increased risk of some human cancers [bib_ref] Environmental pollutants and breast cancer: epidemiologic studies, Brody [/bib_ref]. These compounds possibly come from environment contamination including air, soil and water, in which they are formed during the incomplete combustion of organic materials [bib_ref] Formation of polycyclic aromatic hydrocarbons from tobacco: The link between low temperature..., Mcgrath [/bib_ref]. Benzene derivatives are typically closely related to the odour of cereal. Among the benzene derivatives detected, naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, and 1-ethylnaphthalene have been identified as odour-active compounds in cereal; they are related to naphthalene aromatic aspects [bib_ref] Characterization of volatile aroma compounds in cooked black rice, Yang [/bib_ref] [bib_ref] Comparison of odor active compounds from six distinctly different rice flavor types, Yang [/bib_ref] [bib_ref] Isolation and identification of volatile compounds from wild rice grain (Zizania aquatica), Withycombe [/bib_ref]. Compared with other cereal, such as rice and buckwheat, more benzene derivatives were identified in the millet. These compounds, especially the odour-active benzene derivatives, may contribute to the flavour of millet and distinguish it from other cereal.
## Alcohols and ketones
The amounts and contents of alcohols and ketones detected in the samples were relatively small. In total, four, two and five alcohols were detected in BM, MM and MB. (Z)-2-Octen-1-ol was absent in BM and MM; the missing alcohols in MM were 1-pentanol and 1-octanol. Despite the small amounts of alcohols detected, most of these compounds were relevant to the odour of the cereal. Alcohols are generally formed by the decomposition of the secondary hydroperoxides of fatty acids [bib_ref] Lipid Degradation Products and Flavors, Grosch [/bib_ref]. They generally supply the fruity, floral and grassy aspects to cereal. Among the various alcohols detected, 1-pentanol, 1-hexanol, 1-octen-3-ol and 1-octanol have been identified as the odour-active compounds in rice and have fruity and plastic, green, mushroom and citrus aromatic characteristics, respectively [bib_ref] Characterization of volatile aroma compounds in cooked black rice, Yang [/bib_ref] [bib_ref] Comparison of odor active compounds from six distinctly different rice flavor types, Yang [/bib_ref] [bib_ref] Isolation and identification of volatile compounds from wild rice grain (Zizania aquatica), Withycombe [/bib_ref] [bib_ref] Potent aroma-active compounds of cooked korean non-aromatic rice, Park [/bib_ref].
In total, eight, six and five ketones were present in BM, MM, and MB. The compounds 2,5-octanedione and 2-heptadecanone and 1-octen-3-one were unique ketones in BM and MM, respectively. The missing ketones in MB were 2,5-octanedione and (E,E)-6,10,14-trimethyl-5,9,13-pentadecatrien-2-one. The autoxidation of fatty acids, particularly unsaturated fatty acids, has been proposed to form ketones [bib_ref] Lipid Degradation Products and Flavors, Grosch [/bib_ref]. Ketones typically provide the soapy and fruity characteristics of food. Among the various ketones detected, 2-heptanone (fruity) and 3-octen-2-one (rose) have been identified as odour-active compounds in rice [bib_ref] Comparison of odor active compounds from six distinctly different rice flavor types, Yang [/bib_ref]. Therefore, the contributions of these compounds to millet cannot be ignored.
## Hydrocarbons, acids and esters
A certain number of hydrocarbons, acids and esters were also identified in all of the samples. In total, six, six and four hydrocarbons, two, three and two acids, and two, three and three esters were detected in BM, MM and MB, respectively. Except for hexadecanoic acid, whose content was 8.98-35.77%, most of these compounds were present in small quantities. The hydrocarbons identified in the samples included a homologous series of n-hydrocarbons ranging from C 12 to C [bib_ref] Potent odorants of the wheat bread crumb: Differences to the crust and..., Schieberle [/bib_ref]. Dodecane was absent in MM and MB. The other absent hydrocarbon in BM and MB were nonadecane and tridecane and heptadecane, respectively. These hydrocarbons could result from the decarboxylation and splitting of carbon-carbon chains of higher fatty acids [bib_ref] Lipid Degradation Products and Flavors, Grosch [/bib_ref]. Acids identified in the samples included a homologous series of n-acids ranging from C 14 to C 16 . Among the detected acids, tetradecanoic acid and pentadecanoic acid were absent in MB and BM, respectively. Esters identified in the samples were benzene and hexadecanoic acid esters. Hexadecanoic acid methyl ester was only present in MM, and propyl benzoate and hexadecanoic acid ethyl ester were absent in BM and MM, respectively. Hydrocarbons, acids and esters, typically have relatively high flavour thresholds and likely have little contribution to the odour of the millet. However, some of them are present at high levels in the samples, particularly hexadecanoic acid, and may thus play roles in the overall flavour.
## Heterocycles and sulphur-containing compounds
Some heterocycles and sulphur-containing compounds were also detected in the samples. In addition to 2-pentylfuran, which was present in all of the samples, furfural was detected in MM and MB; other heterocycles were only detected in the bran sample. These compounds, such as 3-furaldehyde (almond) [bib_ref] Effect of temperature and pH on the generation of flavor volatiles in..., Bredie [/bib_ref] , furfural (sweet, woody and almond) [bib_ref] Sensory and instrumental analyses of volatiles generated during the extrusion cooking of..., Parker [/bib_ref] , 2-furanmethanol (sweet and honey) [bib_ref] Compounds contributing to the characteristic aroma of malted barley, Beal [/bib_ref] and 1-(2-furanyl)ethanone (sweet, almondy and nutty) [bib_ref] Compounds contributing to the characteristic aroma of malted barley, Beal [/bib_ref] , have been identified as odour active compounds in some cereals and derived products. In addition, 2-pentylfuran should be noted, as it was detected in all the samples. The compound 2-pentylfuran has a relatively low threshold and a vegetal aromatic note [bib_ref] Gas chromatography olfactometry analysis of the volatile compounds of two commercial Irish..., Machiels [/bib_ref]. The characteristic aromatic notes of sulphur-containing compounds have been described as nutty, roasted and corn-like. Dimethyl sulphide and 2-acetylthiazole contribute a cooked corn and nutty aroma to food, respectively [bib_ref] Changes in volatile compounds of traditional Chinese Nanjing water-boiled salted duck during..., Liu [/bib_ref]. These compounds are less abundant in cereal and may play an important role in millet.
## Experimental section
# Materials
Foxtail millet (Setaria italica), var. JIGU NO19, was obtained from the Institute of Millet Crops, Heibei Academy of Agriculture and Forestry of China, and was stored in a refrigerator at −18 °C until use. After dehusking, 10% of the BM was milled out using an experimental mill (JLMZJ, Shanghai, China), and then the MM and MB were collected. The water, protein, fat, ash and carbohydrate contents were analysed [fig_ref] Table 2: General compositions of BM, MM and MB [/fig_ref]. Water, protein, fat, ash, and carbohydrate content were estimated by standard AOAC Methods.
## Reagents
The water used in the study was purified using a Millipore-Q system (Millipore Corp.
## Simultaneous distillation extraction (sde)
The extraction was performed using a modified Likens-Nickerson SDE apparatus for 2 h. The sample (200 g) and distilled water (2000 mL) were added to a 5000-mL round-bottom flask. Diethyl ether (50 mL) was added to another 100 mL round-bottom flask. Both flasks were connected to the apparatus, and additional distilled water was added into the central arm. The flask containing diethyl ether was heated using a water bath at 40 °C, and the flask containing the millet sample and distilled water was heated to the boiling point using an electric heating jacket. After extraction, the distillate in the 100-mL flask was dried over anhydrous sodium sulphate (5 g), concentrated using vacuum rotary evaporation and stored in headspace vials. Extraction of each sample was performed in triplicate. The results are reported as the mean values of relative peak area percent ± SD (standard deviation).
## Gas chromatography-mass spectrometry (gc-ms)
GC-MS was performed using an HP 5975B quadrupole mass selective detector (Agilent Technologies, Palo Alto, CA, USA). The mass spectral ionisation temperature was set to 230 °C. The mass spectrometer was operated in the electron impact ionisation mode at a voltage of 70 eV. Mass spectra were taken over an m/z range of 30-400. The flow rate of the helium carrier gas on the DB-5 column (30 m × 0.25 mm ID, 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA) was 1 mL/min. The analysis was performed in the splitless mode, and the injector temperature was 250 °C. The column was held at 40 °C for 3 min and then increased from 40 °C to 220 °C at a rate of 4 °C/min, held at 220 °C for 2 min, and finally increased to 230 °C at a rate of 8 °C/min and held for 3 min.
## Identification of components
The volatile components were identified by comparing their mass spectra with the mass spectra from MS libraries (NIST 05, WILEY 7.0). When available, the MS identifications were confirmed by comparing the GC retention times of the analytes with those from pure standards. The linear retention indices (RI) of the compounds were calculated using a series of n-alkanes (C8-C20, Sigma-Aldrich, Germany) injected under the same conditions. When standard chemicals were not available, tentative identification was achieved by matching the mass spectra and RI. The results are provided in [fig_ref] Table 1: Volatile components identified in BM, MM and MB [/fig_ref].
# Conclusions
This work presents the first study on the volatile compound contents of BM, MM and MB. A total of 65 volatile compounds were identified. Aldehydes and benzene derivatives were the most numerous among all of the compounds. Differences were noted in the volatile compounds present in different samples. In total, 34 compounds were detected in the BM, MM and MB. Apart from the aromatic molecules present in all the samples, compounds that were present only in brown millet, milled millet or bran were also identified. Compared with BM and MM, more heterocycles were present in MB. These differences may result in the different overall aromas of BM, MM and MB.
[table] Table 1: Volatile components identified in BM, MM and MB.Table 1. Cont. MS: confirmed by mass spectrum analysis; RT: identified by retention time of standard compounds; RI: identified by retention index; n.d.: not detected. [/table]
[table] Table 2: General compositions of BM, MM and MB. [/table]
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Wuhan mobile cabin hospital
This study aimed to systematically analyze the effect of Wuhan mobile cabin hospitals (WMCHs) on the novel coronavirus-caused pneumonia prevention and control in China. Between February 5, 2020 and March 10, 2020, a total of 16 mobile cabin hospitals were constructed in 3 batches to offer over 13,000 beds and admitted more than 12,000 patients in Wuhan City. The strategy of implementing WMCHs in 3 batches played a key role in fighting against COVID-19 in China. (1) The first batch of WMCHs increased hospital admission capacity of COVID-19 patients in Wuhan, which showed initial effect on COVID-19 epidemic control.(2)The operation of the second batch of WMCHs greatly contributed to the rapid growth in discharged patients. (3) After launching the third batch of WMCHs, the COVID-19 epidemic situation in Wuhan improved considerably. The last batch of WMCHs made a substantial contribution to defeating the COVID-19 epidemic in Wuhan.Abbreviations: COVID-19 = the novel coronavirus-caused pneumonia, MCH = mobile cabin hospital, MCHs = mobile cabin hospitals, TCM = traditional Chinese medicine, WMCH = Wuhan mobile cabin hospital, WMCHs = Wuhan mobile cabin hospitals.
# Introduction
The novel coronavirus-caused pneumonia (COVID-19) played havoc with global health and safety. Many countries in the world have declared national emergency, seriously affecting international security, trade, and economy. From December 2019, a COVID-19 epidemic outbreak occurred in Wuhan City, China.
Wuhan's healthcare system was overwhelmed by patients flooding into local hospitals. As a result of a severe shortage of medical resources, the Chinese government decided to launch the mobile cabin hospital (MCH) construction program, which achieved remarkable results. In order to provide experience and evidence for policy and decision making to be used by other countries fighting the COVID-19 pandemic, this study systematically analyzed the features of Wuhan mobile cabin hospitals (WMCHs') design and management and the operational effect of WMCHs.
# Background
MCH is a field mobile medical platform that can be swiftly transported and built in the form of a medical mobile cabin with an integration of medical services and medical technology support. [bib_ref] China medicine Encyclopedia -disaster medicine, Wang [/bib_ref] [bib_ref] Setup and management of the national emergency medical rescue mobile hospital operating..., Zhang [/bib_ref] According to the data released by Wuhan Health Commission, on January 31, 2020, only 389 out of the 6641 beds in 23 designated hospitals in Wuhan remained available.Hospitals in Wuhan soon reached their capacities. Therefore, it is necessary to launch WMCHs.
# Methods
The operation of 16 WMCHs is systematically summarized, the characteristics of 7 WMCHs are described, and the implementation effect of WMCHs is analyzed by combining official case data.
wards. The government mobilized 22 national medical emergency rescue teams, 3 mobile P3 laboratories, 2 batches of 30 medical teams, 15 nursing teams, and 1 radiographer team from all over China, totaling over 7000 healthcare workers and laboratory technicians.A total of 16 MCHs were constructed to provide more than 13,000 beds and admitted over 12,000 patients in Wuhan. On average, 1 of every 4 confirmed patients received treatment in a WMCH. On February 27, the situation of "a hospital bed being hard to come by" changed fundamentally as the quantity of hospital beds outstripped patient demand in 20 days. "No cross-infection, no deaths, no readmission" were achieved in WMCHs by the time all WMCHs were closed.
## Typical cases of wmchs
## Jianghan mch
Jianghan MCH was one of the first MCHs in Wuhan. It was also the MCH with the largest number of beds and the largest number of admitted and discharged patients. Jianghan MCH was in close proximity of the Union Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology, which enabled timely transfer of critical patients to the hospital. After 48 hours of construction, Jianghan MCH with 120 beds was officially open to receive mildly symptomatic patients at 10 PM on February 5, 2020.
Each WMCH ward was divided into 4 main areas. The first floor was divided into a west, central, and east districts. Each Basic information of 16 MCHs. section was then divided into 8 small sections with 50 to 60 camp beds per small section. The districts were subdivided by high partitions, and beds were subdivided by wooden planks. The second floor was set up as a large area subdivided into 11 sections with passageways specially designed to transport drugs and medical supplies. Each floor had 2 nurses' stations, a fully sealed emergency room, and a dining room for healthcare workers. [bib_ref] Discussion on the Module Hospital Construction Management of COVID-19 Epidemic Situation, Liu [/bib_ref] A 2-meter-wide passageway for garbage and waste disposal was set up at the back door of Wuhan International Convention and Exhibition Center, through which all garbage and waste would be transported to designated locations for disinfection and sterilization. Jianghan MCH also developed an online application that was used by patients to "place orders" to request services such as living, healthcare, and environmental hygiene services on their mobile phones. Healthcare workers or volunteers in the MCH would "accept orders" online and provide services to meet patients' needs.
## Name of wmchs location
## Wuchang mch
Wuchang MCH was the first WMCH launched on February 5, 2020. Twenty-eight patients were discharged from this WMCH 6 days after its launch. It operated continuously for 35 days, and was considered to be the first WMCH launched, the first WMCH establishing a temporary Party Committee, the first WMCH discharging a patient, the first WMCH offering psychological counseling for patients, and the last WMCH closed.
A series of innovative operational and management measures were developed and implemented, and produced positive results. These measures were used for reference by other hospitals. Such measures included conducting level-4 medical rounds and ensuring good patient treatment and care; setting up a comprehensive testing and examination programs that included blood tests, vehicle-mounted CT exams, nucleic acid tests; fully administering treatment of traditional Chinese medicine (TCM) and devising physical exercises; reinforcing efforts of prevention of doctor-patient cross-infection and ensuring goods and materials supply; organizing national psychological counseling expert teams to provide effective psychological counseling services for patients; setting up the first temporary Party committee to promote patient autonomy within MCH.
## Dongxihu mch
Dongxihu MCH was rebuilt from Wuhan Living Room Cultural Expo Center within 3 days. It had 3 cabins, 16 medical workstations, 2 pharmacies, 1 patient entrance, and 1461 beds. It was the largest WMCH in the first batch of MCHs built in Wuhan.
Over 3000 people were in the MCH during peak period, including 1434 patients and 1254 healthcare workers. Four members of the psychological intervention team had to serve 1433 patients and healthcare workers during peak hours. Moreover, its patient satisfaction rate reached 99.44%. Dongxihu MCH became the "cabin of life" that offered a great quantity of beds, admitted a large number patients, and achieved high operational efficiency during the peak period of COVID-19 epidemic.Dongxihu MCH was not only equipped with a "MCH library", but also provided with "MCH digital culture window" application, which contained over 8000 videos, 80,000 e-books, and 420,000 digital audio resources to enrich patients' lives and relieve patient anxiety.
## Jiangxia dahuashan mch
Jiangxia Dahuashan MCH was the only MCH fully taken over by TCM medical team. The national TCM medical team consisted of 209 TCM experts and healthcare workers from first-class hospitals in Tianjin City, Jiangsu Province, Henan Province, Hunan Province, and Shanxi Province. The team used combined therapy of Chinese and Western medicine to treat mildly symptomatic patients with stress on TCM.
Treated with only Chinese medicine decoctions, each patient took one No. 2 and No. 3 decoction in the morning and in the evening. Moreover, according to patient's symptoms, experts also developed 4 routine prescriptions for fever, dry cough, spleen and stomach insufficiency-cold, and anxiety-induced insomnia, assisted by western medicine techniques such as oxygen uptake and fluid transfusion as well as other traditional Chinese nonpharmaceutical treatment such as auricular therapy, moxamoxibustion, and acupoint application. Healthcare workers also led patients to do acupoint flapping exercises, and played Baduanjin qigong and Tai Chi to strengthen muscles and bones and pass on confidence in defeating the disease.
## Huangpi mch
Huangpi MCH was divided into cabin A and cabin B. Cabin A was a 130-bed ward and Cabin B was a 70-beds ward. Five medical teams were stationed there to assist. Within 26 days of operation, a total of 223 patients were admitted and treated and 156 patients were cured.
With the support of remote ultrasound technology from 5G Smart Medical Innovation Lab in Zhejiang General Hospital, "ultrasonic robot" was introduced to carry out remote ultrasonic exams for the patients in Huangpi MCH via 5G and ultrasound technologies. "Ultrasonic robot" could not only report changes in the lungs by assessing pulmonary edema, but also conduct fullbody imaging assessment in a convenient and rapid way. The ultrasonic robot played a crucial part in imaging assessment of changes in pneumonia conditions.
## Hanyang sports school mch
On February 22, 2020, Hanyang Sports School MCH started to officially receive mildly symptomatic patients with COVID-19. The application of HIS remote system for word rounds was a highlight of Hanyang Sports School MCH. Through voice control, doctors in and outside the cabin were able to make ward rounds concurrently and record patient's vital signs and changes in disease conditions. Meanwhile, the physician team outside the cabin could make timely adjustment to treatment plans based on patients' conditions. This system helped reduce physicians' daily rounds time from over 4 hours to 2 hours.
In addition, Hanyang Sports School MCH established a Hanyang Sports School MCH Patient Management Group to motivate patients to participate in daily healthcare management work. The MCH encouraged patients to be "unit leaders" to take charge of distributing boxed meals and fruit, filling medications, among other work. This helped reduce healthcare workers' burden while pacifying patients through mutual aid and mutual support among patients.
## Provincial party school mch
Provincial Party School MCH was equipped with 932 beds and was divided into 8 wards with doctors' offices, nurses' stations, emergency rooms, and hot water rooms. The MCH was also equipped with a mobile imaging trailer and an AI robot for food and drugs delivery. At 9 PM on February 19, 2020, as the first 23 patients being admitted, the AI robot officially went on duty. The robot had the ability of fully autonomous locating and navigation, autonomous route planning, and multi-point cruising delivery. In addition, the robot could also deliver and distribute supplies such as medications, laboratory test reports, protection equipment, and disinfection tools. Using robots for delivery of food, lab test reports, and medications lightened healthcare workers' workload, boosted MCH's operational efficiency, and avoided cross-infection between hospital staff and patients, which could ultimately reduce the risk of epidemic spread. [fig_ref] Figure 1: Figure 1 [/fig_ref] shows trends of cumulative cases overtime and launch times of WMCHs. During the entire period of WHMC operation, the overall growth trend of new cases was retarded in Wuhan, despite a surge in the number of confirmed cases. There was also a sharp rise in the cumulative number of cured cases followed by an
## Implementation effect of wmchs
# Discussion
On February 4 (before launching WMCHs), Wuhan had a total of 8351 confirmed cases, 335 recovered cases, and 362 deaths, with mortality rate reaching 4.33%.Wuhan was seriously and Consequently, many patients were unable to receive timely and effective treatment, which was another important reason for the high percentage of severe cases and high case fatality rate in Wuhan. Meantime, patients being unable to be isolated and treated in time also added uncertainties and posed a great threat to COVID-19 epidemic control. Isolation of infected patients (ie, source of infection) was the most effective way to control the outbreak. After launching WMCHs, the increase of cumulative confirmed cases slowed down in Wuhan as cumulative recovered cases increased significantly. The number of cumulative deaths fluctuated slightly but the general trend was stable. As all WMCHs were closed on March 10, 2020, Wuhan had altogether 49,978 confirmed cases, 33,041 recovered cases, and 2423 deaths.Therefore, the WMCHs played an important role in defeating the COVID-19 epidemic.
## Control effect of the first batch of wmchs
Stage 1: the number of daily new cases in Wuhan decreased slightly, the number of newly recovered cases increased significantly on a daily basis, and the number of new deaths remained stable. The number of daily new cases remained above 1500. The number of newly recovered cases per day increased from less than 200 to over 350. The numbers indicated that the launch of WMCHs brought about effects on raising diagnosis speed and increasing recovered cases. The first batch of WMCHs improved hospital admission capacity for COVID-19 patients in Wuhan, reflecting WMCHs' early effect on epidemic control.
It is important to note that, on February 12, 2020, the number of daily new cases was 13,436 in Wuhan, close to the number 19,558 reported on February 11. This was due to a change in disease diagnosis standard. According to the "COVID-19 Diagnosis and Treatment Guidelines" previously release by China NHS, diagnostic criteria on confirmed cases referred to detection of novel coronavirus nucleic acid in respiratory tract specimen or blood specimen using real-time fluorescent RT-PCR tests (ie, tested positive for novel coronavirus nucleic acid). However, the daily new cases reported on February 11 included clinically diagnosed cases. According to the "COVID-19 Diagnosis and Treatment Guidelines (trial version 5)," Hubei Province added "clinical diagnosis" to diagnostic classifications of COVID-19 cases that suspected cases with pneumonia imaging features could be diagnosed as confirmed cases. Since the epidemic, the shortage of testing kits led to a delay in COVID-19 diagnosis, leaving a large number of suspected cases in Wuhan. Adding clinically diagnosed cases might seem to have increased the number of confirmed cases, but it actually allowed patients to be treated as early as possible, which improved recovery rate and was a necessary step for epidemic control.From February 5 to February 7, adhering to the principle to "admit and treat everyone necessary," Wuchang MCH, Jianghan MCH, and Dongxihu MCH became the first batch of WMCHs launched in Wuhan. Six days later, on February 11, the first group of 34 COVID-19 patients were discharged from Jianghan MCH and Dongxihu MCH. This showed that, to some extent, WMCHs were effective on control of COVID-19 epidemic in Wuhan. The first batch of WMCHs carried out level-4 hospital rounds including inspection rounds by MCH president, and were provided with testing programs to conduct blood tests, CT exams, nucleic acid tests, and others. The WMCHs administered TCM treatment on a full scale and promoted a series of innovative measures such as "patient autonomy" to inform subsequent construction and operation of WMCHs.
## Control effects of the second batch of wmchs
Stage 2: the number of daily new cases decreased, the number of daily newly recovered cases increased significantly, and the number of daily new deaths remained stable. On February 19, the number of daily new cases (615 cases) fell below 1000 for the first time. On February 20, the number of daily newly recovered cases (766 cases) exceeded the number of daily new cases (319 cases) for the first time in Wuhan. With the second batch of WMCHs going into operation, the number of discharged patients in Wuhan presented a rapidly ascending trend, and Wuhan's admission and treatment capacities was quickly improved. This further demonstrated the control effect of the second batch of WMCHs.
The second batch of WMCHs further stressed the "peopleoriented and pragmatical" characteristics,while absorbing experience from constructing the first batch of WMCHs, to provide a full coverage of Wi-Fi service with installation of water heaters and air conditioning, along with boxed meals and service teams. This enabled WMCHs to have better admission conditions so as to raise patients' willingness of being admitted into WMCHs and effectively increase WMCH admission rates, helping to further achieve the goal of "admitting everyone necessary."
The introduction of "ultrasonic robot" to Huangpi MCH allowed clinicians to have a clearer understanding of each patient's condition using technologies like 5G and ultrasound to help make evidence-based follow-up treatment plans. Jiangxia Dahuashan MCH was the first hospital to focus on TCM therapies. It replied on combined therapies of Chinese and Western medicine with stress on traditional Chinese medicine, and carried out training activities such as Baduanjin qigong and Tai Chi. The TCM-oriented therapies played an important role in controlling disease progression for mild and semi-mild cases.
## Control effects of the third batch of wmchs
Stage 3: the number of daily new cases remained stable with slight decrease. On February 27, the number of daily newly recovered cases increased to 2498, which was the largest number of patients discharged during WMCH operation. The number of daily new deaths remained stable with slight decline. On February 24, the number of daily new cases fell below 100 (74 cases) for the first time and the number of daily newly recovered cases (1391 cases) exceeded 1000 for the first time. The number of daily newly recovered cases remained over 1000, except for March 9. During stage 3, Wuhan saw significant improvement in COVID-19 epidemic control. Thus, the last batch of WMCHs accomplished periodic success in COVID-19 epidemic control.
High-tech measures played an important role in the operation of the third batch of WMCHs. The wide adoption of high-tech products, such as integrated cloud platforms, 5G networks, remote diagnosis, unmanned delivery vehicles, and remote monitoring, reduced pressure of medical staff shortage and goods and materials transportation issues, improved hospitals' level of medical precision, and lowered workers' risk of infection.
As of February 25, Hubei Provincial Library, Wuhan City Library, regional libraries in Wuhan, Wuhan Xinhua Bookstore,100:3 Medicine and other organizations jointly established 23 "WMCH Libraries" and set up online cultural service platforms like special column for epidemic information and service, online digital reading platform, and "MCH digital and cultural window" to help relieve patients' psychological pressure and enhance patients' confidence in fighting the disease.
# Conclusion
Launched in 3 batches, the WMCHs have played a key role in defeating the COVID-19 epidemic in Wuhan, China.
(1) Launching the first batch of WMCHs produced effects on speeding up diagnosis and increasing recovered cases, expanding hospital admission capacities of COVID-19 patients in Wuhan. The first batch of WMCHs exhibited initial effects on COVID-19 epidemic control.
[fig] Figure 1: Figure 1. Trends of cumulative cases during WMCH operation in Wuhan. Note: (WMCHs' launch time are shown) Wuchang MCH Jianghan MCH Dongxihu MCH Hanyang MCH Qiaokou MCH Huangpi MCH Jiangan MCH Wuhan Economic Development Zone MCH Qingshan MCH Optical Valley Exhibition Center MCH Zhuankou MCH East Lake Rihai MCH Jianghan Economic Development Zone MCH Hanyang Sports School MCH Jiangxia Dahuashan MCH Provincial Party School MCH. [/fig]
[fig] Figure 2: Daily new case trends during WMCH operation in Wuhan. Note: Main axis refers to daily new cases (left). Secondary axis refers to daily new deaths and daily newly recovered cases (right). MCHs = mobile cabin hospitals, WMCHs = Wuhan mobile cabin hospitals. of infected patients and a shortage of hospital beds. [/fig]
[fig] 2: The second batch of WMCHs led to a rapid increase in the number of discharged patients, and hospital admission and treatment capacities were considerably enlarged in Wuhan, which further demonstrated the control effects of the second batch of WMCHs.(3) With the third batch of WMCHs, the condition of COVID-19 epidemic in Wuhan showed noticeable improvement. The last batch of WMCHs achieved periodic success in epidemic control, playing a key part in the defeat of COVID-19 epidemic in Wuhan. The construction and operational modes of WMCHs may provide valuable experience and evidence for other countries currently affected by the COVID-19 pandemic. Author contributions Conceptualization: Yuyao Zhang, Gang Sun. Data curation: Haiqian Chen, Xiaohan Wang. Formal analysis: Yuyao Zhang. Funding acquisition: Gang Sun. Project administration: Yuyao Zhang, Gang Sun. [/fig]
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Hepatitis B Reactivation During Immunosuppressive Therapy or Cancer Chemotherapy, Management, and Prevention: A Comprehensive Review
Context: Due to the close relationship between the immune system and the hepatitis B virus (HBV) replication, it is essential to monitor patients with current or past HBV infection under any type of immunosuppression. Cancer chemotherapy, immunosuppressive therapies in autoimmune diseases, and immunosuppression in solid organ and stem cell transplant recipients are the major reasons for hepatitis B virus reactivation (HBVr). In this review, the challenges associated with HBVr are discussed according to the latest studies and guidelines. We also discuss the role of treatments with different risks, including anti-CD20 agents, tumor necrosis factor-alpha (TNF-α) inhibitors, and other common immunosuppressive agents in various conditions. Evidence Acquisition: Through an electronic search of the PubMed, Google Scholar, and Scopus databases, we selected the studies associated with HBVr in different conditions. The most recent recommendations were collected in order to reach a consensus on how to manage patients at risk of HBVr.Results: It was found that the positive hepatitis B surface antigen (HBsAg), the high baseline HBV DNA level, the positive hepatitis B virus e antigen (HBeAg), and an absent or low hepatitis B surface antibody (HBsAb) titer prior to starting treatment are the most important viral risk factors. Furthermore, rituximab, anthracycline, and different types of TNF-α inhibitors were identified as the high-risk therapies. By analyzing the efficiency of prophylaxis on the prevention of HBVr, it was concluded that those with a high risk of antiviral resistance should not be used in long-term immunosuppressants. Receiving HBV antiviral agents at the commencement of immunosuppressant therapy or chemotherapy was demonstrated to be effective in decreasing the risk of HBVr. Prophylaxis could also be initiated before the start of therapy. For most immune suppressive regimes, antiviral therapy should be kept up for at least 6 months after the cessation of immunosuppressive drugs. However, the optimal time of prophylaxis keeping should be increased in cases associated with rituximab or hematopoietic stem cell transplants. According to the latest studies and guidelines from different bodies, recommendations regarding screening, monitoring, and management of HBVr are outlined.Conclusions:Identification of patients at the risk of HBVr before immunosuppressive therapy is an undeniable part of treatment. Starting the antiviral therapy, based on the type of immunosuppressive drugs and the underlying disease, could lead to better management of disease.
## Context
It is estimated that more than 2 billion of the world's population have experienced the hepatitis B virus (HBV) infection during their lifetime, and there are approximately 350 million patients with chronic hepatitis B (CHB). Generally, patients with HBV can be divided into four distinct phases: (i) the immunotolerant phase; (ii) the immune active phase; (iii) the low-replication phase; and (iv) the recovery phase. Everyone who has been exposed to HBV infection is in danger of the infection reactivating. In patients with CHB who are under immunosuppressive therapy, HBV replication will increase dramatically due to im-paired cellular and humoral immunity. Following the termination of immunosuppressants, reconstitution of the host immunity results in a serious flare-up of the disease due to cytotoxic activity of the immune cells. This occurrence is considered to be the reactivation of HBV after an increase in HBV replication because of impaired immune responses. HBV reactivation (HBVr) can also occur after immunosuppressive chemotherapy in patients with occult HBV infection (OBI) (HBV DNA and the antibodies to hepatitis B core antigens are present without detectable hepatitis B surface antigens) and resolved HBV (the presence of HBV antibodies without HBV DNA and hepatitis B surface anti-gens). In hepatitis B surface antigen (HBsAg) carriers, immunosuppressant agents that induce weakened immune responses lead to an increase in viral replication as well as removing the immune system balance. This results in the growth of viral replication, which may be followed by rising liver enzymes, liver disease, and even death. Furthermore, HBVr causes premature termination of immunosuppressive chemotherapy or a delay in treatment schedules [bib_ref] Hepatitis B virus reactivation in breast cancer patients receiving cytotoxic chemotherapy: a..., Yeo [/bib_ref]. In individuals who cleared HBsAg, including occult or resolved patients, covalently closed circular DNA (cc-cDNA) can persist. In conditions with declined antiviral immune responses, such as immunosuppressive therapy or chemotherapy, viral core particles that migrated to the hepatocyte nucleus at the time of infection can be repaired to form the cccDNA and rebuild the viral replication cycle. HBV cccDNA acts as the template for viral messenger RNA (mRNA) transcription. mRNA is then translated in the cytoplasm to produce the viral surface, core, polymerase, and X proteins [bib_ref] Hepatitis B virus infection: pathogenesis, reactivation and management in hematopoietic stem cell..., Moses [/bib_ref]. In addition to immunosuppressive therapy or chemotherapy, HBVr may happen spontaneously in the presence of virus mutation.
Immunosuppressant therapy can be performed by multiple agents. Some of them are considered to be the high-risk treatments for OBI/resolved patients in addition to HBV carrier patients. Thus, these treatments should commence with special attention and continuous monitoring of the viral status. Anti-CD20 agents are the most reported ones that are categorized in this group. In these treatments, B cells, which are the main players in suppressing viral replication, will be targeted. Thus, considering the stability of cccDNA in the nucleus, a rapid decrease in B lymphocytes (B cells) population, even in the absence of HBV replication, leads to the reactivation of viral activity. In contrast, some other agents are considered as risk factors only in positive HBsAg patients, but not a serious menace in occult or resolved patients. For example, HBVr is rare in non-HBsAg carriers, due to the use of methotrexate (MTX) or azathioprine (AZA). There is evidence that these treatments manipulate cellular responses more than humoral responses. The suppression of T cell immunity can cause HBVr in HBsAg carriers with low HBV replication. In treatments with MTX and AZA, the levels of different cytokines were significantly decreased, including interferon-γ (IFNγ) and tumor necrosis factor-alpha (TNF-α), which are responsible for the suppression of viral replication.
In order to overcome or at least decrease, the risk of the global menace of HBVr, there are two available options in addition to identifying the patients in a high-risk condition and in continuous monitoring. The first option, which is not practical most of the time and may be followed by irreparable consequences, is the reduction or discontinuation of the responsible therapy. The second option, and the most common, is the initiation of prophylactic therapy before or at the same time of starting immunosuppressant medications. Almost all studies have confirmed the effectiveness of this approach. Despite this consensus, the timing of initiation and optimal duration of antiviral therapy has remained controversial, especially after rituximab administration or hematopoietic stem cell transplant (HSCT) [bib_ref] Prophylactic antiviral therapy in allogeneic hematopoietic stem cell transplantation in hepatitis B..., Liao [/bib_ref]. The risk of drug resistance is another critical factor, which has been warned of by various bodies [bib_ref] Hepatitis B Virus Screening for Patients With Cancer Before Therapy: American Society..., Hwang [/bib_ref]. In this review, the latest findings associated with the risk of HBVr, due to several treatment strategies, were evaluated. Different approaches for the management and prevention of HBV-infected patients in multiple conditions were also discussed.
## Immune system response and hepatitis b virus infection
Host immune response plays a critical role in HBVrelated hepatocyte damage. Strong cellular and humoral immune responses are associated with resolution and long-life antiviral responses in HBV infection [bib_ref] Induced immunity against hepatitis B virus, Said [/bib_ref]. However, weakening of the cellular immune responses could cause an increase in HBV replication that leads to HBVr. Immunosuppressive therapy establishes the weak and narrowly focused immune responses due to a decrease in proliferation of the involved immune cells in inhibition viral replication, including B cells, T cells, and NK cells. It is followed by an increase in HBV replication and then HBVr. In addition to this indirect mechanism on HBVr induction, it was found that glucocorticoid stimulates hepatitis B viral gene expression, which finally results in an increase in viral replication. It is considered to be the direct effect of this type of immunosuppressive therapy.
In HBV infection, multiple cells and cytokines are involved. T cells and B cells are responsible for adaptive immunity, which plays the central role in control and clearing pathogens. NK cells are considered to be the other involved cells during infections. These cells are defined as the effector lymphocytes of an innate immunity endowed with constitutive cytolytic functions. CD4+ T cells play an important role in HBV infection by the secretion of T helper 1 associated cytokines, such as IFN-γ and TNF-α, which down-regulate HBV replication. These are major antiviral cytokines that inhibit HBV replication and promote HBV clearance [bib_ref] Use of tumor necrosis factor alpha inhibitors in hepatitis B surface antigen-positive..., Carroll [/bib_ref]. In some autoimmune diseases, such as rheumatoid arthritis (RA), in which TNF-α is known as the major cause of damage, inhibition of this cytokine with some anti-TNF-α agents, including infliximab, etanercept, and adalimumab, is frequently used [bib_ref] Treatment of rheumatoid arthritis with tumour necrosis factor inhibitors, Mewar [/bib_ref]. It was found that inhibition of this proinflammatory cytokine leads to an increase in viral replication [bib_ref] Tumor necrosis factor (TNF)-alpha and TNF receptors in viral pathogenesis, Herbein [/bib_ref] [bib_ref] Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B..., Kasahara [/bib_ref]. In 2 Hepat Mon. 2016; 16(4):e35810.
fact, there is a deep relation between TNF-α and viral status in HBV infection. Several studies that found a correlation between the TNF-α gene polymorphism, HBV clearance, and CHB support this idea [bib_ref] TNFalpha promoter region polymorphisms affect HBV virus clearance in southern Chinese, Xu [/bib_ref] [bib_ref] Tumour necrosis factor-alpha promoter region polymorphisms affect the course of spontaneous HBsAg..., Kao [/bib_ref] [bib_ref] The relationship between tumour necrosis factor-alpha gene polymorphism and susceptibility and clearance..., Zhang [/bib_ref]. CD4+ T cells also act as the CD8+ T cells' promoters as well as inducers of B cells' activation and differentiation, which results in vigorous immune responses. CD8+ T cells destroy infected hepatocytes in addition to their non-lysis manner. HBV-specific CD8+ T cells inhibit HBV production through the production of some cytokines, such as IFN-γ and TNF-α, which are able to suppress HBV gene expression and replication without destroying the infected cells. Phillips et al.revealed that CD8+ T cells inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. In fact, inhibition of viral replication is mediated predominantly by non-cytolytic mechanisms via IFN-γ and TNF-α contribution. In addition to T cells, B cells are deeply involved during HBV infection. These cells play the role in antigen presentation as well as viral clearance (19). It was found that the hepatitis B core antigen (HBcAg) can also cause the activation of naïve human B cells in vivo in a Tcell-independent way (20). Recently, NK cells have been increasingly valued in their effects on HBV infection. In both the early immune responses and the chronic phase, these types of cells contribute to HBV infection. Wu et al. (21) reviewed the role of NK cells in HBV infection, discussing the mechanisms of action of this arm of the innate immune system in HBV infection.
The lack of, or exhaustion of, one of these cells and cytokines may result in the growth of viral replication and the development of HBVr in HBV inactive carriers, occult, and resolved HBV patients. The weakening or the absence of virus-specific T-cell reactivity is usually observed during human CHB infection (22). This situation causes poor effector cytotoxic activity and impaired cytokine production.
## Hepatitis b virus reactivation definition
In general, during immunosuppressive therapy, HBVr can occur after high HBV replication, which has no clinical sign. Indeed, HBVr starts with immune reconstitution, and has the same symptoms as acute hepatitis. Because HBVr is caused by immune reconstitution, and is not directly due to viral replication, patients will not benefit from the initiation of antiviral therapy during this phase. Recently author discussed about distinction between the HBVr and HBV flare and proper approaches to management of each condition (23).
In the literature, there are several definitions of HBVr, which range from an asymptomatic rise in alanine transaminase (ALT) levels to severe hepatitis, which may result in serious liver injury, liver failure, and death (24).
In some studies, the elevation of ALT and HBV DNA levels > 2000 IU/mL in inactive HBsAg carriers or resolved hepatitis patients is considered to be a factor of reactivation . Reverse seroconversion from the hepatitis B virus e antibody (HBeAb) to the hepatitis B virus e antigen (HBeAg) is also known as another sign of HBVr (26). HBsAg may change from negative to positive during HBVr, which is known as reverse seroclearance. This phenomenon has been considered as HBVr in the literature (27, 28). This could happen in the presence or the absence of HBsAb. Based on the literature, a 10-fold increase in HBV DNA levels (l-log) or more from the baseline level in serum can be considered as evidence of HBVr (24, 29). It is essential to know the similar conditions to HBVr, which are not considered as reactivation. Acute HBV, the immune clearance phase in patients with CHB, drug resistance, co-infection, and hepatotoxins can cause the development of signs, which are the same as HBVr and may be confused with reactivation.
## Conditions with the risk of hepatitis b virus reactivation
HBVr is not limited to positive HBsAg patients. It also may develop during or after immunosuppressive therapy in patients with a positive antibody to the hepatitis B core antibody (anti-HBc) (30-32). Various studies reported clinical and virological reactivation in patients with OBI (30, 31). Furthermore, reports revealed that HBVr may also occur in resolved patients after immunosuppression (32, 33).
All of the discussed reactivation forms were in a situation of past or current infections. However, Feeney et al. (34) reported a case that questions the dependability of anti-HBc testing before immunosuppressant therapy. It was claimed that pre-screening serology indicated negative HBsAg and anti-HBc. However, the patient developed a weak HBsAg with a low HBV DNA level (< 1000 IU/mL) followed by rituximab-based treatment. HBV DNA reached a peak at 1 × 10 6 IU/mL after 5 months, which was treated with tenofovir (TNV). Interestingly, the patient remained negative anti-HBc.
Generally, each agent that causes a weakening of immune responses may lead to HBVr. HBsAg carriers, patients with OBI/resolved HBV infection who receive chemotherapy, those treated for autoimmune diseases, or those undergoing transplantation, are at risk of HBVr.
## Chemotherapy
HBVr is a common problem in patients with CHB or even recovered patients who are under chemotherapy. Several chemotherapeutic agents are associated with HBVr, including anthracyclines, glucocorticoids, and anti-CD20 Hepat Mon. 2016; 16(4):e35810. 3
agents. The rate of reported HBVr during or after the cessation of cancer chemotherapy varies widely and strongly depends on the underlying disease and the treatment regimens. For example, the incidence of HBVr in patients with breast cancer who were under chemotherapy without prophylaxis, was reported to range from 20% to 41% (2, 35-37). However, these rates dramatically decreased via the employment of prophylaxis. Recently, Yang et al. (38) analyzed gastric or colorectal cancer patients with positive HBsAg undergoing chemotherapy. The rate of reactivation was reported to be 14.6% (6 from 35). In a review, the rates of HBVr in chronic carriers with hepatocellular carcinoma (HCC) undergoing chemotherapy were reported to range from 4% to 67% (39). Furthermore, there are several case reports of HBVr in patients with chronic myeloid leukemia under chemotherapy (40, 41).
## Treating autoimmune diseases
The most effective therapy in several autoimmune diseases is corticosteroids, which are widely used in the treatment of these diseases and are associated with HBVr. It has been shown that increasing the dose of corticosteroids can raise the risk of infections (42). The risk of HBVr could significantly increase in patients who received prednisone at high doses (≥ 20 mg/day for at least 4 weeks). In addition to corticosteroids, TNF-α inhibitors also led to the appearance of reactivation signs (43-47). In TNF-α targeted therapy, 39% and 5% reactivation in HBsAg carriers and positive anti-HBc patients, respectively, were reported (47).
The newly emerged therapy, rituximab, which targets B cells, is widely used in chemotherapy as well as in various autoimmune disease therapies. This treatment is considered to be a most serious risk factor of HBVr in autoimmune diseases. Several studies have reported the reactivation of HBV due to rituximab administration in RA patients . In contrast to many other immunosuppressants, HBVr due to rituximab administration may happen after 6 months (up to 12 months). This may be explained by the delay in immune reconstitution in these cases. Thus, in addition to immediate initiation of antiviral therapy, prophylactic antiviral therapy for at least 12 months was recommended after rituximab administration, in various studies (5, 6).
## Transplantation
Although HBVr is defined as the reappearance of HBV, the appearance of HBV after transplantation also has been mentioned as the HBVr in many studies. Various studies confirmed the chance of HBV appearing during solid organ transplantation, bone marrow transplantation, or HSCT (4, 33, 50-57). Blanpain et al. [bib_ref] Reactivation of hepatitis B after transplantation in patients with pre-existing anti-hepatitis B..., Blanpain [/bib_ref] reported three cases of HBV appearing after transplantation in patients with resolved HBV infection (negative HBsAg, positive HB-sAb, and anti-HBc). It is considered to be a serious warning for the awareness of HBVr risk during transplantation in resolved HBV patients. Furthermore, Mikulska et al. [bib_ref] Hepatitis B reactivation in HBsAg-negative/HBcAb-positive allogeneic haematopoietic stem cell transplant recipients: risk..., Mikulska [/bib_ref] reported HBVr within a median of 19 months after HSCT, ranging from 9 months to 77 months. This could be a warning that HBVr can occur even some years after HSCT. In order to prevent HBVr in transplantation, the American society of transplantation and the American society of blood and marrow transplantation released guidelines associated with the reactivation of HBV after transplantation [bib_ref] Infectious Diseases Community of Practice . Viral hepatitis in solid organ transplantation, Levitsky [/bib_ref] [bib_ref] Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global..., Tomblyn [/bib_ref].
In patients receiving allogeneic HSCT, HBVr occurs more frequently than in those who receive autologous HSCT. This may be explained by the reduced suppression of innate cellular immunity in autologous HSCT [bib_ref] Efficacy of antiviral prophylaxis in HBsAg-negative, anti-HBc positive patients undergoing hematopoietic stem..., Yoo [/bib_ref]. However, reverse seroclearance of HBV is not rare in autologous HSCT [bib_ref] Changes in serologic markers of hepatitis B following autologous hematopoietic stem cell..., Uhm [/bib_ref]. The effect of antiviral therapy in cases with HSCT, regardless of donor type, has been confirmed by several authors [bib_ref] Efficacy of antiviral prophylaxis in HBsAg-negative, anti-HBc positive patients undergoing hematopoietic stem..., Yoo [/bib_ref] [bib_ref] Reactivation of hepatitis B virus in hematopoietic stem cell transplant recipients in..., Nakamoto [/bib_ref]. However, recently, it was found that short-term antiviral prophylaxis appeared insufficient to decrease the risk of HBVr. Prophylaxis longer than 24 months was introduced as a more effective way of controlling viral replication for patients under HSCT (54).
## Risk factors
Most people who experienced recovery from HBV infection had an undetectable HBV DNA serum [bib_ref] Persistence of hepatitis B virus DNA in the liver after loss of..., Fong [/bib_ref]. People with this condition are still in danger of HBVr, but the risk is less than in those with positive HBsAg and those with detectable HBV DNA serum. This can be explained with the persistence of HBV in the liver. The reactivation frequency can change among less than 1% and more than 50% which mainly depends on different risk factors.
## Viral status
Viral status is another important factor associated with the risk of HBVr. According to many reports, viral replication in patients with positive HBsAg is higher than in those with negative HBsAg, positive anti-HBc. Indeed, positive HBsAg is considered to be a risk factor of HBVr [bib_ref] Hepatitis B virus infection and risk of non-Hodgkin lymphoma in South Korea:..., Engels [/bib_ref]. Furthermore, HBV DNA serum levels can lead to a significantly higher risk of HBVr compared to the patients with a low or undetectable level of HBV DNA.
Different studies have suggested that low titer or the absence of HBsAb is strongly associated with HBVr [bib_ref] Efficacy of antiviral prophylaxis in HBsAg-negative, anti-HBc positive patients undergoing hematopoietic stem..., Yoo [/bib_ref] [bib_ref] Hepatitis B virus reactivation during anti-cancer chemotherapy in patients with past hepatitis..., Kim [/bib_ref] [bib_ref] Hepatitis B reactivation in patients with previous hepatitis B virus exposure undergoing..., Seto [/bib_ref] [bib_ref] Risk of hepatitis B virus (HBV) reactivation in hepatitis B surface antigen..., Koo [/bib_ref]. However, recently, a meta-analysis containing 578 patients in 15 different studies did not find HBsAb status to be effective on reactivation risk (69). [bib_ref] Prophylactic antiviral therapy in allogeneic hematopoietic stem cell transplantation in hepatitis B..., Liao [/bib_ref] Hepat Mon. 2016; 16(4):e35810.
It was suggested that in patients with HBeAg-positive, HBVr more likely could be developed than in those with HBeAg-negative [bib_ref] Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy:..., Yeo [/bib_ref] [bib_ref] The hepatitis B virus reactivation after transarterial chemoembolization in Chinese hepatocellular carcinoma..., Shao [/bib_ref]. Recently, in HCC patients who experienced HBVr, a significant correlation between HBeAg status in those with low serum HBV DNA level was found [bib_ref] The hepatitis B virus reactivation after transarterial chemoembolization in Chinese hepatocellular carcinoma..., Shao [/bib_ref]. HBV DNA may be considered as the most important factor associated with HBVr [bib_ref] Comprehensive analysis of risk factors associating with Hepatitis B virus (HBV) reactivation..., Yeo [/bib_ref]. In addition to these factors, non-A HBV genotypes are known as the other viral factors associated with a higher risk of reactivation (73).
## Host factors
Being male, especially a younger male, is considered to be a risk factor for HBV in several studies [bib_ref] Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy:..., Yeo [/bib_ref] [bib_ref] Hepatitis B reactivation in patients with hepatocellular carcinoma undergoing systemic chemotherapy, Yeo [/bib_ref]. In a study that introduced this point, 18 patients were found to be HBsAg-positive among 626 patients undergoing cytotoxic chemotherapy. In 15 patients, reactivation was observed: 11 (73%) were male and 4 (27%) were female, which is a significant difference. In HBsAg carriers who did not experience reactivation, there were 27 (43%) male and 36 (57%) female. In that study, a strong relation between the male sex and the risk of HBVr in HBsAg-positive patients was found [bib_ref] Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy:..., Yeo [/bib_ref]. In another study, which reported HBVr in an inactive phase of CHB in Alaska, the male sex was dominant in reactivated cases (22 of 179 patients (12%) in the male group and 14 of 235 patients (6%) in the female group) [bib_ref] Rates and risk factors for hepatitis B reactivation in a cohort of..., Tohme [/bib_ref]. There was no clear association between age and HBVr in that study. Additionally, HBVr developed earlier in the male group.
## Underlying disease and therapy regimens
Based on previous reports, the most common underlying disease associated with HBVr is lymphoma, which is followed by breast cancer [bib_ref] Hepatitis B virus reactivation during anti-cancer chemotherapy in patients with past hepatitis..., Kim [/bib_ref] [bib_ref] Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy:..., Yeo [/bib_ref] [bib_ref] Comprehensive analysis of risk factors associating with Hepatitis B virus (HBV) reactivation..., Yeo [/bib_ref]. This may be the result of marked immunosuppression in lymphoma patients and anthracycline-based chemotherapeutic in patients with breast cancer. It also may be due to the high prevalence of HBV infection among those patients [bib_ref] Hepatitis B infection in patients with lymphomas, Liang [/bib_ref]. The incidence of HBVr in patients with lymphoma is reported to range from 20% to 73%, which could be explained by the frequent use of the anti-CD20 agent (rituximab) to treat this disease (24, 29, 77-79). Furthermore, this risk is high in patients with breast cancer. It has been reported to range from 20% to 41%, which is a significantly high risk . Although there are various reports of HBVr in patients with rheumatic diseases , the rate of reactivation is not as high as the previously mentioned diseases. In a study including 122 HBsAg-positive patients with rheumatic diseases who were under treatment of anti-TNFα agents or disease modifying anti-rheumatic drugs, 15 cases (12.3%) developed HBVr [bib_ref] Hepatitis B virus reactivation in HBsAgpositive patients with rheumatic diseases undergoing anti-tumor..., Lee [/bib_ref]. However, in another report, anti-TNF-α therapy was considered to be a safe option for the treatment of rheumatic diseases in patients with CHB infection when it was combined with antiviral therapy. Also, it was concluded that anti-TNF-α therapy could be safe in resolved HBV patients with rheumatic diseases without using HBV prophylaxis [bib_ref] Long-term safety of anti-TNF treatment in patients with rheumatic diseases and chronic..., Vassilopoulos [/bib_ref].
The risk of HBVr in patients with HCC who are undergoing chemotherapy is very high. It can be explained by the high number of HBV carriers among HCC patients. Various studies have found that approximately 80% of HCC patients in high HBV endemic regions are HBsAg-positive. Furthermore, more than 90% of HCC cases are anti-HBcpositive. Interestingly, population controls typically had rates of HBsAg between 10% and 15%. Because HCC is caused by several HBV-related mechanisms, the prevalence of HBV is significantly higher in HCC patients compared to controls. Considering the high number of HBV carriers among HCC patients, immunosuppressive therapy leads to an increase in viral replication in these patients. Following that, during immune reconstitution, the signs of HBVr will appear. Various studies reported HBVr in patients with HCC who received chemotherapy (39, 71, 74). The incidence of HBVr in patients with HCC varied widely in range with significant mortality. Yeo et al. [bib_ref] Hepatitis B reactivation in patients with hepatocellular carcinoma undergoing systemic chemotherapy, Yeo [/bib_ref] reported a high rate of mortality (30%) between 37 reactivated conditions of 102 HBsAg-carrier patients with HCC under systemic chemotherapy. It seems that the high rate of HBVr among those with HCC is the outcome of increased viral replication, but this is not comparable to HBV reactivation from a state of inactive infection or OBI.
Generally, various treatments, including corticosteroids, anti-CD20 agents, HSCT, TNF-α inhibitors, anthracyclines, transarterial chemoembolization for HCC, methotrexate, ustekinumab, and tyrosine kinase inhibitors, can cause increasing risk of HBVr. [fig_ref] Table 1: The Risk of Hepatitis B Virus Reactivation Due to Some Selected Treatments... [/fig_ref] indicates the HBVr risk of some selected treatments in HBsAg carriers and non-HBsAg carriers.
Rituximab and ofatumumab are the two major B cell inhibitors (anti-CD20), which were identified as very highrisk treatments in HBV-infected patients (risk of reactivation > 20%). Rituximab is a chimeric anti-human CD20 antibody, which was approved by the U.S. Food and Drug Administration (FDA) in 1997 for non-Hodgkin lymphoma treatment. Then its usage was extended to multiple diseases as approved and to off-label groups. It acts directly against the CD20 antigen expression on the surface of B cells. It is becoming recognized that rituximab is strongly associated with an increase in the risk of HBVr (28, 33, 48, 49, 67-69, 83). The first HBVr-associated study on rituximab therapy was published in 2001, which described a patient who had HBsAb, but not HBsAg before rituximab therapy. A meta-analysis was investigated on HBVr in patients with lymphoproliferative diseases who were receiving rituximab from emersion of rituximab through 2009 Hepat Mon. 2016; 16(4):e35810. 5 [bib_ref] Rituximab-associated hepatitis B virus (HBV) reactivation in lymphoproliferative diseases: meta-analysis and examination..., Evens [/bib_ref]. It revealed that the median number of rituximab doses received before HBVr was 6 (between 3 and 10). This analysis indicated the sooner reactivation after the last dose of rituximab in HBsAg-positive patients compared to HBsAg-negative and anti-HBc-positive patients (median of 1 month vs. 5 months). Also, there is limited proof regarding the safety of rituximab therapy in RA patients with CHB [bib_ref] Long-term safety of rituximab in patients with rheumatic diseases and chronic or..., Mitroulis [/bib_ref]. van Vollenhoven et al. [bib_ref] Longterm Safety of Rituximab: Final Report of the Rheumatoid Arthritis Global Clinical..., Van Vollenhoven [/bib_ref] reported no HBVr among 131 anti-HBcpositive patients with RA who received up to 16 courses of rituximab. Despite these studies, it seems that rituximab increases the risk of reactivation in RA patients. This phenomenon was confirmed by several other studies, which reported HBVr development after rituximab therapy between RA patients (33, 48, 83).
Similar to rituximab, ofatumumab is classified as an anti-CD20 drug. It was approved in 2009 and is used for the treatment of chronic lymphocytic leukemia in patients who have the future disease after anti-cancer therapy. Since this is a relatively new drug, the excessive ability of ofatumumab to cause HBVr was not confirmed, although it seems that it can lead to reactivation of HBV as rituximab does. The FDA warned about the risk of HBVr for patients who were administered ofatumumab. In this issue, the FDA recommended the screening of all patients for hepatitis B prior to receiving ofatumumab [bib_ref] Hoofnagle JH. Recent US Food and Drug Administration warnings on hepatitis B..., Bisceglie [/bib_ref].
All the TNF-α inhibitors, including infliximab, adalimumab, certolizumab, golimumab, and etanercept, may cause HBVr in HBsAg carriers in addition to patients with OBI/resolved HBV infection. Anti-TNF-α therapy can be considered as a lower risk factor compared to rituximab or most of the chemotherapy regimens. There are various studies that reported no HBVr in patients during anti-TNFα therapy [bib_ref] Lack of hepatitis B virus reactivation after anti-tumour necrosis factor treatment in..., Biondo [/bib_ref] [bib_ref] Use of anti-tumor necrosis factor alpha therapy in patients with concurrent rheumatoid..., Ballanti [/bib_ref]. However, in some reports, HBVr was observed in cases under anti-TNF-α therapy (44, 91). The first report of HBVr due to TNF-α inhibitors was published in 2003. After that article, HBVr due to anti-TNF-α ther-apy was reported by several other authors (43, 47, 91). After identifying the capability of infliximab to inducing HBVr, the FDA inserted a warning regarding HBVr for the use of infliximab.
## Management of patients with a risk of hepatitis b virus reactivation during immunosuppressive therapy or chemotherapy
Considering that most of the HBV-infected patients are not aware of their infection, it is essential to screen all the candidate patients prior to immunosuppressive therapy or chemotherapy. The urgency of screening may change in various areas with a different prevalence of HBV infection. In HBV-endemic regions, the implementation of HBsAg and anti-HBc is more urgent than regions with a lower prevalence of HBV infection. The screening of anti-HBc may be as important as the screening of HBsAg prior to immunosuppressive therapy or chemotherapy. Guidelines from the international liver society recommended mandatory screening for serum HBsAg and anti-HBc before starting all forms of immunosuppression . Depending on the potency of the drugs, the underlying disease, and the viral factors, the risk of HBVr in those under immunosuppressant therapies can be significantly reduced by HBV antiviral prophylaxis. Receiving HBV antiviral agents at the commencement of immunosuppressive therapy or chemotherapy was demonstrated to be effective in decreasing the risk of HBVr. Prophylaxis also could be initiated before starting therapy. For the most immune suppressive regimes, prophylaxis should continue for at least 6 months after cessation of immunosuppressive drugs. Additionally, it has been recommended that antiviral prophylaxis must continue after 12 months after cessation of rituximab therapy [bib_ref] Rituximab-associated hepatitis B virus (HBV) reactivation in lymphoproliferative diseases: meta-analysis and examination..., Evens [/bib_ref]. Relatively early withdrawal of prophylactic treatment must be strongly [bib_ref] Hepatitis B Virus Screening for Patients With Cancer Before Therapy: American Society..., Hwang [/bib_ref] Hepat Mon. 2016; 16(4):e35810. avoided in cases under HSCT, even in the presence of complete remission [bib_ref] Fatal fulminant hepatitis B after withdrawal of prophylactic lamivudine in hematopoietic stem..., Lin [/bib_ref] [bib_ref] Reactivation of hepatitis B virus in hematopoietic stem cell transplant recipients in..., Nakamoto [/bib_ref]. Thus, prophylaxis longer than 24 months was recommended for these patients [bib_ref] Efficacy of antiviral prophylaxis in HBsAg-negative, anti-HBc positive patients undergoing hematopoietic stem..., Yoo [/bib_ref]. On the issue of HSCT, several studies have reported HBVr after more than 12 months, reaching a peak of 91 months (27, [bib_ref] Efficacy of antiviral prophylaxis in HBsAg-negative, anti-HBc positive patients undergoing hematopoietic stem..., Yoo [/bib_ref] [bib_ref] Hepatitis B reactivation in HBsAg-negative/HBcAb-positive allogeneic haematopoietic stem cell transplant recipients: risk..., Mikulska [/bib_ref] [bib_ref] Reactivation of hepatitis B virus in hematopoietic stem cell transplant recipients in..., Nakamoto [/bib_ref]. Considering the high resistance to lamivudine (LAM), in addition to various reported cases with a risk of reactivation following more than 6 months from the cessation of immunosuppressive therapy or chemotherapy, it is recommended that referenced HBV antiviral agents, such as entecavir (ETV) and TNV should be used in therapies that need long-term prophylaxis. Recommendations of various bodies associated with HBVr are shown in [fig_ref] Table 2: The Most Important Recommendations of Various Bodies Associated With Hepatitis B Virus... [/fig_ref].
In a meta-analysis, LAM prophylaxis in HBsAg carriers with breast cancer under chemotherapy was introduced as an effective option in the reduction of HBVr. Based on a systematic review, LAM prophylaxis had appeared as an effective option in the reduction of HBVr in HBsAg-positive lymphoma patients undergoing chemotherapy. After that, preventing reactivation caused a decrease in the disruption of the chemotherapy [bib_ref] Prophylactic lamivudine to improve the outcome of HBsAg-positive lymphoma patients during chemotherapy:..., Li [/bib_ref].
In another report, anti-HBc-positive patients under immunosuppressive therapy were included [bib_ref] The effects of nucleoside analogue prophylactic treatment on HBV activation in HBcAb+..., Liu [/bib_ref]. These patients suffered from various autoimmune diseases. Among those who had received antiviral therapy, none indicated a rise in HBV DNA levels, while 11.5% of the control group did experience an increase. ALT elevation was significantly lower in the antiviral prophylactic group. Additionally, one patient in the control group showed reverse seroconversion. In contrast, no reverse seroconversion was observed in the prophylactic group.
LAM can be considered as the most common prophylaxis in HBV patients. However, there is a considerable risk of LAM resistance risk in patients with active HBV. Based on the literature, LAM is an appropriate choice in cases with high viral replication that need short-term treatments. In contrast, it was not recommended for HBsAg carriers with detectable HBV DNA levels at baseline, who are candidates for long-term immunosuppressive therapy. Accordingly, it can be concluded that LAM prophylaxis is a reasonable choice for OBI/resolved HBV patients.
LAM is more common in countries with a high prevalence of HBV, where the cost of other antivirals may be prohibitive. Considering the high resistance rate of LAM, it is recommended that it be used for short-term treatment. By LAM resistance, the cessation of immunosuppressive therapy or chemotherapy may be necessary in order to control viral replication. ETV is known as the other antiviral prophylaxis with a lower chance of resistance compared to LAM. ETV was also introduced as a more effective antiviral therapy in preventing HBVr in several studies [bib_ref] Entecavir vs lamivudine for prevention of hepatitis B virus reactivation among patients..., Huang [/bib_ref]. In a study including 121 patients with on treated diffuse large B cell lymphoma receiving R-chop chemotherapy, the rate of HBVr was reported at 13.3% in cases that used LAM in a dose of 100 mg daily. In contrast, no reactivation was observed in the ETV users group with 0.5 mg daily [bib_ref] Entecavir vs lamivudine for prevention of hepatitis B virus reactivation among patients..., Huang [/bib_ref]. It was recommended that ETV be used in preference to LAM in CHB patients who have detectable HBV DNA levels at baseline. There is a positive correlation between the chance of LAM resistance and ETV resistance. Lee et al.conducted a retrospective cohort study to evaluate the probability of developing genotypic resistance to ETV in LAM-exposed patients. They found that patients with previous exposure to LAM developed ETV resistance significantly more frequently than nucleos(t)ide analogue (NA)naive patients. The probabilities of developing ETV resistance in the NA-naïve, LAM peri-exposure group without resistance and with resistance were < 1.0%, 8.0%, and 28.2%, respectively, at month 48. This study revealed the high risk of ETV therapy in patients with prior exposure to LAM, regardless of the presence or absence of LAM resistance. Adefovir and LAM combination therapy was reported as an effective suppression viral replication in patients with LAM resistance (101). Since, TNV was licensed for HBV therapy, it is increasingly being used because of its high efficiency and low rate of resistance. It was reported as the effective alternative for the treatment of patients with LAM resistance [bib_ref] Comparison of adefovir and tenofovir in the treatment of lamivudine-resistant hepatitis B..., Van Bommel [/bib_ref]. Furthermore, TNV was identified as being highly effective and safe in the prophylaxis and rescue treatment of HBVr in patients under immunosuppression therapy (103). indicates our recommendation, based on different studies and reported cases associated with HBVr and using antiviral prophylactic therapy in order to control the disease.
[table] Table 1: The Risk of Hepatitis B Virus Reactivation Due to Some Selected Treatments in Hepatitis B Surface Antigen Carriers and Non-Hepatitis B Surface Antigen Carriers [/table]
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Ann Rheum Dis. 2011;70(6):956-60. doi: 10.1136/ard.2010.144741. [PubMed: 21285116]. 43. Nobili L, Albani L, Gabrielli A, Moroncini G. Reactivation of Hepatitis B Virus Infection Associated with Anti-Tumor Necrosis Factor-a Therapy. J Antivir Antiretrovir. 2014(6):92-101. 44. Ryu HH, Lee EY, Shin K, Choi IA, Lee YJ, Yoo B, et al. Hepatitis B virus reactivation in rheumatoid arthritis and ankylosing spondylitis patients treated with anti-TNFalpha agents: a retrospective analysis of 49 cases. Clin Rheumatol. 2012;31(6):931-6. doi: 10.1007/s10067-012-1960-1. [PubMed: 22349880]. 45. Tamori A, Koike T, Goto H, Wakitani S, Tada M, Morikawa H, et al. Prospective study of reactivation of hepatitis B virus in patients with rheumatoid arthritis who received immunosuppressive therapy: evaluation of both HBsAg-positive and HBsAg-negative cohorts. J Gastroenterol. 2011;46(4):556-64. doi: 10.1007/s00535-010-0367-5. [PubMed: 21246383]. 46. Lan JL, Chen YM, Hsieh TY, Chen YH, Hsieh CW, Chen DY, et al. Kinetics of viral loads and risk of hepatitis B virus reactivation in hepatitis B core antibody-positive rheumatoid arthritis patients undergoing anti-tumour necrosis factor alpha therapy. Ann Rheum Dis. 2011;70(10):1719-25. doi: 10.1136/ard.2010.148783. [PubMed: 21719446]. 47. Perez-Alvarez R, Diaz-Lagares C, Garcia-Hernandez F, Lopez-Roses L, Brito-Zeron P, Perez-de-Lis M, et al. Hepatitis B virus (HBV) reactivation in patients receiving tumor necrosis factor (TNF)-targeted therapy: analysis of 257 cases. Medicine (Baltimore). 2011;90(6):359-71. doi: 10.1097/MD.0b013e3182380a76. [PubMed: 22033451]. 48. Pyrpasopoulou A, Douma S, Vassiliadis T, Chatzimichailidou S, Triantafyllou A, Aslanidis S. Reactivation of chronic hepatitis B virus infection following rituximab administration for rheumatoid arthritis. Rheumatol Int. 2011;31(3):403-4. doi: 10.1007/s00296-009-1202-2. [PubMed: 19830433]. 49. Salman-Monte TC, Lisbona MP, Garcia-Retortillo M, Maymo J. Reactivation of hepatitis virus B infection in a patient with rheumatoid arthritis after treatment with rituximab. Reumatol [/table]
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Proton Exchange Magnetic Resonance Imaging: Current and Future Applications in Psychiatric Research
Proton exchange provides a powerful contrast mechanism for magnetic resonance imaging (MRI). MRI techniques sensitive to proton exchange provide new opportunities to map, with high spatial and temporal resolution, compounds important for brain metabolism and function. Two such techniques, chemical exchange saturation transfer (CEST) and T1 relaxation in the rotating frame (T1r), are emerging as promising tools in the study of neurological and psychiatric illnesses to study brain metabolism. This review describes proton exchange for non-experts, highlights the current status of protonexchange MRI, and presents advantages and drawbacks of these techniques compared to more traditional methods of imaging brain metabolism, including positron emission tomography (PET) and MR spectroscopy (MRS). Finally, this review highlights new frontiers for the use of CEST and T1r in brain research.
# Introduction
Unraveling the biochemical signatures of cellular metabolism and neuronal activity is critical, not only for our basic understanding of brain function, but also for understanding neurological and psychiatric disorders. Positron emission tomography (PET)-based methods and magnetic resonance spectroscopy (MRS) have been established as proven techniques both for clinical routine and investigational neuroscience studies to measure brain metabolism. In this review, we examine two MRI techniques that offer additional possibilities for imaging brain metabolism by tapping into a physical phenomenon known as proton exchange. These MRI techniques are named chemical exchange saturation transfer (CEST), and T1 relaxation in the rotating frame (T1r). Both methods provide whole-brain coverage with high resolution, can be used to monitor pH changes in brain tissues, as well as to measure and map various brain metabolites and neurotransmitters. These techniques provide complementary information to MRS and PET and avoid some of the drawbacks of these techniques. Therefore, proton-exchange imaging may provide useful approaches to address certain research questions and may also hold great potential for clinical applications.
The purpose of this review is threefold. First, we seek to simplify the MRI physics underlying these methods in order to make them understandable to non-experts. Second, we review their current status and use in neuroscience research. Finally, we discuss their potential use to help achieve a better understanding of brain metabolites and neurotransmitters.
## Proton exchange
Proton exchange is a well-known phenomenon in which protons from the bulk water are exchanged with labile protons from soluble molecules (e.g. metabolites and neurotransmitters). MR imaging techniques have been developed that capitalize on the proton exchange phenomenon to indirectly provide information about the concentrations of these molecules. To better understand proton-exchange imaging, a basic overview of MR physics used in traditional MR imaging is provided in Box 1 below for non-MRI experts.
## Proton exchange imaging
Several compounds contain protons that are readily available for proton exchange . Such exchangeable protons are commonly found on amine (-NH 2 ), amide (-NH), thiol (-SH), and hydroxyl (-OH) groups, that are present in a wide variety of molecules including various brain metabolites and neurotransmitters. However, the protons present in these molecules experience slight variations in their Larmor frequency based on their local electromagnetic environment. On the other hand, protons present in bulk water precess at the Larmor frequency of the main magnetic field. Because water protons and exchangeable protons have different Larmor frequencies, they can be selectively marked, manipulated, and monitored using MR imaging techniques. 1 | Proton exchange: Hydroxyl, amine, and amide functional groups attached to molecules contain exchangeable protons (red) that can be readily exchanged with protons in water (blue). When exchange occurs, the proton that was originally part of the solute molecule is attached to the water molecule and viceversa. This exchange process occurs continuously, transferring protons between bulk water and solute molecules.
## Box 1 | basic mri physics
In most MR imaging techniques, the signal comes from hydrogen atoms present in water molecules. The nucleus of the hydrogen atom consists of a single positively charged proton. These protons are highly mobile (i.e. "free protons") and are sensitive to electrochemical and magnetic forces. The magnetic field of the MR scanner, known as the main magnetic field, causes these protons to spin about their axes in specific orientations, either parallel or antiparallel to the main magnetic field . The parallel alignment has a lower energy state causing a slight majority to align in this orientation and producing a net magnetization known as the longitudinal magnetization (M 0 ). M 0 precesses around the main magnetic field at a specific frequency, known as the Larmor frequency (w), which is proportional to the main magnetic field strength (B 0 ) according to the following equation:
[formula] w = g B 0(1) [/formula]
where g is the gyromagnetic ratio, a constant that is specific to the nucleus of interest 577 Mhz/T for 1 H) . Applying radiofrequency (RF) pulses at the Larmor frequency tips the net magnetization away from main field with an angle determined by the product of the RF amplitude and duration.
In traditional MR imaging (e.g. T1-and T2-weighted imaging), RF pulses tip the net magnetization into the transverse plane . Once the RF pulse is turned off, the spins and corresponding transverse magnetization (M xy ) produces measurable signals, which are influenced by two relaxation properties (T1 and T2 relaxation). T1 relaxation is the process by which the net magnetization returns to alignment with the main magnetic field . Assuming that the initial magnetization in the longitudinal plane is M 0 and a 90 degree RF pulse is applied that tips M 0 to the transverse plane, T1 is defined as a time constant whereby the longitudinal component of the magnetization (M z ) recovers exponentially with time (t). The recovery of the magnetization along the main magnetic field over time is characterized by the following equation:
[formula] M z (t) = M 0 * 1 − e − t T1 =(2) [/formula]
Here, T1 is the time it takes for M z to return to approximately 63% of the initial magnetization (M 0 ) in the longitudinal plane. Because the rate of T1 relaxation depends on the tissue structure (e.g. lattice) it is often referred to as "spin-lattice" relaxation and provides an important source of contrast across different types of tissue. For example, in cerebrospinal fluid (CSF) protons are unable to dissipate energy as fast as protons in a more solid tissue, and so they exhibit a longer T1 relaxation time, which ultimately causes CSF to appear darker on an image accentuating T1-dependent contrast, also known as a T1weighted image.
The T2 relaxation process is produced by local magnetic field variations (d i ), which cause subtle differences in Larmor frequencies of individual free protons (w i ) determined by , which is a simple extension of Equation 1.
The difference in Larmor frequency between exchangeable solute protons (w s ) and free protons (w 0 ) is denoted by dw s = w s -w 0, measured in Hz. The frequency difference can also be expressed in parts-per-million (ppm), in which case, the term chemical shift is used to denote the shift between w s and an absolute reference standard (w ref ) normalized to the Larmor frequency of the scanner. RF pulses can be applied at specific Larmor frequencies to selectively manipulate (increase or suppress) the net magnetization of either the water pool or the solute pool. Although the net magnetization of both pools is sensitive to proton exchange, differences in magnetization are easier to measure in the water pool due to its greater abundance. The phenomenon of proton exchange is leveraged by both CEST (1) and T1r imaging, but in somewhat differing ways. In the following sections, we explore these two techniques in more detail.
## Cest acquisition and analysis
CEST imaging uses an RF pulse to selectively suppress the magnetization of a desired pool of solute protons. Specifically, a long RF "saturation" pulse (S sat ) with a narrow bandwidth is applied, that is tuned to match the Larmor frequency of the exchangeable protons (w s ) of a solute of interest. The application of the RF saturation pulse suppresses the magnetization of the solute protons . These saturated solute protons then exchange with the unsaturated water protons causing a measurable reduction in the magnetization from the water pool. The long duration of the saturation pulse allows enough time for the effect of the saturation to reach steady state. This reduction in magnetization can be used to calculate the concentration of the molecule of interest.
One would ideally want the saturation pulse to only influence the solute protons of interest. However, the applied saturation pulse has some direct saturation effects on the water pool as well as a broad effect on other molecules. To minimize the undesired effects of the saturation pulse, a minimum of three measurements are performed where saturation pulses are applied symmetrically
[formula] w i = g (B 0 − d i )(3) [/formula]
Local field variations cause the spins to lose their coherence over time, which in turn causes the associated magnetization in the transverse plane to decay. The decay of the transverse magnetization is commonly referred to as "spin-spin" relaxation. Assuming a 90°tip angle, the decay of the transverse plane magnetization (M xy ) over time (t) is defined by Equation 4
[formula] M xy (t) = M 0 * e − t T 2 = (4) [/formula]
where T2 is a time constant for the relaxation process. That is, T2 is the time it takes for M xy to fall to approximately 37% of its initial value (M 0 ). Using CSF again as an example, the local magnetic field variability is relatively low in CSF as compared to other brain tissues and thus has a long T2 relaxation time.
Therefore, CSF appears bright on T2-weighted images relative to gray matter and white matter, which have greater local magnetic field variations.
As explained above, traditional T1-and T2-weighted imaging capitalize primarily on the magnetization of protons in the bulk water. However, imaging other molecules with protons can provide useful information regarding brain metabolites and neurotransmitters. Sensitizing MRI measurements to the magnetization from the protons of the other molecules requires different acquisition strategies because they resonate at a different frequency from that of the bulk water protons. Moreover, these molecules occur at much lower concentrations (approximately 10 −5 times less) than bulk water. One strategy to generate MRI contrast from these soluble molecules is to measure the influence of their exchangeable protons on the larger bulk water pool through the phenomenon of proton exchange. CEST and T1r are examples of such imaging techniques.
## A b
FIGURE 2 | Effects of an external magnetic field on proton alignment: The nucleus of the hydrogen (H) atom contains a single proton that spins on its axis, generating a small magnetic field (i.e. north (N) to south) represented by the red arrows. (A) In the absence of an external magnetic field, these are randomly oriented, which results in a net magnetization (M 0 ) of zero. (B) When these protons are placed inside a strong magnetic field (B 0 ), their orientations align either parallel or antiparallel to the B 0 field with a slightly more protons aligned parallel to B 0 . This difference between the two alignments results in a small net magnetization (M 0 ) that is parallel to B 0 .
with respect to the water frequency, at +dw s and -dw s, as well as a measurement without any saturation pulse (S 0 ). The two images collected with saturations pulses are subtracted and normalized by the signal acquired without any saturation pulse. This is known as the CEST asymmetry ratio (CEST asym )
[formula] CEST asym (w s ) = S sat ( − Dw s ) − S sat ( + Dw s ) S 0(5) [/formula]
In many applications, even more RF saturation pulses are applied over a broad range of saturation frequencies generating what is known as the Z-spectrum (3). An example CEST data set that targets the exchangeable protons of the amide group is shown inalong with the acquired Z-spectrum. This version of CEST imaging is referred to as amide proton transfer CEST (APTCEST) or simply APT and has been shown to be sensitive to pH.shows the APT CEST asym map A B C FIGURE 3 | The effects of a RF pulse: (A) A typical MR experiment begins with protons in a strong magnetic field (B 0 ) that is aligned with the z axis (down the bore of the MR scanner). As noted in , these protons spin on their axis (grey arrow), generating a small amount of net magnetization (M 0 , red arrow)) that precesses around the z-axis (orange arrow) at the Larmor frequency (w). (B) A 90°radiofrequency (RF) pulse is applied to the system in order to "tip" M 0 into the transverse (X-Y) plane. The net magnetization M 0 will exhibit a spiral trajectory from the initial starting orientation as shown. (C) Immediately after the RF pulse is removed, the net magnetization (M 0 ) is rotating around the z-axis on the transverse plane.
A B D C FIGURE 4 | T1 relaxation: M 0 is the initial longitudinal magnetization (M z =M 0 ) before the application of the RF pulse. M 0 is tipped into the transverse plane by the application of the 90°RF pulse (M z =0). Immediately after removal of the 90°RF pulse (see (A), magnetization will recover from the transverse plane. While this is occurring (B), the net magnetization along the z-axis (M z , blue arrows) will increase while transverse magnetization (M xy , purple arrows) will decrease until the net magnetization returns to its original orientation (C) aligned with the B0 field (M z = M 0 ). The magnitude of the net magnetization along the Z axis follows an exponential recovery curve (D), with T1 relaxation time being the amount of time it takes for magnetization on the z-axis to return to M z . generated at 3.5 ppm corresponding to the amide proton chemical shift. The entire Z-spectrum (red line,between ±6 ppm is shown on the rightas well as the resulting CEST asym curve (blue line,. This data set was collected on 7T MRI to exploit the increased spectral separation at the higher field strengths compared to lower field strengths. It should be noted that the CEST literature has adopted the convention of a chemical shift of 0 ppm corresponding to the water protons when displaying the Z-spectrum.
## Sensitivity and specificity of cest for molecular and metabolite imaging
The CEST technique provides a novel way to detect endogenous compounds with exchangeable protons. These include several molecules that are known to reflect brain metabolism and neurotransmitters such as glutamate, GABA (6), creatine, and lactate. The exchangeable protons on each of these compounds have a unique Larmor frequency. Hence, CEST experiments can be tuned to match the frequency of the desired molecular group. In the CEST literature, it is common to refer to the CEST experiments based on the probed molecule. For example, targeting glutamate with CEST is known as gluCEST while targeting creatine is known as CreCEST.lists several molecules that have been previously measured using CEST imaging as well as the factors proposed by the original manuscript authors thought to contribute to the findings observed. For example, studies have observed intracellular acidosis using APTCEST arising from anaerobic metabolism (9) resulting from stroke by comparing ipsilateral versus contralateral tissue. The specificity of CEST imaging for quantification of molecular concentrations has been extensively explored by many of the studies listed inusing MRS measurements for validation. The CEST effect is known to be confounded by several undesirable factors. These include the contamination of the CEST asym ratio by other saturation exchange mechanisms such as magnetization transfer (MT) and dipolar interactions such as nuclear Overhauser enhancement (NOE)and concentration from other metabolites whose chemical shift is close to the target metabolite. For example, imaging of glutamate is known to be confounded by GABA and Cr concentrations. Because of these confounding factors, the specificity of the CEST measurements are sometimes difficult to be interpret. Several spectral editing techniques are being developed to remove the confounding effects of these source of errors as discussed in Other Confounding Effects. Ignoring these confounding factors for the moment, the reliability of CEST measurements have been shown to have good scan/re-scan reliability.
## Strengths and limitations of cest imaging
The key strength of CEST is that it can quantify several endogenous molecules with high resolution. This quantification can be performed using existing MR hardware and can readily be integrated into a multi-modal MRI study. Other MRI methods, particularly MRS, also provide the ability to study endogenous compounds. Relative to MRS, CEST has the advantage of measuring the endogenous molecules using the water signal, which is 5 orders of magnitude larger than the molecular concentrations directly observed with MRS. This allows CEST imaging to collect data faster and with higher spatial resolution than MRS. However, MRS has at least one significant advantage over CEST, which is its ability to quantify multiple compounds simultaneously, while CEST can typically only acquire one or two compounds at a time that must be defined a priori.
PET is another approach that is often used to study brain metabolism, but instead uses exogenous radioactive contrast agents. This provides PET with superior sensitivity and SNR as compared to CEST. In addition, PET imaging provides the ability to measure rates including blood perfusion, metabolic rate of glucose, and binding rate to a receptor. CEST in contrast is predominantly used to measure metabolite concentration. A detailed comparison of various CEST approaches and their closest PET alternatives was recently provided by Wu and colleagues. It should also be noted that PET and CEST can provide complimentary information. Therefore, the combined acquisition of these two metabolic imaging techniques is an exciting potential research direction facilitated by dual modality PET/MRI scanners, which are now commercially available.
Endogenous CEST imaging has the limitation that it can only be used to study metabolites with exchangeable protons whose exchange rate satisfy the condition that the exchange rate (k) is less than Dw s. Moreover, the chemical shifts of these endogenous species are typically very small (Dw s ≤ 10ppm), which make it prone to noise from undesirable sources. Some of these limitations can be addressed with the administration of exogenous contrast agents which have larger chemical shift (Dw s ≈ 50-700 ppm). Such agents can be extremely useful for FIGURE 6 | Magnetization preparation blocks for CEST imaging sequence: At the beginning of a CEST experiment, protons attached to the solute molecule of interest (red) have a different Larmor frequency from protons in bulk water (blue). A RF saturation pulse is applied at the Larmor frequency of the exchangeable protons of the solute molecule, which causes some of them to become saturated (yellow outline). Over time, the saturated protons exchange with the unsaturated protons in water due to proton exchange while the saturation pulse continues to saturate protons attached to the solute pool, including protons that were originally in the water pool (blue with yellow outline). The presence of the saturated protons in the water results in a reduction of the net magnetization measured from the water over time. The net magnetization at saturation (M sat ) is therefore lower than the initial net magnetization (i.e. M sat < M 0 ). FIGURE 7 | A typical CEST Imaging experiment: In practice, CEST imaging is performed using a series of RF saturation pulses that are applied at different offset frequencies (±Dw) which are measured relative to the Larmor frequency of free water (w). Direct water saturation occurs when CEST is performed at the Larmor frequency of free water (i.e. Dw=0), which will result in a significant reduction in net magnetization (M sat ) relative to the initial net magnetization (M 0 ). The series of RF pulses are typically applied at and near the expected Larmor frequency of a desired solute proton (Dw 1 , Dw 2 , Dw 3 ) and at frequency offsets (-Dw 1 ,-Dw 2 , -Dw 3 ) from water. The presence of a solute proton with a chemical shift (+Dw= Dws) would therefore appear as a dip in the CEST spectrum at that chemical shift (+Dws) relative to its opposite (-Dws). For example, we can see such a dip in Dw 2 relative to -Dw 2 . Ischemic penumbra in strokeSevere intracellular acidosis in ischemic core develops in part due to unopposed anaerobic ATP hydrolysis, with hypoperfusion and reduced bicarbonate buffering at acidic pH exacerbating the acidosis Tumor pH (mouse studies) reported that proportion of APT CEST signal originating from changes in protein concentration was approximately 66%, with the remaining 34% originating from changes in tumor pH.Tumor cells have reversed the pH gradient across the cell membrane with respect to normal cells, with a slightly alkaline intracellular pH (pHi) and an acidic extracellular pH (pHe). Tumors often have regions of acute and chronic hypoxia as a result of both an increased oxygen consumption rate of tumor cells compared with normal cells and hence altered pH.. Proteomic Analysis have revealed significant increase in the cytosolic protein concentration in the tumor, compared to normal brain regions. Tissue grading and classification in tumorThe mean APT asymmetry ratio values highly correlated with tumor grades. Significant differences in APT asymmetry ratio were observed between tumor grades. Increased APT asymmetry associated with increased cell density, gliomas with microscopic necrosis. Alzheimer's Disease vs healthy controlsElevated CEST asymmetry ratio in bilateral Hippocampus in Alzheimer's may be due to increased cytosolic proteins and peptides (accumulation of amyloid plaques, neurofibrillary tangles, and neuronal loss). Parkinson's Disease vs healthy controlsElevated CEST asymmetry in substantia nigra in Parkinson's disease may be due to dopaminergic neuronal loss.
## A b
GluCEST Glutamate Amine group @ 3 ppm
Middle cerebral artery occlusion (MCAO) stroke model (17) MCAO model lead to significant drop in pH resulting in elevated Glutamate concentrations due to increased proton exchange Tumor with BBB disruption and Glutamate injectionGlutamate concentration in tumor cells increased due to glutamate injection Non-lesional Temporal Lobe EpilepsyElevated Glutamate concentration correctly lateralized the temporal lobe seizure foci. Transgenic mouse models Alzheimer's Disease vs wild typeThe excitatory neurotransmitter, Glutamate, is known to decrease in early stages of Alzheimer's disease. Healthy controls vs Psychosis spectrum (4) Abnormal glutamate neurotransmitter levels are implicated in progression of psychosis Differential gray:white (1.6:1 ratio) contrast in healthy brainGlutamate concentration map approximates the Glu receptor distribution CrCEST Creatine Amine group@ 1.8 ppm Plantar flexion exercise within MRI scannerDynamic changes in creatine concentration in reposes to increased ATP consumption during exercise. Post exercise creatine recovery prolonged in mitochondrial disease group Post-exercise; Mitochondrial disease vs healthy controls Creatine concentration in tumor cells varies from normal cells due to abnormal ATP metabolism in tumor MICEST Myo-inositol Hydroxyl group @ 0.625 ppm Transgenic mouse models of Alzheimer's disease vs wild type control mice (23) Elevated expression of activated glial cells from neuroinflammatory responses in Alzheimer's disease pathology leads to increased myoinositol concentrations GABACEST GABA Amine group @ 2.5 ppm Rat models of status epilepticus; pre-post epileptiform activity induced by kainic acid injection.
Epileptic seizure changes GABA concentrations Rat with brain tumor and blood-brain barrier disruption; pre-post GABA injectionThe disrupted blood-brain barrier in tumor region allowed to measure GABA concentration changes
GlucoCEST Glucose Hydroxyl group @ 0.6 to 1.5 ppm pre-post injection of unlabeled glucose in Tumor (mice studies, human studies) (references below)
High rate of glucose uptake in tumors lead to glucose concentration changes Glyco CEST Glycogen Hydroxyl group @ 0.75-1.25 ppm Mouse liver studies with pre-post glucagon administrationGlucagon stimulates glycogenolysis (glycogen to glucose conversion) and depletes glycogen LATEST Lactate Hydroxyl group @ 0.4 ppm Calf muscles pre-post exerciseDynamic lactate changes in exercising muscles tumorup-regulated lactate dehydrogenase (LDH) due to lactate metabolism in tumor ppm, parts per million; APT, amide proton transfer; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ATP, adenosine triphosphate.
several unique applications such as metal ion detection, liposome labeling, nanoparticle/polymer labeling, RNA/DNA/proteinbinding, temperature imaging, detecting enzyme activity, and identification of reporter genes. However, most of these agents have not been approved for use in humans and are currently limited to animal imaging studies. This review will not discuss the use of these CEST contrast agents and instead the reader is referred to Hancu et al..
## Methodological considerations for cest
While CEST has great potential for studying certain molecules that are important to psychiatric research, there is substantial room for improvement in the acquisition and analysis of CEST data.
## B 0 correction
CEST imaging can be sensitive to magnetic field inhomogeneities that shift the Larmor frequency and as a result the saturation pulses may be applied at the wrong frequency offset in some portions of the brain. One method of correcting for this magnetic field variation is to use the acquired Z-spectrum to identify the actual Larmor frequency of water independently for each voxel. This is done by identifying the RF saturation pulse, dw s , that has the strongest saturation of the water signal (instead of assuming that it is at 0 ppm). If the saturation of the water signal is sufficiently broad, a more rigorous approach may be required that involves using an additional scan where a finer sampling of the saturation pulses around the water signal is performed. This acquisition is then used to estimate the effective Larmor frequency on a voxel by voxel basis using a technique called water saturation shift referencing (WASSR). The resulting estimates for S 0 and w 0 are then used to correct the CEST asym at each voxel.
## Other confounding effects
One limitation of using CEST asym to measure metabolite concentrations is that it introduces additional sources of errors due to the fact that macromolecular effects are asymmetric and lipophilic peaks exist on the right side of the water peak (i.e. -Dw s ) that may confound the measurements . It has been shown that these sources of errors can dominate (37-39) the CEST asym quantifications. Several techniques have been proposed to minimize these sources of error including double frequency irradiation, and Lorentzian differences. Lorentzian curve fitting has also been used by several groups to account for these potential sources of error. This technique fits a series of Lorentzian functions accounting for the CEST effect(s) of interest, nuclear Overhauser effect (NOE) or the dipolar interaction of protons with other nuclei species, magnetization transfer (MT), and bulk water to the Z-spectrum.
## Cest imaging scan time
A typical CEST imaging acquisition consists of several measurements performed at different Dw s to sample the Zspectrum and if acquired the WASSR data. The time to collect this data can be long and has often limited CEST measurements to a few slices. While a majority of the CEST imaging studies that have been reported in the literature have focused on limited coverage using 2D acquisitions, it is possible to perform whole brain acquisitions in approximately 10 min by combining fast scan methods such as echo-planar readouts and acceleration methods such as parallel imaging. Image acquisition can also be accelerated using under-sampling techniques such as compressed sensing or machine-learning that minimize the number of measurements needed to reconstruct the data.
Other improvements such as the ability to acquire data from multiple metabolites in a single session, regional specificity, improvements in SNR, and increased sensitivity would help provide a more comprehensive picture of brain metabolism and reduce scan times.
## High field imaging
CEST studies can benefit from high field imaging. A higher magnetic field strength offers greater frequency separation (Dw s measured in MHz) for water and metabolites. This improves quantification of the CEST effect by making it easier to avoid direct saturation effects and leakage from other metabolites with Larmor frequencies close to the target molecule. Since the exchange rate for CEST imaging and must satisfy the condition that the exchange rate must be less than Dw s, CEST experiments at higher field strength scanners can target molecules with faster exchange rates than what is possible at lower fields. Moreover, with higher field strength, the T1 weighting increases making the CEST measurements more time efficient. Stronger magnetic fields also result in increased signal-to-noise-ratio which can be used to improve spatial resolution, increase anatomical coverage, or reduce image acquisition time.
## Other imaging parameters
Like other MR imaging sequences, CEST imaging protocols require optimization of multiple imaging parameters including the number of RF saturation pulses, their step size, saturation pulse duration, repetition time, and flip angle. These parameters need to be tuned for each metabolite and field strength. The specifics of this are outside the scope of this review, however a number of review articles on CEST imaging already exist, which include discussions of parameter optimizations. Finally, optimal approaches for quantification and challenges across field strengths are also not discussed here in the interest of space but have been previously reviewed in Kim et al.and Wu et al..
## Acquisition and analysis of t1r
The phenomenon of proton exchange is also exploited in T1r imaging. In contrast to CEST, which uses a saturation RF pulse to tune to the Larmor frequency of the exchangeable solute protons, T1r relies on generating a secondary magnetic field to become sensitive to proton exchange mechanisms. This sensitization is achieved using an RF pulse known as a "spin-lock" pulse, which generates a secondary weak magnetic field B 1 perpendicular to the main magnetic field B 0 . The spin-lock (SL) RF pulse is applied at the Larmor frequency of the water protons and "locks" the net magnetization in the transverse plane, causing the magnetization to precess around the new B 1 field. The frequency of precession, w sl , about the spin-lock B 1 magnetic field is given by
[formula] w sl = g B 1(7) [/formula]
which is obtained by replacing the scanner magnetic field strength, B 0 , with the spin-lock field strength, B 1 . This new precession frequency about B 1 is typically in the range of 100 Hz to 3 kHz and is much slower than that around the main magnetic field (typically in the range of MHz). The slower precession allows the magnetization to be sensitive to proton exchange processes that also occur in this range. Consequently, the relaxation of the magnetization under the influence of B 1 is made sensitive to proton exchange. The basic T1r imaging scan is implemented using a block of three RF pulses as shown in . The first 90-degree RF pulse tips the longitudinal magnetization into the transverse plane. The second RF pulse, (the spin-lock pulse), is applied in the transverse plane at low amplitude. While the spin-lock (SL) pulse is being applied, the B 1 field itself rotates around the axis of B 0 at the Larmor frequency defined by the main magnetic field. Thus, it is easier to understand the influence of the spin-lock pulse on the magnetization by considering this process in the "rotating frame" from which the imaging technique gets its name. This can also be conceptualized as the perspective from the proton as it precesses around the B 0 field and is similar to a human observing events while standing on earth as it precesses around the sun. In the rotating frame, the B 1 field appears to be stationary (despite the fact that in absolute terms, it is rotating along the transverse plane around the axis of B 0 ), and the net magnetization precesses around the B 1 field. The SL pulse is typically delivered in two segments, with the second segment being delivered at the opposite phase from the first segment. Finally, the third RF pulse returns the magnetization to the longitudinal axis using a −90-degree RF pulse. The T1r preparation block is then followed by a magnetic field gradient that is applied to destroy residual transverse magnetization before using a standard imaging scheme to measure the magnetization stored in the longitudinal axis.
During the spin lock pulse, some relaxation of the magnetization occurs due to proton exchange. This relaxation produces an exponential decay in the signal intensity and is where the technique gets its name (T1 relaxation in the rotating frame). The relaxation time constant, T1r, can be calculated by measuring the change in signal intensity that occurs when applying at least two different spin-lock durations and fitting the data to an exponential decay curve (equation 8,
[formula] S(TSL) = S 0 e −TSL=T1r(8) [/formula]
In Equation 8, S 0 is the signal with no spin-lock pulse applied; TSL is the time duration of the spin-lock pulse. While the A B FIGURE 9 | Measurement of T1r relaxation is performed using a magnetization preparation block (A) consisting of a series of RF pulses. The first RF pulse tips the magnetization 90˚from the Z axis into the transverse plane. The second RF pulse is the spin-lock pulse (SL) which generates a small magnetic field (B 1 ) in the rotating (transverse) plane. The spin-lock pulse is often broken into two equal duration RF pulses that are 180˚out of phase. The third RF pulse is a −90°pulse, which restores the magnetization to the Z axis. During this spin-lock pulse, the net magnetization of the protons precesses around the B 1 field. Because the strength of B 1 is relatively small, this precession occurs at a frequency w SL , which is substantially slower than precession around B 0 and that is similar to the frequency of several slow processes including proton exchange. Because of this, over time, a loss of magnetization occurs in the transverse plane. (B) Measuring the loss of signal resulting from T1r relaxation requires that at least two different spin lock durations (TSL) are applied during the experiment (i.e. the steps in A are repeated). T1r follows an exponential decay curve and is calculated by using these different TSL values to fit the exponential decay function: M TSL = M 0 e -TSL/T1r . quantification of T1r requires a minimum of two measurements with different TSL values , acquiring data from more than two TSL values increases the accuracy of the T1r quantification. Since the spin-lock pulse slows the relaxation process in the transverse plane, the T1r relaxation time is longer than the T2 relaxation time for a given sample. As the amplitude of the spin-lock pulse (B 1 ) approaches zero, T1r relaxation times will get shorter and will approach T2 relaxation times. Similarly, as the amplitude of the spin-lock pulse increases the T1r relaxation times will increase.
## Sensitivity and specificity of t1r for metabolic and molecular imaging
T1r imaging can be used to measure low-frequency biological processes that occur on the same time frequency as the applied spin-lock pulse that is not feasible using conventional T1 or T2 imaging. Since the frequency of precession resulting from the spin-lock pulse is typically in the range of 100 Hz to 3 kHz, T1r imaging is sensitive to exchange processes that fall in this range. These include chemical exchange as well as spin-spin coupling, dipole-dipole interactions, and diffusion. Importantly, the frequency of precession can be "tuned" to favor a particular process. This is analogous to how diffusion gradients are used in diffusion weighted MR imaging where the sensitivity to water motion is dependent on the amplitude of the gradients applied.
Notably, T1r may be sensitive to a broader range of exchange processes than CEST, since it is not tuned to a particular group of exchangeable protons. Proton exchange with amide, hydroxyl, and amine groups are all thought to contribute to T1r relaxation. Thus, the T1r relaxation time is affected by changing the concentrations of these solutes and the rate at which they can exchange with water protons. T1r values has been shown to be sensitive to the concentrations of molecules including glucosein phantom studies. T1r imaging is also sensitive to pH(56, 59, 60), the amount of water that is present in tissue, macromolecular density (60), and temperature. Several studies have assessed the reliability of T1r relaxation times at 3T and found good scan/re-scan reliability with differences on the order of 2% or lessbetween scans.lists previous studies that have used T1r imaging in the context of brain imaging. Most of these studies have been in human subjects and observed T1r relaxation time differences when comparing various psychiatric and neurological disorders to control subjects. This has included increased T1r relaxation times observed in subjects with mild cognitive impairment and Alzheimer's Disease as compared to controls that the authors attributed to differences in macromolecular content, oxidative stress, and pH secondary to the disease pathophysiology. In Parkinson's disease T1r relaxation times in the substantia nigra were increased compared to control subjects likely due to tissue degeneration. In multiple sclerosis three studies have all reported increased T1r relaxation times in cerebral white matter likely reflecting the associated de-myelination. And in pre-manifest Huntington's disease (HD), T1r relaxation times in the striatum were increased compared to controls and correlated with proximity to predicted symptom onset, likely reflecting degeneration-induced changes in pH and/or glucose. 10 | Axial slice from a T1r data set collected from a human participant using a 3D spin-echo sequence with a spin-lock amplitude of 14.1uT. Images A and B show the same axial slice collected using spin-lock durations of 10 and 80 ms respectively, which illustrates the decay due to T1r relaxation that occurs while applying the spin-lock pulse. The resulting T1r map is shown in C) and the color scale on the far right corresponds to the relaxation times in C.
## Strengths and weaknesses of t1r imaging
T1r imaging shares many of the same strengths as CEST; namely that it can be performed using typical MRI hardware, does not require injected contrast agents, and the SNR of the technique relies on the signal from water which exists in high concentrations. However, T1r can also be acquired with a higher spatial resolution and more quickly than CEST since a relatively small number of measurements are needed. Quantitative whole brain T1r measurements can be obtained in 6 min or less with a 2-mm isotropic resolution. Another strength of T1r imaging is the relative short preparation block on the order of 100 ms as compared to CEST imaging where the long saturation pulses can often last a second or more. As a result, the T1r preparation block can be added to fast imaging techniques such as echo-planar imaging and used to assess functional brain activity. Studies of functional T1r have shown that the signal peaks more quickly following a stimulusand that the T1r signal is more spatially Relationship altered between functional T1r and BOLD signals in bipolar disorder (77) 39 BD I, 32 HC Functional T1r and BOLD were strongly related during flashing checkerboard task, but this relationship was weaker in BD PD, Parkinson's disease; HC, healthy control; BD, bipolar disorder; AD, Alzheimer's disease; MCI,-mild cognitive impairment; MS, multiple sclerosis; CSF, cerebrospinal fluid; TBI, traumatic brain injury; WM, white matter constrained than the blood-oxygen-level-dependent (BOLD) contrast that is typically used for functional imaging. These characteristics hold the potential to increase the temporal and spatial resolution of functional imaging. Furthermore, the T1r signal reaches a plateau during stimulation rather than dropping off and lacks a post-stimulus undershoot, both of which are present in BOLD response. Furthermore, it is possible that the source of functional T1r is more directly linked to neuronal activity than the BOLD signal which relies on a hemodynamic response. Because T1r is highly sensitive to pH in the physiological range, one possibility is that the functional T1r signal reflects local pH dynamics. Alternative possibilities include changes in cerebral blood volume (CBV), which accompany the functional hemodynamic response, and may also contribute to the functional T1r signal. However, two studies that suppressed the blood signal using contrast agents (60) and saturation pulsesfound that suppressing the blood signal reduced only a portion of the functional T1r signal leaving most of the signal unchanged. Thus, CBV likely underlies some of the functional T1r signal, but approximately 2/3 of the signal seems to come from other sources that may include glutamine, glucose, and pH. A weakness of T1r is likely its lack of specificity. Given the potential broad sensitivity of T1r imaging to multiple metabolites as well as pH, it may be best to currently consider T1r as an overall marker for metabolic activity and not a quantification of any single metabolite. Hence, its utility in determining changes driven by a single molecule must be performed in a highly controlled experimental condition. While, it has been shown that collecting multiple spin-lock amplitudes as well spin-lock durations can help to identify individual metabolic contributions (54), more work is still needed to identifying how different imaging parameters effect the sensitivity of T1r to different mechanisms or molecules especially in vivo. The factors contributing to T1r relaxation time is a highly relevant topic that is still being investigated by several groups.
T1r imaging should be considered complementary to PET and MRS techniques. PET imaging is specific regarding the metabolic processes or receptors that it is targeting based on the radiotracer administered. Similarly, MRS can quantify several brain metabolites that contain hydrogen or phosphorus atoms with well-known chemical shifts near the Larmor frequency of the particular metabolite. T1r is not specific and instead is influenced by factors that may influence proton exchange. However, T1r is a low-cost technique to screen for potential metabolic changes that could then be followed up with either PET or MRS. For example, finding a T1r difference in the cerebellum could be followed up with targeted single voxel or multi-voxel 1 H and 31 P measurements in that region to help understand the underlying metabolic differences. Likewise, PET imaging could be used to probe differences in glucose metabolism. With the commercial availability of combined MR/PET scanners, simultaneous T1r and PET studies are now possible and could provide new insights into the biological processes that drive the T1r differences that have been observed to date.
## Methodological considerations for t1r imaging
## Magnetic and rf field inhomogeneities
T1r imaging is susceptible to B 1 and B 0 field inhomogeneities. B 1 inhomogeneity leads to a deviation of the expected flip angles and result in banding artifacts in the acquired images. Common approaches for avoiding this include a rotary approach where the phase of the second half of the spin-lock pulse is reversed. However, this approach makes the imaging sensitive to relaxation in the plane perpendicular to the spin-locking RF pulse, which needs to be accounted for using multi-exponential decay models. Phase cycling methodsand adiabatic RFpulses have also been proposed to compensate for the B 1 inhomogeneities. B 0 compensation can be achieved by using a high-amplitude spin-lock pulse. However, this results in tissue heating and elevated specific absorption rate (SAR) issues. A solution that overcomes both B 1 and B 0 inhomogeneity is to insert a 180°RF pulse in the middle of the spin-lock pulse to compensate for off-resonant effects. Some residual B 1 effects may still exist and more complex spin-lock preparation blocks have been proposed.
## T1r imaging time
Multiple images with different spin-lock times (TSLs) are needed to quantify T1r relaxation times, and the preparatory nonselective spin-lock pulse precludes use of many rapid multi-echo and multislice strategies due to heating and saturation constraints. Several techniques have been investigated to reduce acquisition times, including rapid pulse sequences, parallel imaging, keyhole imaging, and reduction of the number of TSLs required for specific applications (53). Johnson et al. proposed an optimal sampling of the spin-lock durations when the approximate values of the T1r relaxation times are known in advanceto improve the T1r imaging efficiency. Owusu et al. have also recently shown that a spin-lock amplitude of 14.1µT (600Hz) may be optimal for in vivo imaging the brain at 3T when considering tissue heating / SAR constraints.
## T1r in functional mr imaging
There are several ways in which functional T1r imaging could be improved. For example, it remains unknown whether there is a stereotypical response function for functional T1r that would serve a similar purpose as the hemodynamic response function does for BOLD imaging. Such a function could be used to improve data collection, allow for more complicated task designs, and would improve statistical analysis of functional T1r data. T1r imaging sequences are also currently less developed, which limits spatial coverage as compared to BOLD imaging due to time needed for the spin-lock pulses in the imaging sequence. However future innovations in sequence development such as the integration of simultaneous multislice (SMS) acquisition may help to overcome this limitation.
## High field imaging
High field imaging can be used to improve the SNR for T1r studies. Furthermore, detection of exchange from certain molecules such as glutamine and glucose (56) may require ultrahigh magnetic fields and large spin-lock amplitudes (e.g. 9.4T; SL amplitudes: 125-4,000 Hz) in order to achieve sufficient signal or to appropriately match their exchange rates, which may be difficult to achieve in vivo due to subject heating / SAR constraints.
## Prior uses of cest and t1r to study psychiatric disorders
Proton-exchange imaging has been used in the study of neurological disorders. CEST and T1r have detected pathophysiological changes in stroke, multiple sclerosis, epilepsy, Alzheimer's disease, Huntington's disease, and Parkinson's disease. These studies are listed in Tables 1, 2 to illustrate the broad utility for proton exchange imaging in neuroscience. In addition, because many of the compounds detectable by CEST and T1r have been suggested to be abnormal in psychiatric illnesses, there is also great potential for CEST and T1r imaging in psychiatric research. Below we summarize how proton exchange techniques have been used thus far in psychiatric research and highlight potential future opportunities for development of proton exchange techniques that would enhance their use in the field. CEST 1 H-MRS studies have suggested that glutamate and glutamine levels in the brain may be abnormal in patients suffering from schizophrenia; both increases and decreases have been reported. Recent studies have thus tested whether CEST might also detect differences in glutamate levels in schizophrenia. One study used CEST to measure glutamate in research participants with psychosis, including 5 with schizophrenia, 14 at familial high risk for developing psychosis, and 17 healthy controls (4). Lower glutamate concentrations were detected in both the schizophrenia and high-risk participants in both cortical and subcortical brain regions. Moreover, the severity of clinical symptoms correlated with the low glutamate concentrations. A second study used CEST to test glutamate concentrations in the olfactory cortex, and found glutamate was increased in participants with schizophrenia compared to a control group. Because sample sizes in these studies were so small, their results remain inconclusive; however, they highlight the potential utility of CEST imaging in psychiatry research.
## T1r
A few studies have tried T1r to probe underpinnings of psychiatric illnesses. For example, Johnson et al. used wholebrain quantitative T1r imaging to study participants with bipolar disorder in the euthymic state compared to healthy control participants. Interestingly, T1r relaxation times were elevated in participants with bipolar disorder, specifically in cerebral cortex (white matter) and cerebellum (white and gray matter). The white matter abnormalities were identified in regions that had not been previously implicated in bipolar disorder by other imaging modalities, including corpus callosum, sagittal striatum, superior longitudinal fasciculus, and cerebellar peduncle. Brain volumes in these regions were normal, suggesting that cellular loss was not the source of the T1r abnormalities. A comparison with inflammatory markers further suggested that inflammation was not likely the source of the increased T1r signal. One potential cause might be altered metabolism, as previous studies in bipolar disorder using MRSbased techniques have found decreased pH, increased glucose concentrations, and evidence of altered cellular metabolism. Interestingly, Johnson et al. found that the elevated T1r signal in the cerebellum in bipolar disorder was absent in participants taking lithium, suggesting the intriguing possibility that lithium may correct the pathophysiology underlying the abnormal T1r signal. Finally, Johnson et al. have also observed differences in T1r relaxation times that were associated with mood. When comparing subjects with bipolar disorder in depressed and manic mood states to those in a euthymic mood state, the subjects in depressed and manic mood states had shorter T1r relaxation times in the basal ganglia and thalamus. These different patterns of T1r signal with different mood states suggest that T1r may be useful for pinpointing specific changes in brain function and/or metabolism during specific mood states. Further study using a longitudinal design would help to better understand these changes.
## Functional t1r
Functional T1r imaging has been explored recently as well. In a study by Magnotta et al., participants with panic disorder and healthy controls underwent both functional T1r and BOLD imaging during a flashing checkerboard task. T1r responses were significantly increased in the visual cortex and significantly decreased in the anterior cingulate cortex in participants with panic disorder compared to healthy controls. Increased T1r responses correlated with panic symptom severity quantified using the Beck Anxiety Inventory. Interestingly these differences were not detected in the BOLD data, suggesting that activity-evoked T1r responses reflect mechanisms distinct from the hemodynamic response. Consequently, activity-evoked T1r responses may help identify abnormalities in brain function that BOLD cannot. Consistent with this possibility, a recent study investigated the relationship between the activity-evoked T1r response and the BOLD signal in research participants with bipolar disorder. Interestingly, the functional T1r -to-BOLD relationship was weaker in people with bipolar disorder compared with healthy controls, suggesting that the mechanisms underlying the functional T1r and BOLD responses are different and somehow differentially affected by the illness. These observations suggest that for some applications functional T1r may be better than BOLD. Moreover, combining the two methods may provide a more thorough functional assessment of brain activity.
## Potential uses for cest and t1r in psychiatric imaging
Given the strengths of proton exchange imaging techniques, they may serve as powerful tools for imaging important aspects of psychiatric illness. Outlined below are a few of the areas where proton exchange imaging could be applied to studies of psychiatric disorders and potential new insights that could be gained.
## Acidosis (or ph sensitive neuroimaging)
Abnormally acidic pH in brain tissue can result from a variety of complex physiological and pathophysiological processes affecting proton generation and/or pH buffering (100-102). These processes include respiration, blood flow, metabolism, and inflammation. Thus, finding pH abnormalities in the brain could be an important step toward understanding an illness and might suggest a variety of potential causes. Because a discussion of the many pathophysiological causes of abnormal pH is too broad for this review, we instead point to specific examples of psychiatric illnesses for which altered brain pH has been implicated. These include panic disorder (103-106), bipolar disorder, and schizophrenia. MR spectroscopy-based studies have confirmed pH changes and lactate changes in schizophrenia and bipolar disorder. Abnormal pH buffering and metabolism have been suggested to cause the acidosis in panic disorder. Altered pH or pH dynamics have the potential to alter physiology and behavior through pH sensitive receptors and channels (100) or by directly affecting macromolecules. The ability to detect these pH differences in the functioning brain may be key to gaining insight into physiology and pathophysiology of psychiatric disorders. Moreover, localized changes in pH might serve as a valuable biomarker. CEST imaging is sensitive to pH since the exchange rates are influenced by pH. Taking advantage of this sensitivity, Zhou et al. demonstrated the utility of APTCEST for pH sensitive imaging studies. Several studies have since then employed APTCEST to detect pH reduction in acute ischemic acidosis. While pH sensitivity of proton exchange is detectable via other flavors of CEST including GluCEST and LATEST, APTCEST remains the most widely employed pH-sensitive CEST technique because it can be readily performed at 3T. Absolute quantification of pH is not currently possible in vivo without the use of a pH reporting CEST contrast agent.
T1r imaging is also sensitive to pH. Systemic manipulation of pH by using CO 2 inhalation caused increase in T1r relaxation times. Similarly, the T1r has shown to increase in human brain in response to visual flash checkerboard (82), a task that has been shown to decrease pH detected by 31 P spectroscopy and to increase lactate/creatine ratio by 1 H MR Spectroscopy.
## Neurotransmitters
Glutamate and GABA are the most common excitatory and inhibitory neurotransmitters in the brain and are thought to have a role in several psychiatric disorders. Postmortem and MRS studies have reported altered glutamate levels in diverse brain areas in individuals with mood disordersand glutamatergic abnormalities are also thought to have a role in schizophrenia. Several novel classes of antidepressants and mood stabilizers target glutamatergic activity. However, it is difficult to study glutamate concentrations either dynamically or throughout the brain using MRS. GluCEST may provide an alternative method for measuring differences in glutamatergic function and to assess relationship between glutamate levels and clinical symptoms or in response to therapies. Similarly, GABA has been implicated in schizophrenia, bipolar disorder and major depression through several postmortemand MRS studies. GABA plays an important role in inhibition in the brain and is thought to regulate functions that have been disrupted in psychiatric illness including oscillatory rhythms, information processing, and sensory gating. GABACEST may therefore be a useful tool in measuring the activity of GABAergic neurons in psychiatric illness.
## Neuroinflammation
MICEST is sensitive to myoinositol, a glial cell marker that can be used as an indicator for neuroinflammatory response. Neuroinflammatory microglia activity has been reported in autism, mood disorders, and schizophrenia. Gene expression studies in postmortem brain tissue have also indicated glial abnormalities, including reduced expression of astrocyte related genes in the cerebral cortex of individuals with major depressionand oligodendrocyte related transcripts in bipolar disorder. Postmortem studies have also observed reduced glial number in the hippocampusand dorsolateral prefrontal cortex in alcoholism, with and without comorbid mood disorder. Therefore, MICEST may be a useful neuroimaging tool for the study of glial cells in vivo, particularly their development and response to treatments.
## Metabolism
Creatine (Cr) is an important molecule for energy homeostasis and metabolism and altered creatine levels have been reported in schizophrenia, bipolar disorder, and other mood disorders. While creatine is difficult to distinguish from phosphocreatine (PCr) using 1 H MRS, phantom studies and muscle energetics studies using pre and post-exercise have shown that the contribution of PCr is minimal in the signal measured by CrCEST, allowing for more accurate measurement of Cr. Similarly, the direct measurement of glucose (GlucoCEST), lactate (LATEST) and glycogen (Glycogen CEST) can also be used to accurately and quickly measure brain metabolism. GlucoCEST could also be used in conjunction with exogenous 3-O-methyl-D glucose, a method that has been shown to be useful for identifying tumors in rodent modelsand in human glioma patients, but that could also be used to measure the uptake of glucose in psychiatric disorders. 3-O-methyl-D-glucose is non-radioactive and biodegradable, and the dynamic imaging is shown to provide comparable results to that of FDG PET studies. These approaches are currently limited to ultra-high field strengths (18).
## Multimodal imaging
Because CEST and T1r can be performed in the same imaging session as other common MRI techniques, it may be useful to use it as part of a multi-modal approach. For instance, combining T1r imaging with T1 and T2-weighted structural images may provide insight into the neurobiological underpinnings for the anatomical differences that have been identified in a number of psychiatric disorders. Likewise, the use of quantitative T1r may improve the interpretability of DWI data and may be used to help clarify whether differences in tissue diffusivity are related to altered tissue integrity (i.e. fewer/smaller cells), inflammation (more fluid), or tissue organization. Perhaps most interestingly, the use of functional T1r imaging in conjunction with BOLD imaging may allow researchers to separate metabolic and hemodynamic components of the functional response to task stimuli.
Ultimately, further work is needed to identify when to use T1r or CEST imaging most effectively. Currently, for example, T1r could be used to identify whether metabolic differences are present in a psychiatric disorder with image-level resolution. These findings could then be followed up with MRS to identify specific metabolites that may be abnormal in regions-of-interest identified using T1r. Similarly, CEST imaging could be used to study differences in certain metabolite concentrations with high spatial resolution. Taken together, this potential for high resolution metabolite maps may allow for rapid, whole-brain screening for metabolic abnormalities in psychiatric illness and may also serve as a novel biomarker for assessing treatment response. Identifying such opportunities; and also identifying new ways to use chemical exchange imaging techniques is an area of ongoing research.
# Conclusion
Proton exchange is a novel target for measuring certain aspects of brain metabolic changes in vivo. Two brain imaging methods, CEST and T1r are sensitive to proton exchange and have been successfully implemented in clinical studies in human populations. However, their use to study psychiatric disorders has been minimal. CEST provides the ability to measure the concentration of specific molecules including a number of metabolic products and neurotransmitters that are thought to be relevant to psychiatric disorders. This selectivity suggests that CEST can be used as an complementary approaches to positron emission tomography (PET) or magnetic resonance spectroscopy (MRS) for certain experiments. T1r is less specific than CEST but is also more sensitive and can be carried out more quickly allowing better spatial and temporal resolution as well as for functional imaging. Therefore, T1r may be an alternative to PET and functional blood-oxygen-level-dependent (BOLD) contrast imaging. Continued improvement in these imaging techniques to shorten their acquisition time, sensitivity, and specificity may provide new insight into the neurobiology of these devastating diseases that to date have shown relatively subtle differences using MR imaging and have not yet provided a diagnostic test.
# Author contributions
JS and MM were principally responsible for authoring the content of this review and are co-first authors. SS was responsible for the artistic creation of several figures and was substantially involved in the editing process. JX and NO provided technical knowledge regarding CEST and T1rho imaging and assisted with the editing process. DW assisted with editing and revising the manuscript. VM and JW are the senior researchers and provided feedback and were substantially involved in the editing and revision process. All authors contributed to the article and approved the submitted version.
# Funding
Several of the co-authors of this manuscript are funded by NIH (R01EB022019 and R01MH111578) to study the brain using T1r imaging. Some of the data shown in this manuscript was conducted on an MRI instrument funded by NIH (S10RR028821). |
Crystal structure of the red light-activated channelrhodopsin Chrimson
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# Results
Structural determination. As the expression level of wild-type Chrimson is very low in insect cells, we tested two Chrimson variants in which the extracellular N-terminal sequence was replaced by either the corresponding sequence of the Chloromonas subdivisa channelrhodopsin (CsChrimson) or the Nterminal sequence of CrChR1 (C1Chrimson) (Supplementary . Although CsChrimson showed improved expression and excellent stability in detergent, the purified protein yielded only poorly diffracting crystals, with low reproducibility. In contrast, C1Chrimson yielded crystals that diffracted X-rays to the maximum resolution of 2.6 Å, and the structure was determined by molecular replacement, using the C1C2 structure as the template (PDB: 3UG9) . The photocurrent properties of Chrimson were minimally affected by the different N-terminus, and red light activation, high proton selectivity, and fast pHdependent photocurrent kinetics were preserved in all three Chrimson variants (Supplementary Figure 4a-e). For simplicity, we will therefore refer to C1Chrimson as Chrimson in the following sections. Overall structure of Chrimson. a The dimeric structure of Chrimson is shown in ribbon models, with the two protomers in different colors. b Chrimson (purple) and C1C2 (orange) are superimposed, viewed from within the membrane plane (left), the intracellular side (center) and the extracellular side (right). The TM numbering is indicated on each helix, and the deviation of the helix termini for TM2 and TM7 are indicated by red arrows Gate interactions. Although Chrimson is distantly related to the CrChRs (about 50% sequence identity over transmembrane region) (Supplementary , the overall structure of Chrimson superimposes well with those of C1C2 . The two protomers in the asymmetric unit form a dimer, with each protomer composed of the extracellular N-terminal domain, 7TM helix bundle and the C-terminal β-hairpin loop region. The CrChR1-derived N-terminal residues mediate the crystal packing contacts between the layers in the lipidic cubic phase crystal and facilitate the dimeric interaction by forming the inter-protomeric β-sheet and three disulfide bonds (Supplementary [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref]. While the outer membranous regions are partially disordered, the TM region is clearly visible in the electron density map, including the covalently attached all-trans retinal (Supplementary [fig_ref] Figure 3: Counterion configuration of Chrimson and adjacent residues [/fig_ref]. The structures of the two protomers (mol A and B) are essentially the same (RMSD = 0.66) (Supplementary [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref] , but due to their different crystal packing contacts, mol A presents better electron density. Therefore, our discussion below is based on the mol A structure of Chrimson. In direct superposition of Chrimson with C1C2 and CrChR2, we observed slight differences in the helix orientations, especially at TM2 and TM7, which deviate by about 2 Å on the extracellular side and 2.5 Å on the intracellular side, respectively . As these helices are directly involved in the ion pore formation in ChRs, by mediating conformational changes during the photocycle 21 , these variations might be partly responsible for the differences in ion permeation of Chrimson and CrChRs 22 . In Chrimson, the putative ion pathway, formed by TM2, 3, 6, and 7, is lined by five highly conserved negatively charged residues, Glu124 (E1), Glu125 (E2), Glu132 (E3), Glu139 (E4), and Glu143 (E5) (residue numbering is according to wild-type Chrimson 16 ) [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref]. In the current ground state structure of Chrimson, the ion-conducting pore is closed by three constriction sites that restrict ion permeation, on the intracellular side (inner gate), at the middle of the membrane (central gate), and on the extracellular side (outer gate) [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref]. While the inner and central gates are also observed in C1C2 and CrChR2 7,13 , their constituents differ in several aspects [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref] and Supplementary [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. The inner gates of C1C2 and CrChR2 are mainly formed by ionic interactions between the acidic residues (E1 and E2) and the basic residues (His173 and Arg307 in C1C2 and His134 and Arg268 in CrChR2) [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref]. In Chrimson, this histidine is substituted with lysine (Lys176), and both Glu124 (E1) and Glu125 (E2) are oriented toward the cytoplasmic solvent, with only E2 providing an ionic interaction with Arg310. The central gate is located adjacent to the retinal Schiff base [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref] middle panels), where three hydrophilic residues (Ser102, Glu129 (E3) and Asn297 in C1C2, and Ser63, Glu90 (E3) and Asn258 in CrChR2) occupy the pore and thereby hinder ion permeation. In Chrimson, two of the three residues are substituted, resulting in a rather loose packing. In addition, Ala105 (serine in C1C2 and CrChR2) is no longer involved in the gate formation, and instead, Glu300 forms a hydrogen bond with the backbone carbonyl of Ala101, which is one helical turn below the involved residues in C1C2 and CrChR2. Finally, we observed remarkable structural diversity at the extracellular end of the ion-conducting pathway among all three ChR structures. In Chrimson, direct hydrogen bonding between Glu139, Tyr159, and Ser288 interconnects TM2, TM3, and TM7 and constitutes the outer gate constriction [fig_ref] Figure 2: Ion pores of Chrimson and ChRs [/fig_ref] lower panels). In contrast, C1C2 has an open tunnel that allows water access to the middle of the pathway, and CrChR2 has an extended water-filled cavity (EC2), which is interrupted at the extracellular end of TM2 by hydrogen bonds between Glu101, Gln117 and Thr246 (EG: Extracellular gate, Supplementary [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. Mutations of the three pore-lining glutamates involved in the interhelical hydrogen bonds in Chrimson, namely, Glu125 (E2) in the inner gate, Glu300 in the central gate and Glu139 (E4) in the outer gate, caused dramatic decelerations in the photocurrent kinetics [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref] , whereas the homologous mutations of E2 and E4 in CrChR2 did not affect the channel kinetics [bib_ref] Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2, Sugiyama [/bib_ref]. Therefore, the observed variations in the gating interactions may account for the fast gating kinetics of Chrimson 22 . Unique counterion configuration in Chrimson. The electrostatic interaction between the protonated Schiff base (PSB) and the counterion is the major determinant for the absorption spectrum of the retinal chromophore [bib_ref] Light-induced helix movements in channelrhodopsin-2, Muller [/bib_ref] [bib_ref] Electrostatic potential at the retinal of three archaeal rhodopsins: implications for their..., Kloppmann [/bib_ref] [bib_ref] The opsin shift and mechanism of spectral tuning in rhodopsin, Rajamani [/bib_ref]. In general, negative charges in close proximity to the PSB stabilize the ground state and lead to a higher energy gap between the ground and excited states, resulting in blue-shifted absorption, while neutralization or spatial separation of the counterion charge leads to a lower energy gap and red-shifted absorption. In C1C2, the positive charge of the PSB is stabilized by two anionic counterion residues, Ci 1 and Ci 2 (Glu162 and Asp292, respectively), which are highly conserved among ChRs, including Chrimson (Glu165 and Asp295) [bib_ref] Independent optical excitation of distinct neural populations, Klapoetke [/bib_ref] [bib_ref] Diversity of Chlamydomonas channelrhodopsins, Hou [/bib_ref] [fig_ref] Figure 3: Counterion configuration of Chrimson and adjacent residues [/fig_ref]. Recent studies have indicated that the highly red-shifted absorption of Chrimson is partly due to the protonation of the counterion residue Glu165 [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref] [bib_ref] Photochemical properties of the red-shifted channelrhodopsin Chrimson, Urmann [/bib_ref] , which weakens the stabilization effect for the PSB, thus causing red-shifted absorption similar to that of the acidic form of BR (BR 605 ) [bib_ref] Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in Channelrhodopsins, Grimm [/bib_ref]. Consistent with these studies, the purified Chrimson protein showed a large pH-dependent spectrum shift (Supplementary and b). The crystals of Chrimson were obtained under low pH conditions (Methods), and the blue color of the crystals indicates that the current structure represents its red lightabsorbing (protonated) state (Supplementary and d).
Regarding the two counterion residues, Glu165 is in 3.0 Å proximity to the PSB, while Asp295 is farther away, at 3.6 Å [fig_ref] Figure 3: Counterion configuration of Chrimson and adjacent residues [/fig_ref]. Glu165 resides just beside the PSB, a location corresponding to the hydrated water molecule in the bacteriorhodopsin (BR) dark state [fig_ref] Figure 3: Counterion configuration of Chrimson and adjacent residues [/fig_ref] , and the two counterion residues Glu165 and Asp295 are only 3.3 Å apart from each other (Supplementary . Therefore, the Glu165 proton might be partly shared by both counterion residues, via hydrogen bond interactions. Most importantly, the counterion complex is shielded from the extracellular bulk solvent by the hydrophobic side chain of Phe135, which is essential to stabilize the protonated form of the carboxylate, as reported in previous studies [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref] [bib_ref] Photochemical properties of the red-shifted channelrhodopsin Chrimson, Urmann [/bib_ref] [bib_ref] Role of a helix B lysine residue in the photoactive site in..., Li [/bib_ref]. . This can be explained by the decreased pK a and consequent deprotonation of the remaining counterion residue (Asp295 for the Glu165 mutants and Glu165 for the Asp295 mutants), consistent with previous studies of Chrimson [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref] [bib_ref] Photochemical properties of the red-shifted channelrhodopsin Chrimson, Urmann [/bib_ref] and other channelrhodopsins [bib_ref] Role of a helix B lysine residue in the photoactive site in..., Li [/bib_ref]. The blue-shift was more prominent when Asp295 is mutated, whereas the mutation of Glu165 to Gln or Ala had a moderate effect. This is also consistent with the counterion configuration in the current structure. Since the PSB is closer to Glu165 than to Asp295, the neutralization of Asp295 should focus the negative charge exclusively on Glu165, which is much closer to the PSB, thus causing a larger blue-shift. Whereas blue-shift was equally pronounced for all Asp295 mutants, it varied largely for different mutants of Glu165 action (e, f) and absorption (g, h) spectra of the counterion residue mutants (Action spectra represent Mean ± SD; n = 5-7; symmetric 110 mM NaCl, pH e,i 7.2 and −60 mV. Datasets indicated by asterisk are quoted from a previous study [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref]. Same mutants are indicated by the same color codes. The fitted curves for the D295N and D295A action spectra were adjusted to the photocurrents activated by light of 440 nm or higher wavelength. Absorption spectrum of the D295A mutant could not be measured, due to its instability in the detergent solubilized form, and thus is not included in the panel (h)
In addition to the counterion residues, mutations of the neighboring carboxylates, such as Glu132 (E3), Glu139 (E4), and Glu300, located within the extracellular cavity, also led to strong blue-shifts [fig_ref] Figure 4: Outer gate and the extracellular ion pore [/fig_ref] and Supplementary Figure 7d-f), although these residues are not in the proximity of the Schiff base. The effects of these mutants are more prominent, as compared to the counterion mutants (E165Q and D295N), although these mutants still retained the pH-dependency and showed small fractions of red-light sensitive components under the low pH conditions (Supplementary . Therefore, we conclude that both mutations predominantly affect the protonation of the counterion residues, via either the hydrogen-bonding network or long-range electrostatic interactions. In contrast, the mutation of Glu143, located outside the extracellular cavity, had minimal effects on the absorption spectrum and the pH-dependent shift (Supplementary . These results indicated that the cluster of carboxylate residues that are separated from the extracellular solvent by the outer gate might also contribute to the protonation of the counterion residues. Consistently, mutations that destabilize the outer gate hydrogen-bonding network, such as Tyr159 and Glu139 (E4), caused a largely blue-shifted activation spectrum [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref].
We further investigated the residues constituting the extracellular ion-conducting pathway. At the exit of this pathway, Arg162 partly participates in the extracellular ion pore, together with Tyr159 and Phe135 [fig_ref] Figure 4: Outer gate and the extracellular ion pore [/fig_ref]. Stabilized by a hydrogen bond with Asn287, Arg162 is slightly oriented toward the negatively charged residue Glu277, which is exposed to the solvent. The mutation of Arg162, as well as those of Asn287 and Glu277, caused a large blue-shift of the activation spectrum [fig_ref] Figure 4: Outer gate and the extracellular ion pore [/fig_ref] , indicating that Arg162 electrically affects the PSB environment, and its precise arrangement is important for the protonation of Glu165. By analogy to its functional significance in other microbial rhodopsins [bib_ref] The role of protein-bound water molecules in microbial rhodopsins, Gerwert [/bib_ref] , we assumed that Arg162 is directly involved in channel opening and thus characterized its role in more detail. Whereas the R162A mutant is completely non-functional, as previously shown in C1C2 and CrChR2 7,31 , mutations to positively charged residues preserved the channel function, although the mutants showed significantly reduced c Mutations in the extracellular ion pore caused blue-shifting of the action spectrum. Normalized peak photocurrents after 10 ms excitation at different wavelengths of equal photon count (Mean ± SD; n = 5-7; symmetric 110 mM NaCl, pH e,i 7.2 and −60 mV) are shown. d Representative photocurrent traces of the Arg162 and Asn287 mutants at different voltages illuminated either with 550 nm light (green line) or with 580 nm light (orange line) depending on peak absorption. The inset shows representative photocurrent traces of wild type (upper) and R162H (bottom) upon replacement of Na + with guanidinium. The purple arrows indicate the photocurrent increase or decrease upon cation exchange in the R162H mutant and Chrimson WT, respectively. e Current-voltage dependence of normalized R162H photocurrents in extracellular solutions of different cation or proton composition (mean ± SD; n = 6-9; intracellular 110 mM NaCl, pH i 7.2). R162H is still selective for protons, as the current-voltage dependence drastically shifts under the alkaline conditions (two-sample t-test with p = 0.025), while it is not affected by decreasing the Na + ion concentration (p = 0.75). In contrast, the current-voltage dependence is drastically shifted when Na + is replaced by guanidinium (p = 0.0002), indicating that R162H is permeable to guanidinium. f Normalized photocurrents at the ionic conditions of (e) and −60 mV compared to WT (Mean ± SD, n = 5-10; intracellular 110 mM NaCl, pH i 7.2, junction potential corrected). g Normalized photocurrent amplitudes of WT and N287 mutants at different extracellular pH (Mean ± SD; n = 2-10; intracellular 110 mM NaCl, pH i 7.2; −60 mV). Photocurrents of both mutants and WT were equally reduced at low proton concentration indicating comparable high proton selectivity (two-sample t-test compared to WT with p < 0.0001 for N287A and p = 0.9 for N287E). At extracellular pH 5.0 photocurrent increase of the N287E mutant was significantly higher than for WT or the N287A mutant (p = 0.5 for N287A and p = 0.0005 for N287E) NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06421-9 ARTICLE NATURE COMMUNICATIONS | (2018) 9:3949 | DOI: 10.1038/s41467-018-06421-9 | www.nature.com/naturecommunications amplitudes and impaired kinetics [fig_ref] Figure 4: Outer gate and the extracellular ion pore [/fig_ref] and . Unexpectedly, a photocurrent analysis in HEK cells indicated that the ion-conducting pore of the R162H mutant is permeable to the large cation guanidinium, which is only poorly conducted by wild-type Chrimson [fig_ref] Figure 4: Outer gate and the extracellular ion pore [/fig_ref] [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref]. Furthermore, the photocurrent amplitudes were significantly reduced by the N287E mutation, which may stabilize the Arg162 orientation in the current dark state structure, whereas the photocurrents were partly restored in pH 5, where this effect should be weakened [fig_ref] Figure 4: Outer gate and the extracellular ion pore [/fig_ref]. As previous studies have shown that two neighboring carboxylate residues, E4 (Glu139) and E5 (Glu143), located at the extracellular end of TM2, are responsible for the proton selectivity in Chrimson [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref] , these results are in support of Arg162 being directly involved in the formation of the ionconducting pore and possibly contributing to the ion selective filter of Chrimson.
[formula] E165 D295 E132 E139 F135 Q98 S288 E143 E300 A101 ATR Y159 N287 E277 Y163 R162 K299 A105 a b c * ns WT N287E N287A WT WT WT WT R162H R162H R162H N287E N287A [/formula]
Retinal binding pocket. As described above, the outer gate and the consequential protonation of the counterion residues essentially contribute to the red-shifted absorption of Chrimson. However, even at high pH with a deprotonated counterion, Chrimson absorbs a longer wavelength (530 nm) than C1C2 (480 nm), implying an additional mechanism contributing to the large red-shifted absorption. Compared to C1C2, about half of the residues constituting the retinal binding pocket are substituted in Chrimson, and the structural comparison revealed that Chrimson resembles BR rather than C1C2, with respect to the residues surrounding the polyene chain and the β-ionone ring [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. We constructed Chrimson mutants with residues mutated to the corresponding residues of C1C2, and measured their action spectra [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. The spectra of M201T, S223G, Y231F, Y268F, and A298S were all blue-shifted by [bib_ref] Conversion of channelrhodopsin into a light-gated chloride channel, Wietek [/bib_ref] The residue numbering of Chrimson is indicated above the sequence. Chrimson resembles BR in TM5-7, surrounding the polyene chain and β-ionone ring, while it resembles C1C2 (or CrChRs) in TM3, located near the ion-conducting pore. Residues common to BR and C1C2 are depicted in cyan and red, respectively. e Mutations that affect action spectrum. Normalized peak photocurrents after 10 ms excitation at different wavelengths of equal photon count (Mean ± SD; n = 5-8; symmetric 110 mM NaCl, pH e,i 7.2 and −60 mV) are shown. f Apparent photocurrent off-kinetics of retinal binding pocket mutants (τ apparent off ; Mean ± SD; n = 5-11; symmetric 110 mM NaCl, pH e,i 7.2 and −60 mV). Empty columns for M201T and M201N represent conductance measurements by short 20 ms voltage pulses to −60 mV at 0.2 and 0.5 Hz, respectively, and at a holding potential of 0 mV in order to reduce kinetic artefacts imposed by intracellular acidification due to continuous proton influx as previously reported 22 (Mean ± SD; n = 5-6). g, h Absorption spectra of wild type and mutant Chrimson (g) and C1C2 (h). Mutations at the same positions are indicated by the same color codes. Peak shifts caused by mutation is indicated by blue or red arrow. i Representative photocurrent traces of the M201T, C170A, and C198D mutants, measured at different voltages in symmetric 110 mM NaCl, pH e,i 7.2 and illuminated with 580 nm light (orange line) red-shifted activation of Chrimson. According to the general mechanism of color tuning in retinal proteins, negative polarity near the β-ionone ring stabilizes a light-excited intermediate that involves charge movement toward the β-ionone ring during retinal isomerization and thus leads to the red-shifted absorption, while negative polarity near the Schiff base leads to the blue-shifted absorption [bib_ref] The opsin shift and mechanism of spectral tuning in rhodopsin, Rajamani [/bib_ref]. Consistently, polar residues such as Ser223 and Tyr231 are located near the β-ionone ring, while the non-polar residue Ala298 is located near the Schiff base [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. All of these residues are conserved in other red-shifted variants such as VChR1 and ReaChR, but they are substituted with the non-polar residues Gly220 and Phe228, and the polar residue Ser295, respectively, in C1C2 [fig_ref] Figure 5: Retinal binding pocket [/fig_ref] and . The contributions of these residues to the red-shifted absorption were further confirmed by measuring the absorption spectra of the above mutants (S223G, Y231F, and A298S) [fig_ref] Figure 5: Retinal binding pocket [/fig_ref] , which though showed slightly larger blue-shifts as compared to the action spectra. Conversely, the introduction of these residues to C1C2 (G220S, F217Y, and S295A) caused a redshift of the absorption peak by about 10-30 nm [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. These results show that the biased distribution of the polar residues throughout the retinal binding pocket contributes to the redshifted absorption of Chrimson and other red-shifted variants. Meanwhile, Tyr268 is the exception, as the non-polar substitution Y268F caused blue-shifted absorption by about 10 nm, although it is located near the Schiff base [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. As Tyr268 is engaged in the hydrogen bond with the counterion residue Asp295 [fig_ref] Figure 3: Counterion configuration of Chrimson and adjacent residues [/fig_ref] , the mutation to phenylalanine probably allows Asp295 to approach toward the PSB and stabilizes its positive charge, thus causing the blue-shift.
Additional structural differences were found in TM4 and TM5, which flank the retinal binding pocket [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. Chrimson has several bulky residues on these helices. In particular, the bulky side chain of Met201 tightly packs against the polyene chain of retinal [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. As previously shown for C1C2 and archaerhodopsin-3, the residue at this position is particularly important for the torsion angle at the C6 = C7 bond [bib_ref] Atomistic design of microbial opsin-based blue-shifted optogenetics tools, Kato [/bib_ref]. In agreement with this study, the mutation of Met201 to the smaller residue, threonine, caused a large blue-shift in the absorption peak by about 50-80 nm [fig_ref] Figure 5: Retinal binding pocket [/fig_ref] and . In C1C2, the reverse mutation T198M caused a redshifted absorption [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. These observations suggest that tight packing against the polyene chain is important for the red-shifted absorption, probably by affecting the π-electron conjugation.
Remarkably, the M201T mutant further showed extremely slow closing kinetics, and photocurrents were sustained for minutes [fig_ref] Figure 5: Retinal binding pocket [/fig_ref] , indicating an additional important role of Met201 in channel gating kinetics. Also in bacteriorhodopsin, the alanine mutation of the corresponding methionine (Met118) or the adjacent methionine (Met145) leads to a slower photocycle and a consequent reduction in the proton pump activity, as well as a large blue-shift in the absorption [bib_ref] Hydrophobic amino acids in the retinal-binding pocket of bacteriorhodopsin, Greenhalgh [/bib_ref] [bib_ref] Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs, Ihara [/bib_ref]. These results indicate that the structural rigidity around the β-ionone ring is especially crucial for the proper photocycle process. Therefore, the slow kinetics of the M201T mutant is probably associated with the loose interaction at the retinal binding pocket, which decelerates conformational propagation from the isomerized retinal to the protein. The effect of the M201T mutation is reminiscent of the step function opsin (SFO) mutants of the CrChRs, in which the mutation of the DC-pair (Asp195 and Cys167 in C1C2 and Asp156 and Cys128 in CrChR2) caused extremely slow channel kinetics, by affecting proton exchange reaction at the Schiff base [bib_ref] Bistable neural state switches, Berndt [/bib_ref] [bib_ref] Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond, Bamann [/bib_ref] [bib_ref] Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins, Hososhima [/bib_ref]. Although Met201 occupies a position close to the DCpair in CrChRs [fig_ref] Figure 5: Retinal binding pocket [/fig_ref] , the Asp residue of DC-pair, which is the putative internal proton donor for the deprotonated Schiff base in CrChR2 37 , is substituted with cysteine (Cys198), suggesting a completely different mechanism between the Chrimson M201T mutant and the CrChR SFO mutants. In Chrimson, mutation of the Cys128 homolog (C170A) had minimal impact on the photocurrent kinetics, and the reintroduction of the DC-pair (C198D) caused nearly complete loss of the photocurrent, albeit accelerated photocurrent kinetics [fig_ref] Figure 5: Retinal binding pocket [/fig_ref] , further supporting a different gating mechanism of Chrimson compared to the previously proposed gating model for CrChRs. The observation in Chrimson is more in agreement with the general importance of the retinal binding pocket stability for the overall photocycle 38 , which is indeed different between Chrimson and CrChRs [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. This notion is also consistent with the recent study showing that mutations near the β-ionone ring (TM6) affect the channel kinetics in Chrimson and other ChRs 39 .
Rational design of a more red-shifted Chrimson. The current structure and concomitant extensive mutation analysis revealed three important factors underlying the large red-shift of Chrimson: the unique protonation state of the counterion residues, the highly biased distribution of the polar residues toward the βionone ring, and the structural rigidity of the retinal binding pocket. These notions allowed us to rationally design a more redshifted Chrimson variant. As two of the three factors, the electrostatic stabilization of the PSB and the tight association around the retinal pocket, are already highly optimized in Chrimson, we engineered the polarity distribution around the retinal binding pocket and successfully obtained a red-shifted mutant by decreasing the polarity near the Schiff base (S169A) [fig_ref] Figure 5: Retinal binding pocket [/fig_ref]. The absorption and action spectra peaks of this mutant are at 608 nm and 605 nm, respectively, which are red-shifted by about 20 nm as compared to wild-type Chrimson and . Surprisingly, this mutant displayed 10-fold accelerated channel closing kinetics, as compared to wild-type Chrimson , which is comparable to the recently reported engineered Chrimson variants with fast closing kinetics (f-Chrimson and vf-Chrimson) [bib_ref] High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics, Mager [/bib_ref]. Due to the red-shift and the accelerated closing kinetics, we named this new mutant Chrim-sonSA (S169A, Super red-shifted and Accelerated). To validate its usefulness for optogenetic applications, we tested ChrimsonSA in hippocampal neurons . Although the photocurrent density of the S169A mutant was reduced in HEK cells, as compared to wild-type Chrimson (12 ± 4 pA/pF for S169A versus 90 ± 60 pA/pF for WT) , its photocurrent size is comparable to the CrChR2 stationary currents measured in HEK cells under the same conditions (15 ± 7 pA/pF) [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref]. In neurons, photocurrents of ChrimsonSA were smaller than WT Chrimson but sufficient to trigger action potential firing with 635 nm light at intensities of 5 mW/mm 2 or less , confirming its utility for optogenetic applications. Neurons expressing ChrimsonSA further showed significantly smaller blue-lightevoked photocurrents, as compared to red light, than neurons expressing wild-type Chrimson, resulting in a higher red/bluelight-activation ratio . As a consequence, the action potentials were selectively evoked by red light, with a significantly decreased probability of blue-light-evoked action potential firing at similar intensities . Furthermore, due to the accelerated channel closing kinetics of this mutant, the recovery of the membrane potential after each spike was quicker as compared to neurons expressing wild-type Chrimson, leading to a more natural waveform of light-evoked action potentials . Thus, ChrimsonSA is a useful optogenetic tool for neuronal spiking with far-red light and therefore of high value for dual-color optogenetic experiments.
# Discussion
Chrimson is widely used in optogenetic experiments, since its red-shifted action spectrum allows deep tissue penetration of the excitation light, activation in a spectral range beyond the natural sensitivity of native photoreceptors in many animal species and combination with blue-light-activated tools for dual-color experiments. In the present study, we have reported the crystal structure of Chrimson, which revealed substitutions and structural differences in the putative ion pore, as compared to the CrChRs, which contribute to the different gating kinetics and the elevated proton selectivity of Chrimson. While the putative ion pore shares similarity to other ChRs, the structural motifs of Chrimson around the retinal binding pocket are more similar to those of BR. Since ChRs presumably evolved from prokaryotic light-driven pumps [bib_ref] New insights into the evolutionary history of type 1 rhodopsins, Ruiz-Gonzalez [/bib_ref] , Chrimson might therefore reflect an early branching of ChRs during the molecular evolution from its prokaryotic ancestors. We conducted an extensive mutational analysis, which revealed the structural features in the counterion region and retinal binding pocket that enable the red light absorption and fast channel gating transitions in Chrimson. Chrimson-S169A in rat CA1 pyramidal cells. a, b Red-shifted absorption spectrum (a) and action spectrum (b) of Chrimson S169A (ChrimsonSA) (Mean ± SD; n = 7; 10 ms illumination of equal photon counts, symmetric 110 mM NaCl, pH e,i 7.2 and −60 mV). c, d Photocurrents of ChrimsonSA in HEK cells. Representative photocurrent traces (c) and channel closing kinetics (d) at the same ionic conditions as in (b) are shown. e CA1 pyramidal neuron heterogeneously expressing ChrimsonSA-mCerulean, 5 days after electroporation (stitched maximum intensity projections of two-photon images). The inset shows a magnified view of the apical dendrite. The scale bars indicate 10 μm. f Representative photocurrent traces of Chrimson-WT (top) and ChrimsonSA (bottom) expressing CA1 pyramidal cells evoked with either 635 nm (red trace) or 460 nm (blue trace, 1 mW/ mm 2 ) light. The ChrimsonSA traces were scaled to WT Chrimson g Relative photocurrent responses of WT Chrimson and ChrimsonSA to blue light (460 nm, 10 ms, 1 mW/ mm 2 ). Values were normalized to the response to red light (635 nm, 10 ms, 1 mW/ mm 2 ). Dots represent single cells. Lines show mean ± SEM (n WT = 6; n S169A = 7). h Quantification of the red/blue activation ratio. Bar plots show mean ± SEM. i, j Action potentials triggered by red light pulses at a frequency of 10 Hz in cells expressing WT Chrimson (i) and ChrimsonSA (j) at threshold light intensities of 0.5 and 5 mW/mm 2 , respectively. Using the same light intensities, the action potentials were triggered by blue light in WT, but not in the ChrimsonSA. Dashed lines represent the membrane resting potential. k, l The light intensities at 460 nm and 635 nm required to reach the action potential threshold in neurons expressing WT Chrimson (k) or ChrimsonSA (l) Based on these findings, we engineered ChrimsonSA, with further red-shifted peak absorption beyond 600 nm and strongly accelerated photocurrent closing kinetics. ChrimsonSA represents the most red-shifted microbial rhodopsin known to date that preserves protein function. Due to its reduced blue-light sensitivity, it is an excellent optogenetic actuator for dual-color applications.
# Methods
Expression and purification of Chrimson. The C1Chrimson was constructed by replacing the N-terminal sequence of CsChrimson with that of Clamydomonas reinhardtii CrChR1 , by In-Fusion Cloning Kit (Clontech). The construct was subcloned into a modified pFastBac1 vector for expression in Sf9 insect cells (11496015, Thermo Fischer Scientific), with the tobacco etch virus (TEV) protease cleavage site, the enhanced GFP (EGFP), and the FLAG-tag (EGFP-FLAG) fused at the C-terminus. Baculovirus-infected Sf9 cells were cultured in Sf900II (Invitrogen) at 27°C for 24 h, and 100 µl all-trans retinal (SIGMA-ALDRICH) was added at 24 h post-infection. Cells were harvested 48 h after infection by centrifugation at 6000× g for 10 min. The pellets were disrupted by sonication and resuspended in buffer containing 150 mM NaCl, 50 mM HEPES, pH 7.0, 5% glycerol and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The cell debris was cleared by centrifugation at 10,000× g for 30 min, and the crude membrane fraction was collected by ultracentrifugation (Ti45 rotor, 215,000× g, 1 h). This fraction was solubilized in buffer containing 150 mM NaCl, 50 mM HEPES, pH 7.0, 5% glycerol, 0.1 mM PMSF, 2.5% n-dodecyl-β-D-maltoside (DDM), and 0.5% cholesteryl hemisuccinate (CHS). The insoluble material was removed by ultracentrifugation (Ti70 rotor, 208,000× g, 30 min), and the supernatant was mixed with ANTI-FLAG M2 Agarose Affinity Gel(SIGMA-ALDRICH). After binding for 1.5 h, the flow-through was discarded. Following the cleavage of EGFP-FLAG by TEV protease (homemade), the flow-through containing Chrimson was collected, concentrated, and further purified by size-exclusion chromatography in 150 mM NaCl, 50mM HEPES, pH 7.0, 5% glycerol, 0.05% DDM, and 0.01% CHS. Peak fractions were pooled and concentrated to 7.0 mg/ml for crystallization.
Crystallization. The purified C1Chrimson protein was mixed with monoolein (SIGMA-ALDICH) in a 2:3 protein to lipid ratio (w/w). Aliquots (50 nl) of the protein-LCP mixture were spotted on a 96-well sitting-drop plate and overlaid with 800 μl of precipitant solution by the crystallization robot, Crystal Gryphon (Art Robbins Instruments). Crystals were obtained in 28-33% (w/v) PEG500DME, 100 mM Na citrate, pH 5.0, 100 mM Na malonate, pH 7.0, and 10-40 mM sarcosine. All crystals were incubated for 4 weeks in the dark. The crystals were harvested using micromounts (MiTeGen) and flash-cooled in liquid nitrogen without any additional cryoprotectant.
Structure determination. X-ray diffraction datasets were collected at the SPring-8 BL32XU beamline. Data processing and merging of multiple crystals were performed using the program KAMO (https://github.com/keitaroyam/yamtbx/blob/ master/doc/kamo-en.md) [bib_ref] KAMO: towards automated data processing for microucrystals, Yamashita [/bib_ref]. Each dataset was indexed and integrated using XDS. Since the crystals were anisotropic, with varied lattice parameters, datasets with a caxis less than 173 Å and a b-axis less than 82 Å were selected. Finally a group of outlier-rejected datasets was scaled and merged using XSCALE. The structure was solved by the molecular replacement method implemented in Phenix Phaser-MR 43 , using the model of C1C2 (PDB: 3UG9). The structure was manually modified to fit into the electron density maps, using the program Coot 44 , and then refined with the programs Phenix 43 and Refmac5 in the CCP4 suite [bib_ref] REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use, Vagin [/bib_ref] Ultraviolet/visible spectroscopy. C1Chrimson was fused with the C-terminal FLAG-tag sequence, and expression and purification were performed as described above. The peak fraction of the size-exclusion chromatography was collected and concentrated for the measurement. The pH was adjusted by the addition of 20 μl 1 M buffer (pH5:Na-acetate, pH6:Na-citrate, pH7:HEPES-NaOH, pH8:Tricine-NaOH, pH9:Tris-HCl) to 100 μl of the protein solution. Ultraviolet/visible absorption spectra were recorded with an Ultrospec 3300 Pro ultraviolet/visible spectrophotometer (Amersham Biosciences), using 1-cm quartz cuvettes.
HEK-cell electrophysiology. The coding sequences of Chrimson (KF992060.1), CsChrimson (KJ995863.2), and C1Chrimson were cloned into the pmCerulean-C1 vector (using the NheI and AgeI restriction sites for Chrimson and CsChrimson or the Nhe1 and Kpn2I restriction sites for C1Chrimson) and expressed under the control of the CMV-promotor. Site-directed mutagenesis was performed using a QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA), according to the manufacturer's instructions. As the Chrimson photocurrents were nearly unaffected by the targeting strategy [fig_ref] Figure 4: Outer gate and the extracellular ion pore [/fig_ref] , we employed CsChrimson as the backbone for the electrophysiological mutant analysis.
HEK cell preparation and whole-cell patch-clamp experiments were performed as previously described [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref] [bib_ref] Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in Channelrhodopsins, Grimm [/bib_ref]. HEK293 (human embryonic kidney) cells (85120602, Sigma-Aldrich) were cultured in Dulbecco's Modified Medium (DMEM) with stable glutamine (Biochrom, Berlin, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS Superior; Biochrom, Berlin, Germany), 1 μM all-trans retinal and 100 µg/ml penicillin/streptomycin (Biochrom, Berlin, Germany). The cells were seeded on coverslips at a concentration of 0.75 × 10 5 cells/ml and transiently transfected using the FuGENE® HD Transfection Reagent (Promega, Madison, WI) 28-48 h before measurement.
Patch pipettes were prepared from borosilicate glass capillaries (G150F-3; Warner Instruments, Hamden, CT) using a P-1000 micropipette puller (Sutter Instruments, Novato, CA) and were subsequently fire polished. The pipette resistance was between 1.8 and 3.0 MΩ. Single fluorescent cells were identified using an Axiovert 100 inverted microscope (Carl Zeiss, Jena, Germany). Monochromatic light (±7 nm) was provided by a Polychrome V monochromator (TILL Photonics, Planegg, Germany), attenuated by a motorized neutral density filter wheel (Newport, Irvine, CA) for equal photon fluxes at different excitation wavelengths, and temporally controlled by a VS25 and VCM-D1 shutter system (Vincent Associates, Rochester, NY). Recorded signals were filtered at 2 kHz using an AxoPatch 200B amplifier (Molecular Devices, Sunnyvale, CA) and digitized using a DigiData 1440 A digitizer (Molecular Devices, Sunnyvale, CA) at a sampling rate of 5-10 kHz. The reference bath electrode was connected to the bath solution via a 140 mM NaCl agar bridge. The extracellular buffer exchange was performed manually by adding at least 5 ml of the respective buffer to the recording chamber (500 µl chamber volume) while a Ringer Bath Handler MPCU (Lorenz Messgerätebau, Katlenburg-Lindau, Germany) maintained a constant bath level. Standard bath solutions contained 110 mM NaCl, 1 mM KCl, 1 mM CsCl, 2 mM CaCl 2 , 2 mM MgCl 2 , and 10 mM HEPES at pH e 7.2 (with glucose added up to 310 mOsm). Standard pipette solution contained 110 mM NaCl, 1 mM KCl, 1 mM CsCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM EGTA, and 10 mM HEPES at pH i 7.2 (glucose added up to 290 mOsm). For ion selectivity measurements, NaCl was replaced by either 110 mM GuanidiniumCl or 110 mM NMDGCl with 1 mM NaCl remaining, and HEPES was substituted with citric acid or TRIS for pH 5 or pH 9 respectively. For the measurement of ultra slow closing kinetics (empty columns [fig_ref] Figure 5: Retinal binding pocket [/fig_ref] cells were clamped at 0 mV and channel conductance was probed by short 20 ms voltage pulses to −60 mV at the slow frequency of 0.2 or 0.5 Hz (for M201T or M201N, respectively) in order to reduce kinetic artefacts imposed by intracellular acidification due to continuous proton influx as previously reported [bib_ref] Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin, Vierock [/bib_ref]. The light intensities were measured after passing through all of the optics using a P9710 optometer (Gigahertz-Optik, Türkenfeld, Germany) and normalized to the water Plan-Apochromat 40 × /1.0 differential interference contrast (DIC) objective illuminated field (0.066 mm 2 ). The maximum light intensity was 2.28 mW × mm −2 at 580 nm and 2.47 mW × mm −2 at 530 nm. All electrical recordings were controlled by the pCLAMP™ software (Molecular Devices, Sunnyvale, CA). The whole-cell recordings had a minimum membrane resistance of 500 MΩ (usual > 1 GΩ) and an access resistance below 10 MΩ.
Electrical recordings were analyzed using the Clampfit 10.7 software (Molecular Devices, Sunnyvale, CA), Microsoft Excel and Origin 2017® (OriginLab, Northampton, MA). Photocurrent traces were baseline corrected, filtered, and reduced in size for display purposes. Photocurrents were normalized to peak photocurrents at −60 mV under standard conditions of symmetric 110 mM NaCl pH e/i 7.2. Action spectra were fitted using a parametric Weibull function (y = y0 + A*((w2-1)/w2)^((1-w2)/w2)*S^(w2-1)*exp(-s^w2 + (w2-1)/w2) with S = (x-xc)/ w1 + ((w2-1)/w2)^(1/w2) and the estimated parameters A, y0, w1, w2, and xc). The photocurrent kinetics were estimated by biexponential fits and simplified by an apparent time constant (τ apparent ) calculated as (A 1 *τ 1 + A 2 *τ 2 )/(A 1 + A 2 ). The exact number of biological replicates for each measurement is provided in the figure legend. To compare the data, we performed two-sample t-tests with Welch's correction in Origin 2017®. The significance thresholds were set at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).
Neuronal recordings in hippocampal slice cultures. Chrimson wild type and the ChrimsonSA mutant were subcloned into neuron-specific expression vectors (pAAV backbone, human synapsin promoter). Organotypic slice cultures of rat hippocampus were prepared as described [bib_ref] Preparation of slice cultures from rodent hippocampus, Gee [/bib_ref] and transfected by single-cell electroporation 15 after 14 days in vitro (DIV). Animal procedures were in accordance with the guidelines of local authorities and Directive 2010/63/EU. Plasmids were each diluted to 1 ng/μl in K-gluconate-based solution consisting of (in mM): 135 K-gluconate, 4 MgCl 2 , 4 Na 2 -ATP, 0.4 Na-GTP, 10 Na 2 -phosphocreatine, 3 ascorbate, 0.02 Alexa Fluor 594, and 10 HEPES (pH 7.2). An Axoporator 800 A (Molecular Devices) was used to deliver 50 hyperpolarizing pulses (−12 mV, 0.5 ms) at 50 Hz. At DIV 18-20, targeted patch-clamp recordings of transfected neurons were performed under visual guidance using a BX-51WI microscope (Olympus, Hamburg, Germany) equipped with Dodt-gradient contrast and a Double IPA integrated patch amplifier controlled with SutterPatch software (Sutter Instrument, Novato, CA, USA). Patch pipettes with a tip resistance of 3-4 MΩ were filled with (in mM): 135 K-gluconate, 4 MgCl 2 , 4 Na 2 -ATP, 0.4 Na-GTP, 10 Na 2 -phosphocreatine, 3 ascorbate, 0.2 EGTA, and 10 HEPES (pH 7.2). Artificial cerebrospinal fluid (ACSF) consisted of (in mM): 135 NaCl, 2.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 Na-HEPES, 12.5 D-glucose, 1.25 NaH 2 PO 4 (pH 7.4). Synaptic currents were blocked with 10 µM CPPene, 10 µM NBQX, and 100 µM picrotoxin (Tocris, Bristol, UK). Measurements were not liquid junction potential (LJP) corrected. The LJP was -15.5 mV. A 16-channel pE-4000 LED light engine (CoolLED, Andover, UK) was used for epifluorescence excitation and delivery of light pulses (ranging from 365 to 660 nm). Light intensity was measured in the object plane with a 1918-R power meter equipped with a calibrated 818-ST2-UV/D detector (Newport, Irvine CA) and divided by the illuminated field (0.134 mm 2 ) of the LUMPLFLN 60XW objective (Olympus).
Two-photon microscopy. The custom-built two-photon imaging setup was based on an Olympus BX-51WI upright microscope upgraded with a multiphoton imaging package (DF-Scope, Sutter Instrument, Novato, CA, USA and Rapp OptoElectronic, Wedel, Germany), and controlled by ScanImage (Vidrio Technologies, Ashburn, VA, USA). Fluorescence was detected through the objective (LUMPLFLN 60XW, Olympus, Hamburg, Germany) and the oil-immersion condenser (1.4 NA) using GaAsP-PMTs (Hamamatsu Photonics, Hamamatsu, Japan). A tuneable Ti:Sapphire laser (Chameleon Vision-S, Coherent, Dieburg, Germany) was set to 810 nm to excite cerulean. 46. Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. .
[fig] Figure 2: Ion pores of Chrimson and ChRs. a Water accessible cavities are illustrated in the Chrimson structure, with the putative ion pathway indicated by an arrow. Five glutamic acid residues lining the ion pore (E1-5) and two counterion residues (Ci 1 and Ci 2 ) are indicated by sticks, and the three constriction sites for the inner, central and outer gates are indicated by boxes. b Comparison of the constriction sites of Chrimson (left panels), C1C2 (center panels), and CrChR2 (right panels), for the inner (upper panels), central (middle panels), and outer (lower panels) gates. The constituent residues are shown as sticks, and the TM helix number is indicated on each helix [/fig]
[fig] Figure 3: Counterion configuration of Chrimson and adjacent residues. a-d Hydrogen-bonding interactions around the protonated Schiff base are shown for Chrimson (a), BR (b), C1C2 (c), and CrChR2 (d). Proposed hydrogen-bonding interactions are indicated by yellow dotted lines, and additional possible interactions with longer distances are indicated by gray lines, with the distance (Å) indicated beside each line. e-h [/fig]
[fig] Figure 4: Outer gate and the extracellular ion pore. a Residues around the outer gate are shown as sticks, with the interactions indicated by yellow lines. Water accessible cavities are indicated by blue meshes. b Absorption spectra of the carboxylate mutants within the extracellular ion pore and the outer gate. [/fig]
[fig] Figure 5: Retinal binding pocket. a-c Structural comparisons of the retinal binding pockets of Chrimson (a), C1C2 (b), and BR (c). As the retinal pockets of C1C2 and CrChR2 are quite similar, C1C2 is shown as the representative structure of the CrChRs. Residues constituting retinal binding pocket are shown in sticks (left panels). CPK model representations shows different association in the retinal binding pocket (right panels). d Sequence comparison of Chrimson with C1C2 and BR. [/fig]
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Association between immunosuppressants and poor antibody responses to SARS-CoV-2 vaccines in patients with autoimmune liver diseases
The antibody and B cell responses after inactivated SARS-CoV-2 vaccination have not been well documented in patients with autoimmune liver disease (AILD). Therefore, we conducted a prospective observational study that included AILD patients and healthy participants as controls between July 1, 2021, and September 30, 2021, at the Second Affiliated Hospital of Chongqing Medical University. All adverse events (AEs) after the COVID-19 vaccination were recorded and graded. Immunoglobulin (Ig)-G antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (anti-RBD-IgG) and neutralizicadng antibodies (NAbs) were tested following full-course vaccination (BBIBP-CorV or CoronaVac). In addition, SARS-CoV-2-specific B cells were detected by flow cytometry. In total, 76 AILD patients and 136 healthy controls (HCs) were included. All AEs were mild and self-limiting, and the incidences were similar between the AILD and HCs. The seropositivity rates of anti-RBD-IgG and NAbs in AILD were 97.4% (100% in HCs, p = 0.13) and 63.2% (84.6% in HCs, p < 0.001), respectively. The titers of anti-RBD-IgG and NAbs were significantly lower in AILD patients than those in HCs. After adjusting for confounders, immunosuppressive therapy was an independent risk factor for low-level anti-RBD-IgG (adjusted odds ratio [aOR]: 4.7; 95% confidence interval [CI], 1.5-15.2; p = 0.01) and a reduced probability of NAbs seropositivity (aOR, 3.0; 95% CI, 1.0-8.9; p = 0.04) in AILD patients. However, regardless of immunosuppressants, the SARS-CoV-2-specific memory B cells responses were comparable between the AILD and HC groups. Our results suggest that inactivated SARS-CoV-2 vaccines (BBIBP-CorV and CoronaVac) are safe, but their immunogenicity is compromised in patients with AILD. Moreover, immunosuppressants are significantly associated with poor antibody responses to the SARS-CoV-2 vaccines. These results could inform physicians and policymakers about decisions on screening the populations at Frontiers in Immunology frontiersin.org 01 (2022) Association between immunosuppressants and poor antibody responses to SARS-CoV-2 vaccines in patients with autoimmune liver diseases. Front. Immunol. 13:988004.
# Introduction
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a significant global public health threat. To date, more than 528 million people have been diagnosed with COVID-19, and more than 6 million deaths have been confirmed worldwide [bib_ref] An interactive web-based dashboard to track COVID-19 in real time, Dong [/bib_ref]. Numerous studies have shown that people with comorbidities, including chronic liver disease, are highly vulnerable and have worse outcomes from COVID-19 than those without underlying liver disease [bib_ref] Prevalence of chronic liver disease in patients with COVID-19 and their clinical..., Kovalic [/bib_ref]. Therefore, liver societies have recommended vaccination against SARS-CoV-2 for patients with chronic liver diseases. However, a series of case reports suggest that mRNA-based vaccines may induce autoimmune liver disease (8- [bib_ref] A prospective multicenter study assessing humoral immunogenicity and safety of the mRNA..., Vera-Lastra [/bib_ref]. This has caused concern among hepatologists as well as patients with autoimmune liver diseases (AILD) (17, 18).
AILD are chronic immune-mediated liver diseases that includ a wide range of disorders, such as autoimmune hepatitis (AIH), primary biliary cholangitis (PBC) and AIH-PBC overlap syndrome, which are frequently treated with either broad or targeted immunosuppressants. Moreover, some AILD patients usually have other autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus and Sjögren's syndrome, which also require lifelong immunosuppressive drug therapy. Existing data have shown that immunosuppressants can significantly reduce the antibody response to COVID-19 mRNA vaccines or the Johnson & Johnson vaccine in liver transplant patients (19) and immune-mediated inflammatory disorders .
In China, inactivated vaccines (BBIBP-CorV or CoronaVac) are widely used COVID-19 vaccines. Systematic evaluation of the safety and immunogenicity of these vaccines in people with AILD has been rare. Here, we aim to evaluate the safety and antibody responses after the whole-course COVID-19 vaccination and explore the association between immunosuppressants and antibody responses to inactivated SARS-CoV-2 vaccines in patients with AILD.
# Patients and methods
## Study design and participants
Between July 1, 2021, and September 30, 2021, we performed a prospective observational study at the Second Affiliated Hospital of Chongqing Medical University, China. We included participants aged older than 18 years, diagnosed with any of the prespecified immune-mediated liver disorders (AIH or AIH-PBC overlap syndrome) (22-24), no SARS-CoV-2 infection before receipt of the first vaccine dose (determined based on either a negative anti-SARS-CoV-2 IgM/IgG test or the absence of a positive polymerase chain reaction assay result for SARS-CoV-2, with no history of suspected clinical SARS-CoV-2 infection), completed whole-course COVID-19 vaccination (2 doses of BBIBP-CorV or CoronaVac vaccine), and were able to understand and complete questionnaires. Healthy participants were included as healthy controls (HCs). Participants with known pregnancy during study entry, those who did not complete the full course of vaccination, and those who provided incomplete vaccination information (including the date of first vaccine dose and complete vaccination and vaccine manufacturers) were excluded. Blood samples were collected for serological assays for SARS-CoV-2 at least 21 days after the whole-course vaccination from AILD patients and HCs.
This study was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University and in accordance with the ethical guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants. This study has been registered at ClinicalTrials.gov (NCT05007665).
## Variables and definitions
Clinical characteristics, including age, sex, body mass index (BMI), comorbidities, history of diseases and concomitant medications, of all patients were collected via a standardized questionnaire. The presence or absence of cirrhosis was confirmed using clinical or biochemical evidence, FibroScan, liver imaging (ultrasound, CT, or MRI) and endoscopy. The concomitant medications, especially types and doses of immunosuppression, were further confirmed through the prescribing information system in the hospital.
All adverse events (AEs) within 7 days and 30 days after COVID-19 vaccination were recorded and graded according to the National Medical Products Administration of China (version 2019). AEs related to vaccination were judged by investigators. Safety was evaluated by determining the overall incidence of AEs.
## Sars-cov-2 antibody test
The SARS-CoV-2 antibody against the spike protein receptor-binding domain (anti-RBD-IgG) was detected by indirect ELISA using the SARS-CoV-2 RBD antibody detection kit (Sino Biological, Beijing, China). The lower limit of quantification is 5.0 arbitrary units per mL (AU/mL). The neutralizing antibodies (NAbs) were detected by the competitive ELISA method using the SARS-CoV-2 neutralizing antibody detection kit (Sino Biological, Beijing, China). The details were described in our previous study (25).
## Sars-cov-2-specific b cells responses
Previous studies have described the selection of markers of memory B cells (MBCs) and their subsets [bib_ref] Immune memory in convalescent patients with asymptomatic or mild COVID-19, Long [/bib_ref]. For SARS-CoV-2-specific B cells detection, biotinylated SARS-CoV-2 spike RBD protein (Sino Biological, 40592-V08H2-B) was mixed with streptavidin BV421 (Biolegend, 405225) at a 4:1 molar ratio for one hour at 4°C to obtain the antigen probe. According to the manufacturer's instructions, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood by Histopaque (Sigma-Aldrich, 10771) density gradient centrifugation. After washing with FACS buffer (PBS+2% FBS), PBMCs were stained for 30 minutes at 4°C using an antigen probe (1:33.3) and the following conjugated antibodies: anti-human CD3 (300430, Biolegend, 1:50), anti-human CD19 (302212, Biolegend, 1:50), anti-human CD21 (354918, Biolegend, 1:50), anti-human CD27 (356406, Biolegend, 1:50), anti-human IgG Fc (410722, Biolegend, 1:50), and anti-human IgM (314524, Biolegend, 1:50). After staining, the cells were rewashed and resuspended in 200 µl FACS buffer. Samples were then evaluated by flow cytometry (Beckman Coulter, CytoFLEX) and analyzed using FlowJo (Treestar, 10.0.7r2).
# Statistical analysis
Data are presented as the median (interquartile range, IQR) for continuous variables and proportions for categorical variables. Continuous variables were compared using Student's t test for the variables of age and BMI. Categorical variables were compared using Fisher's exact test or the chi-square test for sex, cirrhosis, comorbidities, immunosuppressants, and vaccine types. One-way analysis of variance (ANOVA) was used to compare the results of multiple groups, and Tukey's correction was used to correct for comparisons between groups. Negative responses and relatively low antibody levels following SARS-CoV-2 vaccination were classified as poor antibody responses in previous studies [bib_ref] Poor antibody response after two doses of severe acute respiratory syndrome coronavirus..., Mazzola [/bib_ref] [bib_ref] Factors associated with poor antibody response to third-dose SARS-CoV-2 vaccination in patients..., Connolly [/bib_ref]. In this study, a poor antibody response was defined as an anti-RBD-IgG titer less than the median value (≤ 34.0 AU/ml) or negative for NAbs. Multiple logistic regression was used to explore the independent variables associated with poor antibody responses and presented as odds ratios (ORs) (95% confidence intervals, CIs) with adjustment for potential confounding factors. Statistical analysis was performed using EmpowerStats (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA, USA) and R (http://www. Rproject.org, the R Foundation). All statistical tests were twosided, and p < 0.05 was considered statistically significant.
# Results
Characteristics of participants 76 eligible AILD patients and 136 HCs were included in the study. The characteristics of the participants are shown in [fig_ref] TABLE 1: Characteristics of the Participants [/fig_ref] after COVID-19 vaccination was slightly higher in AILD patients than in the HC group (p = 0.20) [fig_ref] TABLE 2: Adverse events of COVID-19 vaccination in participants [/fig_ref]. All AEs were mild, and none of them had any serious AEs. The common AEs in AILD patients in local AEs were pain at the injection site (4.0%, 3/76); in systemic AEs were fatigue (5.3%, 4/76) and headache (5.3%, 4/76). The most common AE in HC was local pain at the injection site (8.1%, 11/136) [fig_ref] TABLE 2: Adverse events of COVID-19 vaccination in participants [/fig_ref]. Notably, one patient increased the serum gamma-glutamyl-transpeptidase (GGT) level from 20 U/L to 90 U/L (upper limit of normal: 45 U/L) after vaccination and returned to normal after continuing the original treatment strategy during the follow-up. Another patient's antinuclear antibody (ANA) was positive at a titer of 1:320 before vaccination. It increased to 1:1000 after vaccination, but the liver function test, serum IgG, anti-liver-kidney microsomal, anti-smooth muscle, anti-mitochondrial antibodies, and anti-soluble liver antigen were normal, and the patient had no symptoms of discomfort.
## Covid−19 vaccination safety
## Antibody responses after covid-19 vaccination
The seropositivity for anti-RBD-IgG was 97.4% (74/76) in AILD patients, which was similar to that in the HC group (100%) (p = 0.13) . However, anti-RBD-IgG levels were significantly lower in AILD patients than those in HCs (mean: 49.1 AU/mL vs 71.9 AU/mL, p = 0.02) . Regarding NAbs, seropositivity (63.2% [48/76] vs 84.6% [115/ 136]) and antibody titers were both significantly lower in AILD patients than those in HCs (p < 0.001) . Compared with controls, anti-RBD-IgG and NAbs levels seemed to decrease slightly over time after the second dose vaccination in AILD patients .
## Effect of immunosuppressants on antibody responses
Analysis of clinical features showed no significant correlations between poor antibody responses and parameters such as age, sex, BMI, cirrhosis, and comorbidities (all p > 0.05) [fig_ref] TABLE 3: Distribution of clinical characteristics by serum antibody titers to SARS-CoV-2 vaccine in... [/fig_ref]. However, low-level antibodies were significantly related to the types of vaccine (p = 0.01) [fig_ref] TABLE 3: Distribution of clinical characteristics by serum antibody titers to SARS-CoV-2 vaccine in... [/fig_ref] , Supplementary and the use of immunosuppressants (p = 0.04) [fig_ref] TABLE 3: Distribution of clinical characteristics by serum antibody titers to SARS-CoV-2 vaccine in... [/fig_ref] ; , and these results were consistent with univariate analysis [fig_ref] TABLE 1: Characteristics of the Participants [/fig_ref]. Furthermore, after adjusting for potential confounding factors (age, BMI, sex, cirrhosis, comorbidities, types of vaccine, and days between final dose and antibody test) in multiple logistic regression analysis, the use of immunosuppressants remained significantly related to low-level antibody levels . Compared with patients with no immunosuppressive medication, the crude odds ratio (OR) of lowlevel antibody response risk among patients who used immunosuppressants was 3.3 (95% CI, 1.3-8.5; p = 0.01), and their adjusted OR (aOR) increased to 4.9 (95% CI, 1.5-15.6; p = 0.01). Notably, the risk trend does not seem to increase with the number of immunosuppressants. The aORs of the use of one and more immunosuppressive medications were 5.0 (95% CI, 1.1-23.1; p = 0.04) and 4.9 (95% CI, 1.1-19.2; p = 0.03), respectively . Similar results were also observed for NAbs responses in the AILD patients. Negative NAbs were significantly associated with the types of vaccine (p = 0.01) (Supplementary and immunosuppressants (p = 0.02) (Supplementary , except for age, sex, BMI, cirrhosis, and comorbidities [fig_ref] TABLE 1: Characteristics of the Participants [/fig_ref]. After adjusting for confounding factors, immunosuppressants were associated with a reduced probability of NAbs seropositivity (aOR, 3.0; 95% CI, 1.0-8.9; p = 0.04), especially when ≥2 immunosuppressive medications were used (aOR, 4.4; 95% CI, 1.3-15.3; p = 0.02) .
Concisely, the results suggested that immunosuppressive therapy was an independent risk factor for poor antibody responses to COVID-19 vaccination in patients with AILD.
## Specific b cells responses after covid-19 vaccination
The mean frequencies of B cells (CD3 -CD19 + ) in AILD patients with and without immunosuppressive therapy were 9.42% and 8.63%, respectively, with no statistically significant difference compared with those in HCs (8.82%). The frequency of total MBCs (CD3 -CD19 + CD27 + ) was significantly higher in HC group than in patients with AILD who were not treated with immunosuppressants (37.6% vs 29.7%, p = 0.01), while the frequency in patients treated with immunosuppressants was at an intermediate level . To further investigate the humoral immune response to the SARS-CoV-2 vaccine, the frequency and phenotype of specific B cells were also detected. As expected, the percentage of specific B cells was very low in the peripheral blood of AILD patients and the HC group. No significant difference was found in the frequency of RBD-specific B cells (CD3 -CD19 + RBD + ) and IgG RBD-specific memory B cells (IgG + CD3 -CD19 + RBD + CD27 + ) between the AILD and HC groups, regardless of immunosuppressants . However, the frequency of IgM RBD-specific MBCs (IgM + CD3 -CD19 + RBD + CD27 + ) was significantly lower in AILD patients with (17.2% vs 25.3%, adjusted p < 0.01) or without immunosuppressants (19.4% vs 25.3%, adjusted p = 0.03) than in the HC group . To better understand the functional phenotype of RBD-specific MBCs, we further compared RBD-specific resting MBCs (CD3 -CD19 + RBD + CD21 + CD27 + ), RBDspecific activated MBCs (CD3 -CD19 + RBD + CD21 -CD27 + ), RBDspecific atypical MBCs (CD3 -D19 + RBD + CD21 -CD27 -), and RBDspecific intermediate MBCs (CD3 -CD19 + RBD + CD21 + CD27 -) between AILD patients and HC groups. Compared with HCs, AILD patients without immunosuppressants had a lower frequency of RBD-specific activated MBCs (13.0% vs 16.9%, adjusted p = 0.03) and a higher frequency of RBD-specific intermediate MBCs (47.1% vs 39.9%, adjusted p = 0.02), but not in patients with immunosuppressants. Moreover, there was no significant difference in RBD-specific resting MBCs and RBD-specific atypical MBCs between AILD patients and the HC groups . These results indicate that patients with AILD may develop humoral immunity as robust as in a healthy population when receiving a booster dose or against SARS-CoV-2 infection despite ongoing immunosuppression.
# Discussion
AILD is a chronic disease characterized by immune-mediated disorders. Safety and immunogenicity have been of concern in patients with AILD since vaccination against COVID-19. In this prospective observational study, we found that inactivated SARS-CoV-2 vaccines achieved a favorable safety profile, but their immunogenicity is compromised in patients with AILD. The use of immunosuppressants had an estimated 3-to 5-fold increased risk of poor antibody responses to the SARS-CoV-2 vaccine. Moreover, the specific MBCs responses were comparable between patients in the AILD and HC groups despite ongoing immunosuppression.
Similar to the general population and other chronic liver diseases, such as NAFLD, liver transplantation, chronic hepatitis B, and liver cirrhosis, adverse events related to the COVID-19 vaccine in patients with AILD were mild and self-resolved within a few days after vaccination (19, [bib_ref] Efficacy and safety of an inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac): interim results..., Tanriover [/bib_ref] [bib_ref] Safety and immunogenicity of COVID-19 vaccination in patients with non-alcoholic fatty liver..., Wang [/bib_ref] [bib_ref] Safety and immunogenicity of a SARS-CoV-2 inactivated vaccine in patients with chronic..., Xiang [/bib_ref]. However, after the whole-course vaccination, one patient experienced an increased serum GGT level, and another experienced a sharply increased Data are presented as n (%) or median (IQR). *Stratified by the median level of anti-RBD-IgG to SARS-CoV-2 vaccine. AILD, autoimmune liver disease; BMI, body mass index; IQR, interquartile range; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 ANA titer. Because they refused liver biopsy, it is unclear whether this phenomenon reflects the fluctuation of the disease itself or whether the vaccine induces new-onset autoimmune diseases. Fortunately, we did not observe any evidence of clinical deterioration in neither patients during follow-up over 6 months. Herein, we believe that the COVID-19 inactivated vaccine is safe in patients with AILD. Among the AILD patients, the COVID-19 inactivated vaccine showed an efficient antibody response of anti-RBD-IgG (97.4%). This was similar in a multicenter study of NAFLD patients in China (95.5%) but much higher than the reported seropositivity of SARS-CoV-2 RBD-specific antibodies in patients with chronic hepatitis B virus infection who were also vaccinated with inactivated COVID-19 vaccines (87.25%) [bib_ref] Safety and immunogenicity of COVID-19 vaccination in patients with non-alcoholic fatty liver..., Wang [/bib_ref] [bib_ref] Safety and immunogenicity of a SARS-CoV-2 inactivated vaccine in patients with chronic..., Xiang [/bib_ref]. This can be attributed to the large percentage of patients in our study were female (85.5% vs 27.5%).
Xiang et al. found that female patients exhibited higher seropositivity for SARS-CoV-2 RBD-specific antibodies than males with chronic hepatitis B (95.1% vs 84.3%) [bib_ref] Safety and immunogenicity of a SARS-CoV-2 inactivated vaccine in patients with chronic..., Xiang [/bib_ref]. A similar finding that female vaccine recipients showed more robust antibody responses to COVID-19 vaccination was also reported in a clinical trial in Turkey [bib_ref] Efficacy and safety of an inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac): interim results..., Tanriover [/bib_ref]. Similar to chronic hepatitis B, the NAbs seropositivity was lower than that of anti-RBD-IgG in patients with AILD. We found that immunosuppressants have an estimated 3-to 5-fold increased risk of poor antibody responses to the SARS-CoV-2 vaccine. In a nationwide multicenter prospective cohort study of 125 patients with multiple sclerosis, Bsteh et al. reported that immunosuppressive therapy could significantly reduce the probability of NAbs seropositivity after symptomatic COVID-19 (OR, 0.51; 95% CI, 0.17-0.82) [bib_ref] Humoral immune response after COVID-19 in multiple sclerosis: A nation-wide Austrian study, Bsteh [/bib_ref]. This finding might partly explain why the seropositivity and titer of NAbs in AILD patients B A Association analysis between immunosuppressant and poor antibody responses to SARS-CoV-2 vaccine in patients with AILD. Odds ratio (95% CI) of poor response to RBD-IgG (A) and NAbs (B) in immunosuppressant-treated AILD patients in crude (unadjusted) and adjusted models compared with healthy controls. The adjusted model was adjusted age, BMI, sex, cirrhosis, comorbidities, types of vaccine, and days between final dose and antibody test. AILD, autoimmune liver diseases; BMI, body mass index; CI, confidence interval; OR, odds ratio; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
are lower than those in HCs. However, a cohort study in patients with immune-mediated inflammatory disorders on immunosuppressants showed that only certain specific immunosuppressants attenuated the humoral responses after SARS-CoV-2 mRNA vaccine. More studies are needed to compare the effects of multiple types of immunosuppressants on different types of SARS-CoV-2 vaccines.
This study found that the RBD-specific MBCs responses were comparable between patients with AILD and HCs despite ongoing immunosuppression. It's consistent with Kirchner et al. in a small study showing that patients with AIH receiving immunosuppressive therapy still developed strong humoral and cellular immunity to SARS-CoV-2 (34). Given the role of MBCs, it is speculated that patients with AILD may develop humoral immunity as robust as those in a healthy population when receiving a booster dose or against SARS-CoV-2 infection.
There are several main limitations in this study. First, a lack of longitudinal serial antibody testing limits the possibility of measuring a change in antibody levels individually. Second, the relatively small sample size of the study and the lack of subgroup analyses, such as strength of immunosuppression and antibody response, may reduce the reliability and limit the generalizability of the findings. Third, the antibody response is only part of the immunogenicity of the COVID-19 vaccine, so there is a need to explore the T-cell response. However, given the unprecedented nature of the COVID-19 pandemic and the low prevalence of AILD, we believe that our study offers valuable insights into the management of these patients to clinicians.
In conclusion, the COVID-19 inactivated SARS-CoV-2 vaccines (BBIBP-CorV and CoronaVac) are safe, but their immunogenicity is compromised in patients with AILD. In addition, immunosuppressants are significantly associated with poor antibody responses to the SARS-CoV-2 vaccines. These results could inform physicians and policymakers about decisions on screening the populations at higher risk of poor antibody responses to SARS-CoV-2 vaccines and providing additional vaccinations in patients with AILD.
# Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.
# Ethics statement
The studies involving human participants were reviewed and approved by ethics committee of the Second Affiliated Hospital of Chongqing Medical University. The patients/participants provided their written informed consent to participate in this study.
# Author contributions
The authors DC, DZ, and HR contributed to the conception and design and critical revision of important intellectual content. Data collection was performed by YW, LA, MK, ZC, MC, MP, NL, and PH. Statistical analysis was performed by HL and YW. The first draft of the manuscript was written by HL. All authors approved the final version and agreed to be accountable for all aspects of the work.
# Funding
This work is supported by the National Science and Technology Major Project of China (2017ZX10202203-007, 2017ZX10202203-008, 2018ZX10302206-003) and a pilot project of clinical cooperation between traditional Chinese and western medicine for significant and complicated diseases of the National Administration of Traditional Chinese Medicine: hepatic fibrosis.
[table] TABLE 1: Characteristics of the Participants. [/table]
[table] TABLE 2: Adverse events of COVID-19 vaccination in participants. [/table]
[table] TABLE 3: Distribution of clinical characteristics by serum antibody titers to SARS-CoV-2 vaccine in patients with AILD. [/table]
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The Influenza A Virus Endoribonuclease PA-X Usurps Host mRNA Processing Machinery to Limit Host Gene Expression
Graphical Abstract Highlights d Influenza A virus PA-X targets the majority of host mRNAs for destruction d Downregulation by PA-X correlates with the number of splice sites in a transcript d Splicing renders RNAs susceptible to PA-X d The cellular CFIm complex interacts with PA-X and contributes to PA-X activity Correspondence [email protected] (C.M.), [email protected] (D.A.K.), [email protected] (M.M.G.) In Brief Gaucherand et al. uncover a unique relationship between RNA degradation by the influenza A virus ribonuclease PA-X and host RNA splicing, which allows PA-X to selectively target host RNAs for destruction.
# Introduction
Despite their small genomes, influenza A viruses (IAVs) dedicate multiple proteins to the suppression of host gene expression, or ''host shutoff,'' which limits host antiviral responses. One of these IAV host shutoff proteins is the endoribonuclease PA-X, which selectively degrades host RNAs [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref] and limits innate immune responses in vivo. PA-X-deficient viruses induce stronger innate immune and inflammatory responses in mice, chickens, and pigs [bib_ref] The contribution of PA-X to the virulence of pandemic 2009 H1N1 and..., Gao [/bib_ref] [bib_ref] PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured..., Gong [/bib_ref] [bib_ref] Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of..., Hayashi [/bib_ref] [bib_ref] PA-X decreases the pathogenicity of highly pathogenic H5N1 influenza A virus in..., Hu [/bib_ref] [bib_ref] PA-X-associated early alleviation of the acute lung injury contributes to the attenuation..., Hu [/bib_ref] [bib_ref] An overlapping protein-coding region in influenza A virus segment 3 modulates the..., Jagger [/bib_ref] [bib_ref] PA-X protein contributes to virulence of triple-reassortant H1N2 influenza virus by suppressing..., Xu [/bib_ref]. In some IAV strains, the immune-evasion activity of PA-X reduces inflammationinduced pathology, thereby protecting the host and reducing mortality [bib_ref] The contribution of PA-X to the virulence of pandemic 2009 H1N1 and..., Gao [/bib_ref] [bib_ref] PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured..., Gong [/bib_ref] [bib_ref] PA-X decreases the pathogenicity of highly pathogenic H5N1 influenza A virus in..., Hu [/bib_ref] [bib_ref] PA-X-associated early alleviation of the acute lung injury contributes to the attenuation..., Hu [/bib_ref] [bib_ref] An overlapping protein-coding region in influenza A virus segment 3 modulates the..., Jagger [/bib_ref]. While the role of PA-X in immune evasion is well established, its molecular mechanism of action remains poorly understood.
PA-X is produced by ribosomal frameshifting during the translation of the polymerase acidic protein (PA) mRNA [bib_ref] An overlapping protein-coding region in influenza A virus segment 3 modulates the..., Jagger [/bib_ref]. The frameshift generates a protein with the PA amino-terminal ribonuclease (RNase) domain fused to a unique carboxy-terminal domain known as the X-open reading frame (X-ORF). The X-ORF is required for PA-X function [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref]. Despite this non-canonical production mechanism, PA-X is encoded by all IAV strains [bib_ref] Evolutionary conservation of the PA-X open reading frame in segment 3 of..., Shi [/bib_ref]. We previously reported that PA-X selectively degrades RNAs transcribed by host RNA polymerase II (Pol II), but not other polymerases . This characteristic leads to the protection of viral RNAs created by the viral RNA-dependent RNA polymerase (RdRp) . However, the mechanism for PA-X targeting of Pol II transcripts is not known.
Other viruses encode host shutoff RNases that selectively target Pol II transcripts, including alphaherpesviral vhs proteins, gammaherpesviral SOX/BGLF5 proteins, and the severe acute respiratory syndrome-related coronavirus (SARS-CoV) nonstructural protein 1 (nsp1) [bib_ref] The herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target RNAs..., Elgadi [/bib_ref] [bib_ref] Lytic KSHV infection inhibits host gene expression by accelerating global mRNA turnover, Glaunsinger [/bib_ref] [bib_ref] Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by..., Kamitani [/bib_ref] [bib_ref] Host shutoff during productive Epstein-Barr virus infection is mediated by BGLF5 and..., Rowe [/bib_ref]. However, SARS nsp1 and the herpesviral host shutoff proteins operate in the cytoplasm and only degrade transcripts that are bound by components of the protein synthesis machinery [bib_ref] Herpes simplex virus virion host shutoff protein is stimulated by translation initiation..., Doepker [/bib_ref] [bib_ref] mRNA decay during herpesvirus infections: interaction between a putative viral nuclease and..., Feng [/bib_ref] [bib_ref] mRNA decay during herpes simplex virus (HSV) infections: protein-protein interactions involving the..., Feng [/bib_ref] [bib_ref] A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1..., Kamitani [/bib_ref]. By contrast, PA-X accumulates in the nucleus, and the protein synthesis machinery has no role in RNA targeting and degradation [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref]. Our previous analysis of select transcripts suggests that not all Pol II transcripts are equally susceptible to PA-X degradation , similar to reports for other viral host shutoff RNases [bib_ref] The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is..., Esclatine [/bib_ref]. In agreement with this, a recent study of the host transcriptome in IAV-infected cells showed that certain functional classes of RNAs were spared from shutoff, although no specific link to PA-X activity was established. By contrast, in the context of studying the relative contribution of IAV PA-X and NS1 proteins to host shutoff, Toru Takimoto's group recently reported that host mRNAs targeted by PA-X do not clearly belong to specific functional classes, whereas there is functional specificity among NS1 targets. These findings suggest that PA-X may have a unique mechanism to selectively target host RNAs in the nucleus, perhaps in conjunction with RNA processing and the assembly of functional messenger ribonucleoprotein (mRNP) complexes.
Here, we report transcriptome-wide analysis of PA-X targets in human lung A549 cells, in both de novo infection and ectopic expression models. This analysis revealed that PA-X susceptibility is tightly linked to Pol II transcript splicing. Moreover, we identified host proteins involved in mRNA processing that associate with the C-terminal X-ORF, suggesting that PA-X target selection may involve physical interactions with components of the host mRNA processing machinery.
# Results
## Pa-x causes global changes in rna levels during infection
To determine the scope of PA-X specificity for host Pol II transcripts, we profiled RNA levels in cells infected with wild-type (WT) and PA-X-deficient IAVs. To generate PA-X-deficient mutants in the well-characterized strain A/PuertoRico/8/1934 H1N1 (PR8), we introduced 2 mutations in the frameshifting site and a nonsense mutation in PA-X, L201Stop, that truncated the X-ORF after 9 amino acids (aa); we called this virus PA(DX) [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref]. These mutations were designed to be silent in the PA ORF. We previously used a strain with only the frameshifting mutations, IAV PA(fs) [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] , but we created IAV PA(DX) to ensure that any residual frameshifting would produce a non-functional PA-X. We confirmed that the 9-aa truncated PR8 PA-X was largely inactive, as it lost the ability to degrade a b-globin reporter, whereas an X-ORF truncation to 15 aa retained activity [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref]. The b-globin reporter includes the two introns of the native b-globin gene, and expresses an mRNA that is spliced. The results in [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] recapitulate previous findings using truncations in PA-X variants from other strains [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref]. We also generated a virus with only the L201Stop mutation and called it ''X9'' [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref]. We chose to use the PR8 strain because it lacks two other known IAV host shutoff mechanisms; its NS1 protein does not block host mRNA processing [bib_ref] Structural basis for suppression of a host antiviral response by influenza A..., Das [/bib_ref] [bib_ref] Effects of influenza A virus NS1 protein on protein expression: the NS1..., Salvatore [/bib_ref] , and its RdRp does not trigger Pol II degradation [bib_ref] Attenuated strains of influenza A viruses do not induce degradation of RNA..., Rodriguez [/bib_ref].
Using high-throughput RNA sequencing (RNA-seq), we found that the infection of cells with WT IAV caused a dramatic global decrease in transcript levels compared to mock-infected cells [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] ; Data S1). However, a small fraction of transcripts escaped shutoff (right tail end of distribution; [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref]. By contrast, shutoff was substantially attenuated in IAV PA(DX)infected cells. Cells infected with strains carrying either the PA(X9) or PA(fs) mutations also displayed attenuated host shutoff, and the defect was similar in all three mutants . This demonstrates that X-ORF truncation disrupts PA-X function during infection, as predicted from ectopic PA-X expression studies [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref]. Infection rates by WT and mutant viruses were comparable, based on immunofluorescence staining and viral protein levels [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. We also measured the nuclear accumulation of cytoplasmic poly(A) binding protein (PABP), a well-described consequence of host shutoff [bib_ref] Influenza a virus host shutoff disables antiviral stress-induced translation arrest, Khaperskyy [/bib_ref] [bib_ref] Nuclear import of cytoplasmic poly(A) binding protein restricts gene expression via hyperadenylation..., Kumar [/bib_ref] [bib_ref] Importin a-mediated nuclear import of cytoplasmic poly(A) binding protein occurs as a..., Kumar [/bib_ref] [bib_ref] Aberrant herpesvirus-induced polyadenylation correlates with cellular messenger RNA destruction, Lee [/bib_ref]. Infection with PA(X9) and PA(DX) viruses resulted in significantly lower rates of PABP nuclear accumulation compared to WT, confirming the impairment of host shutoff [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. Lastly, our RNA-seq results strongly correlated with those from a previous transcriptome profile of IAV PR8-infected cells, despite the differences in multiplicity of infection (MOI) and time course of analysis [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref]. These data demonstrate that PA-X controls the levels of the majority of host RNAs during infection.
## Pa-x causes global downregulation of host rnas in an ectopic expression model
To simplify our system, we also examined changes in RNA levels after ectopic PA-X expression. We used a doxycycline-inducible PA-X expression system, ''iPA-X'' cells , to induce the expression of WT PA-X or the catalytically inactive D108A mutant in A549 cells. Because iPA-X cells were clonally selected, we analyzed 2 independently generated cell lines for each variant. As expected from previous results with targeted RT-qPCR and metabolic labeling [bib_ref] Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of..., Hayashi [/bib_ref] [bib_ref] An overlapping protein-coding region in influenza A virus segment 3 modulates the..., Jagger [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref] , WT PA-X robustly downregulated steady-state transcript levels [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref] ; Data S2). The degree of host shutoff correlated with the levels of PA-X (percentage of total reads mapping to PA-X: WT #1 = 0.005%-0.006%, WT #10 = 0.023%-0.026%) and was dependent on RNase activity, because expression of the PA-X catalytic mutant had no effect [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. A substantial minority of transcripts was unaffected by PA-X expression (right tail end of distributions; [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. Furthermore, we observed a highly significant correlation between the PA-X-dependent downregulation of RNAs in the ectopic PA-X expression system and in virusinfected cells [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. This indicates that PA-X largely targets the same RNAs in the absence of other viral proteins and that the ectopic expression model accurately reflects the contribution of PA-X to host shutoff during infection. To further validate these findings, we selected representative RNAs, choosing RNAs that were strongly downregulated (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], glucose-6-phosphate dehydrogenase [G6PD]) or largely unaffected (heterogeneous nuclear ribonucleoprotein A0 [HNRNPA0], TATA-box binding protein associated factor 7 [TAF7], enhancer of polycomb homolog 1 [EPC1]) in both de novo infection and ectopic expression models. We then validated the change in RNA levels in iPA-X cells by RT-qPCR. The RT-qPCR results agreed with the RNA-seq data in terms of the selective effects on the tested transcripts [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. By contrast, doxycyline-inducible expression of the catalytically inactive PR8 PA-X D108A mutant or the PA-X RNase domain (aa 'N term'') did not affect the level of any of the tested transcripts [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. Expression of PA-X or the RNase domain was confirmed by western blotting [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. Because the RNase domain alone is active in vitro [bib_ref] The novel influenza A virus protein PA-X and its naturally deleted variant..., Bavagnoli [/bib_ref] [bib_ref] The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit, Dias [/bib_ref] [bib_ref] Crystal structure of an avian influenza polymerase PA( N ) reveals an..., Yuan [/bib_ref] , this result confirms that it is specifically the activity of PA-X and not the overexpression of any active RNase that controls RNA levels in the iPA-X cells. Moreover, the mRNA levels were similarly affected by the expression of PA-X from the A/Udorn/72 H3N2 (Udorn) strain, suggesting that target selection by PA-X is conserved among virus strains [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref]. These data demonstrate that PA-X broadly targets RNA for degradation, while a subset of RNAs remains unaffected.
Specific Functional Classes of Host RNAs Are Differentially Sensitive to PA-X Although most RNAs were downregulated by PA-X, the levels of $25% of RNAs remained largely unchanged . To identify resistant RNAs, we used k-means clustering to group RNAs with similar patterns of regulation (Gasch and Eisen, [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref] , and S1B). Two sets of RNAs making up 55% of the RNAs that were detected in all conditions were identified as true PA-X targets [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] ; Data S1a and S2a). These RNAs were downregulated in a PA-X-dependent manner both during infection and by PA-X ectopic expression. The RNAs in the first set were completely PA-X-specific, as their levels were largely unchanged in IAV PA(DX)-infected cells [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] , left). The (C) Levels of several endogenous mRNAs were measured by RT-qPCR in cells expressing the indicated PA-X variants. After normalization to 18S, mRNA levels are plotted relative to uninduced cells. Values represent means ± SDs. n R 3. **p < 0.01 and ***p < 0.001. ANOVA followed by Dunnett's multiple comparison test versus PR8 PA-X D108A.
(D) A representative western blot using anti-myc antibodies to detect myc-tagged PA-X and a total protein stain as loading control (blot section from 25 to 35 kDa) shows successful induction of PA-X in each cell line (corresponds to one of the experiments shown in C). (legend continued on next page) RNAs in the second set were PA-X sensitive, but were partially downregulated by other mechanisms during IAV PA(DX) infection [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] , right). By contrast, 28% of the RNAs were PA-X resistant and were not downregulated by infection or PA-X expression [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] ; Data S1b and S2b). In addition, the k-means algorithm identified a group of RNAs that were downregulated during infection by a PA-X-independent mechanism and yet were PA-X sensitive in PA-X-expressing cells ; Data S1c and S2c). The levels of these transcripts may be substantially decreased by other regulatory mechanisms during infection, such that their targeting by PA-X is masked. Based on Gene Ontology (GO) term analysis, the host shutoff-resistant RNAs were significantly enriched for genes involved in transcription and translation, including ribosomal RNA processing, ribosomal proteins, and membrane protein synthesis [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref]. This result is consistent with the IAV requirement for host biosynthetic machinery and observations by. These results suggest that while PA-X can target many RNAs, it retains some specificity for functional classes of RNAs.
In addition to this unbiased analysis, we examined how PA-X expression affected the levels of interferon-stimulated genes (ISGs), which are induced in infected cells and function in antiviral defense. We observed that although ISGs were induced during IAV infection, as shown by their higher expression compared to all of the detected RNAs, their levels were even higher in the absence of PA-X [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref]. While the activity of PA-X is clearly not limited to ISGs, these data indicate that PA-X can contribute to dampening the cell-intrinsic response to infection.
## Pa-x strongly and preferentially downregulates spliced pol ii transcripts
We previously showed that PA-X selectively degrades RNA transcribed by Pol II and spares Pol I and Pol III transcripts . Although all cellular transcripts are modified post-synthesis, only Pol II transcripts can be spliced. The process of RNA splicing is mechanistically linked to transcription, as recruitment of the spliceosome is mediated by the C-terminal domain of the large subunit of Pol II [bib_ref] CTD serine-2 plays a critical role in splicing and termination factor recruitment..., Gu [/bib_ref]. However, a subset of Pol II transcripts naturally lacks introns. When we analyzed spliced versus intronless RNAs separately, we found that PA-X downregulated spliced RNAs more than intronless RNAs [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. Our targeted validation also showed that two intronless mRNAs, TAF7 and HNRNPA0, were not downregulated by PA-X [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref] , and, as mentioned above, the b-globin reporter used in [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] encodes a spliced mRNA. Moreover, during infection, the downregulation of spliced RNAs was clearly dependent on PA-X, whereas most of the downregulation of intronless RNAs was PA-X independent . For these analyses, we only included intronless RNAs longer than 300 nt, excluding small non-coding RNAs and ensuring that the length distribution was similar between spliced and intronless RNAs. We also analyzed how the number of exons affected RNA downregulation, as the number of splice sites varies dramatically among spliced RNAs. There was a significant negative correlation between the number of exons in a transcript and its steady-state levels in PA-X-expressing and infected cells (PA-X-expressing cells: Spearman's r = À0.52, [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref] ; IAVinfected cells: Spearman's r = À0.47, [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref]. This result suggests that RNAs with more exons are more susceptible to PA-X degradation. However, the number of exons in an RNA is often proportional to RNA length. A prior study reported a relation between IAV host shutoff and transcript length. Likewise, there was a correlation between degradation and RNA length in our data (Spearman's r = À0.38 for both PA-X-expressing [ [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref] ] and IAV-infected cells [ [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref]. To determine whether exon number or transcript length was important, we examined RNAs of similar length or with a specific number of exons. We still found a robust negative correlation between relative RNA levels in the presence of PA-X and exon number among RNAs of similar length (length = 3.5-4.0 kb, Spearman's r = À0.42, N = 674; [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. Similar correlations were also seen for RNAs of other lengths . By contrast, there was only a small correlation between degradation and RNA length among RNAs with the same number of exons (number of exons = 6, Spearman's r = À0.16, N = 642; [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. Again, similar correlations were seen for other exon numbers [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref]. We also tested another key characteristic of RNAs, guanine-cytosine (GC) content, and found no correlation with PA-X activity in our dataset .
The results from the clustering and GO analyses (Figures 3A-3D) suggest that PA-X differentially regulates RNAs from specific functional groups, whereas Figures 4A-4F suggest a difference based on the structure of the nascent transcript. Interestingly, we found a connection between the structural and functional specificity. RNAs classified as resistant by k-means clustering (shown in [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] had fewer exons than those classified as PA-X targets (shown in [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. These results suggest that targeting by PA-X is connected to RNA splicing and that this preference has consequences for the selection of functionally relevant targets.
Splice Sites Confer Susceptibility to PA-X Endogenous RNAs with different numbers of exons also have different sequences, lengths, and post-transcriptional modifications. To investigate the effect of splicing in a more controlled system, we examined the same RNA in both spliced and intronless forms. We used interferon l2 (IFN-l2) mRNA as a model transcript. IFN-l2 is a type III IFN that contributes to IAV immune responses [bib_ref] Lambda interferon is the predominant interferon induced by influenza A virus infection..., Jewell [/bib_ref]. In the RNA-seq, IFN-l2 transcripts were detectable only in IAV-infected cells and were downregulated by PA-X [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. We cloned the IFN-l2 cDNA and the full IFN-l2 genomic sequence containing introns (D) DAVID was used to identify overrepresented Gene Ontology (GO) terms for biological processes and molecular functions among PA-X-resistant RNAs. Fold enrichment is plotted for GO terms that had corrected p < 0.01. into plasmid expression vectors [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] and co-transfected them into HEK293T cells with PR8 PA-X. In these transfection experiments, the detection of PA-X protein is hindered by auto-cleavage of the PA-X Pol II transcript. As an alternative control to ensure comparable PA-X activity between transfections, we also measured the RNA levels of a co-transfected introncontaining luciferase reporter ; luciferase downregulation thus serves as a positive control for PA-X activity [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] , and 5G). We confirmed that IFN-l2 mRNA was expressed and exported to the cytoplasm at similar levels, irrespective of the construct used [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. We also checked that the 5 introns were properly spliced by PCR analysis [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] , and S4D). As expected, we found that PA-X downregulated the IFN-l2 mRNA expressed from the full genomic region [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. However, the levels of the same IFN-l2 mRNA expressed from an intronless cDNA construct were only minimally reduced [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. In both conditions, the control luciferase reporter was downregulated by PA-X, confirming that the activity of PA-X was similar in all of the samples. PA-X proteins from the 2009 pandemic H1N1 strains (e.g., A/California/7/09H1N1 [CA/7] and A/Tennessee/1-560/2009) and the Udorn H3N2 strain also preferentially degraded the spliced mRNAs [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. These results confirm the prediction that splicing is important for PA-X targeting. In addition, we tested the downregulation of IFN-l2 mRNAs expressed from chimeric constructs that contained only 1 of the 5 introns from the original genomic sequence to determine whether a single splicing event was sufficient to restore PA-X targeting. Addition of a single intron increased susceptibility to PA-X [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. Downregulation of the luciferase reporter indicated that these changes were not due to varying PA-X activity in the samples. We found that while the addition of introns 1, 3, or 5 alone restored PA-X susceptibility, introns 2 or 4 had little effect. The difference between the introns was also apparent when we normalized IFN-l2 to luciferase mRNA levels [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. Intron 4 was not spliced efficiently in the absence of other introns [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] , but intron 2 was still efficiently spliced [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. Therefore, splicing efficiency alone does not explain the differential effects of the introns. More in-depth examination of the IFN-l2 sequence revealed that the 5 0 splice sites for introns 1, 3, and 5 match the consensus 5 0 splice site sequence, AGjGT (where j marks the splice site). By contrast, the 5 0 splice sites for introns 2 and 4 are an imperfect match (TAjGT and GTjGT, respectively). 5 0 splice-site quality scores calculated using the MaxEntScan::score5ss program were also higher for introns 1, 3, and 5 (8.8-10.5) than introns 2 and 4 (5.8) [bib_ref] Maximum entropy modeling of short sequence motifs with applications to RNA splicing..., Yeo [/bib_ref]. When we mutated these two 5 0 splice sites to match the consensus sequence AGjGT and increase the splice site quality score (mutated introns 2 and 4 = 10.7), we found that the mutated introns 2 and 4 could restore PA-X susceptibility (Figures 5G and S4F normalized to luciferase mRNA levels) and intron 4 splicing efficiency [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. This result further strengthens the link between splicing and PA-X susceptibility. Despite these results linking mRNA splicing and PA-X degradation, one unresolved issue is that we and others have previously used intronless reporters to study PA-X, and they appeared to be efficiently degraded. To investigate this issue, we compared intronless and spliced luciferase reporters . The spliced reporter, which we used as a control in [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] , contains a portion of the b-globin intron . PA-X had a more robust effect on the spliced mRNA, although it could also downregulate intronless luciferase mRNA [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref]. These results suggest that the addition of a single splicing event further promotes degradation by PA-X. Reporter constructs are selected for their robust expression; it is possible that certain intronless reporters also associate with cellular factors involved in PA-X targeting. Nevertheless, our findings lead us to recommend the use of introncontaining reporters for future cell-based studies of PA-X.
## The x-orf mediates interaction with proteins involved in rna metabolism
As shown in previous studies [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref] and the PR8 PA(X9) RNA-seq results [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] , the C-terminal X-ORF is required for PA-X activity. We hypothesized that the X-ORF interacts with cellular proteins that mediate the association of PA-X with target mRNAs, especially in light of our results connecting PA-X targeting with splicing [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref] and mRNA 3 0 end processing . To identify cellular X-ORF-interacting proteins, we used BioID, a proteomic technique that relies on non-specific proximity biotinylation of lysine residues by a modified Escherichia coli biotin ligase, BirA* [bib_ref] A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in..., Roux [/bib_ref]. Since there are two major classes of PA-X isoforms that differ in X-ORF length [bib_ref] Evolutionary conservation of the PA-X open reading frame in segment 3 of..., Shi [/bib_ref] , we fused BirA* to X-ORFs representative of each class: the 61-aa PR8 X-ORF (X61) and the 41-aa CA/7 X-ORF (X41) . We used BirA* alone and BirA* fused to a mutated PR8 X-ORF in which 4 positively charged residues were replaced by alanine (X61(4A)) as negative controls. These mutations prevent the nuclear localization of a GFP-X-ORF fusion and disrupt mRNA degradation by PA-X . As expected from our previous studies , fusion to the WT X-ORFs, but not X61(4A), led to the accumulation of BirA* in the nucleus [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. Moreover, all BirA* fusions efficiently biotinylated many cellular proteins [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] is plotted. The two groups of PA-X targets [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] are plotted together. p < 0.01, Kolmogorov-Smirnoff test. See also [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref]. To identify cellular proteins that bind both the X61 and X41 X-ORFs, we affinity-purified biotinylated proteins from HEK293T cells expressing BirA*-X-ORF fusion proteins and prepared them for quantitative mass spectrometry using reductive dimethylation. Reductive dimethylation exploits formaldehyde variants with different molecular weights due to substituted carbon-13 or deuterium atoms to label captured proteins with stable isotope tags and allow quantitative comparisons among samples . We performed the experiment 3 times-twice with BirA*-X61 and once with BirA*-X41-comparing them in each case to the BirA* alone and BirA*-X61(4A) controls . A total of 156 candidate X-ORF-interacting proteins were represented by at least 2 unique peptides and were present in all of the runs . Among these, we selected 29 highconfidence interacting proteins with higher relative peptide abundance in the test (BirA*-X61 or -X41) versus control (BirA*-X61(4A) or BirA* alone) conditions (>2-fold higher than controls in at least 2 experiments or >1.5-fold in all 3 experiments; ; . depicts the relative peptide abundance of proteins in X61 samples compared to the X61(4A) control or the BirA* alone control, with black and red open circles identifying the high-confidence hits. Red circles indicate 2 proteins (nucleolin [NCL], nucleophosmin [NPM1]) that were enriched >2-fold compared to controls across all 3 runs. Because NCL and NPM1 are abundant proteins that traffic in and out of nucleoli, these interactions may explain the apparent nucleolar accumulation of biotinylated proteins [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] , even though neither the BirA-X-ORF fusion [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] nor the full-length PA-X [bib_ref] Influenza a virus host shutoff disables antiviral stress-induced translation arrest, Khaperskyy [/bib_ref] accumulate in nucleoli. A STRING protein-protein interaction network analysis of the hits revealed several physical and functional interaction nodes, including protein trafficking, transcription, translation, and mRNA processing . Similarly, GO term analysis revealed a strong association with mRNA processing, RNA splicing, and mRNA metabolic process functions among the high-confidence hits . These data show that the PA-X C-terminal X-ORF is physically recruited to protein complexes involved in nuclear Pol II RNA processing (commonly referred to as mRNA processing), which likely explains the preferential degradation of RNAs that have undergone co-or post-transcriptional processing.
## The cfim complex may regulate pa-x activity
The BioID screen identified several proteins involved in RNA splicing (RNA binding motif protein 39 [RBM39], poly(U) binding splicing factor 60 [PUF60], and pre-mRNA processing factor 4 [PRPF4]) and/or polyadenylation (nudix hydrolase 21 [NUDT21]/ cleavage and polyadenylation specificity factor 5 [CPSF5]/ cleavage factor Im 25 [CFIm25] and CPSF6/CFIm68) as X-ORF-interacting proteins. We conducted co-immunoprecipitation experiments to validate these interactions using nuclear extracts derived from an HEK293T iPA-X cell line that produces high levels of a myc-tagged PA-X . We recapitulated the interaction between full-length PA-X and endogenous NUDT21, suggesting that this is a stable interaction that can survive affinity isolation procedures . NUDT21 and CPSF6 assemble into a functional heterotetrameric CFIm complex [bib_ref] Evidence that cleavage factor Im is a heterotetrameric protein complex controlling alternative..., Kim [/bib_ref] that enhances polyadenylation and guides polyadenylation site choice [bib_ref] Molecular Mechanisms for CFIm-Mediated Regulation of mRNA Alternative Polyadenylation, Zhu [/bib_ref]. In addition, the CFIm complex is present in the spliceosome and has been proposed to link splicing to polyadenylation during RNA processing [bib_ref] Large-scale proteomic analysis of the human spliceosome, Rappsilber [/bib_ref] [bib_ref] Comprehensive proteomic analysis of the human spliceosome, Zhou [/bib_ref]. To test whether the interaction with the CFIm complex was required for PA-X activity, we used small interfering RNAs (siRNAs) to deplete NUDT21 and CPSF6 alone or in combination . Partial silencing of CFIm proteins reduced PA-X downregulation of IFN-l2 upon co-transfection in HEK293T cells . Moreover, in A549 cells, silencing of the CFIm complex reduced PABP nuclear localization during IAV PR8 infection , a hallmark of PA-X dependent host shutoff [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref] ; [bib_ref] Influenza a virus host shutoff disables antiviral stress-induced translation arrest, Khaperskyy [/bib_ref]. PABP relocalization was quantified on a per-cell basis to control for unrelated effects of the knockdowns on cell viability and infection rates. In fact, we found that NUDT21 silencing reduced cell viability, and CPSF6 silencing dramatically reduced infection rates . We also tested two additional potential interaction partners, NCL and RBM39, in the PABP relocalization assay . While NCL silencing had little effect on cell viability, infection rates, and host shutoff, RBM39 silencing reduced cell viability and infection rates . We conclude that NCL is unlikely to have a role in PA-X-mediated host shutoff, whereas the effects of RBM39 silencing on cell physiology are too severe to assess its role in PA-X host shutoff. Nonetheless, these data suggest that the RNA processing and spliceosome-associated CFIm complex is required for at least some of the activity of PA-X in cells.
# Discussion
A thorough understanding of the molecular mechanism of action of PA-X is required to determine how it selectively degrades host RNAs and limits innate immune response. In this study, we discovered a key aspect of the PA-X mechanism of action: its selectivity for transcripts that are spliced. This coupling to RNA processing sets PA-X apart from other viral host shutoff RNases that target mRNAs in the cytoplasm in association with translation [bib_ref] Herpes simplex virus virion host shutoff protein is stimulated by translation initiation..., Doepker [/bib_ref] [bib_ref] mRNA decay during herpesvirus infections: interaction between a putative viral nuclease and..., Feng [/bib_ref] [bib_ref] mRNA decay during herpes simplex virus (HSV) infections: protein-protein interactions involving the..., Feng [/bib_ref] [bib_ref] A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1..., Kamitani [/bib_ref]. Our transcriptomic results show that, as expected, PA-X downregulates many host RNAs, both on its own and in the context of infection. However, some RNAs are less susceptible to PA-X activity, and a key characteristic of these resistant RNAs is that they are the indicated introns to test splicing. Amplified PCR products are shown (image is representative of 4 experiments). A 1:1 mix of the IFN-l2 cDNA and genomic constructs was included to check that unspliced and spliced products could be simultaneously amplified. (H) Cells were transfected with an intronless (À) or an intron-containing (+ intron) luciferase reporter and PR8 PA-X. Luciferase RNA levels were measured by RT-qPCR, normalized by 18S rRNA, and were plotted relative to vector-transfected cells.
Values represent means ± SDs; n R 4. *p < 0.05, **p < 0.01; For (B) and (C), ANOVA followed by Tukey's pairwise test; (D) ANOVA followed by Dunnett's test, p values relative to cDNA construct; (H) Student's t test. See also [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref].
intronless or have fewer introns. Moreover, the C-terminal X-ORF of PA-X interacts with many proteins involved in cellular RNA metabolism. We propose a model whereby PA-X associates with a discrete set of RNA metabolism proteins that allows selective targeting of RNAs during transcription or early processing. In this model, RNAs that are not canonically processed, including viral RNAs, are spared.
Our transcriptomic study confirms that the PA-X-dependent downregulation of host protein production [bib_ref] Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of..., Hayashi [/bib_ref] [bib_ref] An overlapping protein-coding region in influenza A virus segment 3 modulates the..., Jagger [/bib_ref] is due to a reduction in RNA levels, and defines PA-X-dependent and PA-X-independent components of RNA downregulation during infection. The PA-Xindependent component is likely due to a recently described generalized reduction in cellular transcription (Bauer et al., . The X-ORF Interactome Is Enriched for Proteins Involved in mRNA Processing (A) Schematic diagram of X-ORF-BirA* fusion baits used in the BioID mass spectrometry experiment. The numbers indicate independent runs using each construct set. Light, medium, and heavy = light, medium, or heavy isotope tags. (B) Overlap between proteins identified by mass spectrometry by R2 unique peptides in 3 BioID runs. (C) Average relative abundance of 286 proteins identified in at least 2 BioID experiments, plotted as log2 ratio of medium versus light (x axis, X-ORF/À) and medium versus heavy (y axis, X-ORF/X61(4A)). Green dots represent proteins with >1.5-fold enrichment over both negative controls; blue dots represent proteins with >1.5-fold enrichment over BirA*-myc alone; black and red open circles represent high-confidence hits (>2.0-fold over BirA*-myc in R2 experiments or >1.5-fold over BirA*-myc and BirA*-X61(4A)-myc in 3 experiments); red open circles represent nucleolin (NCL) and nucleophosmin (NPM1), which were enriched >2.0-fold over both negative controls in all 3 experiments. (D) STRING protein-protein interaction network of high-confidence hits. Apparent nodes were differentially colored (only 1 annotation per protein is shown for simplicity). (E) Gene Ontology (GO) enrichment analysis of X-ORF BioID hits (black and red circles in C). All enriched functional classes are presented (excluding parental subclasses for each term). Note that the >100-fold enriched functional classes contain only 2 proteins each. See also [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. . The CFIm Complex Is Involved in PA-X Activity (A) Proteins were extracted from the nuclei of uninduced or doxycycline-treated HEK293T cells expressing inducible WT PR8 PA-X, and incubated with myc-trap beads to immunoprecipitate PA-X-myc (myc) or control beads (ctrl). Input and immunoprecipitation (IP) samples were resolved by SDS-PAGE and analyzed by western blotting for PA-X-myc, NUDT21, and CPSF6. The image is representative of 3 independent experiments. (B) NUDT21 and CPSF6 were knocked down by siRNA, separately or in combination, in HEK293T cells. For NUDT21, siRNA #2 was used (see STAR Methods). For CPSF6, siRNA #1 was used for knockdown in combination with NUDT21. Cells were then transfected with a reporter expressing IFN-l2 mRNA from the genomic locus, with and without WT PR8 PA-X. The levels of IFN-l2 mRNA and 18S rRNA were measured by RT-qPCR. The expression of IFN-l2 mRNA is plotted relative to vector-transfected cells, after normalization to 18S rRNA.
(legend continued on next page) 2018; [bib_ref] Transcription Elongation Can Affect Genome 3D Structure, Heinz [/bib_ref] [bib_ref] Influenza virus infection causes global RNAPII termination defects, Zhao [/bib_ref] , because other known modalities of IAV host shutoff are not active in the PR8 strain [bib_ref] Structural basis for suppression of a host antiviral response by influenza A..., Das [/bib_ref] [bib_ref] Attenuated strains of influenza A viruses do not induce degradation of RNA..., Rodriguez [/bib_ref] [bib_ref] Effects of influenza A virus NS1 protein on protein expression: the NS1..., Salvatore [/bib_ref]. The mechanism of reduced host transcription in IAV-infected cells remains a matter of debate [bib_ref] Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription, Bauer [/bib_ref] [bib_ref] Transcription Elongation Can Affect Genome 3D Structure, Heinz [/bib_ref] [bib_ref] Influenza virus infection causes global RNAPII termination defects, Zhao [/bib_ref]. However, it is most likely PA-X independent, because transcription is also reduced during infection with influenza B viruses [bib_ref] Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription, Bauer [/bib_ref] , which do not encode PA-X [bib_ref] Evolutionary conservation of the PA-X open reading frame in segment 3 of..., Shi [/bib_ref]. Our clustering analysis also revealed that some functional classes of RNAs are spared from PA-X degradation, including mRNAs for proteins involved in translation, which agrees with previous results from [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref]. Our new results suggest that the small number of exons of these mRNAs, particularly RNAs for ribosomal proteins, may explain this phenomenon.
Another general conclusion of our RNA-seq analysis is that PA-X with a 9-aa truncated C-terminal X-ORF is essentially non-functional in the context of infection. The shutoff impairment of IAV PA(X9) is very similar to that of the PA(fs) and PA(DX) viruses [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] , which presumably have reduced PA-X production. This finding validates the results of multiple studies using ectopic PA-X expression models that concluded that at least 15 aa of the X-ORF is required for full RNA degrading activity in cells [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref] , despite in vitro activity of the RNase domain in isolation [bib_ref] The novel influenza A virus protein PA-X and its naturally deleted variant..., Bavagnoli [/bib_ref] [bib_ref] The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit, Dias [/bib_ref] [bib_ref] Crystal structure of an avian influenza polymerase PA( N ) reveals an..., Yuan [/bib_ref]. Similarly, a 1918 H1N1 chimeric virus with a stop codon after 15 aa had an intermediate host shutoff phenotype between IAV WT and PA(fs) . The finding that truncating the X-ORF is sufficient to block PA-X activity in the virus is important because single-point mutations in the X-ORF sequence are less disruptive than frameshifting mutations. Thus, viruses carrying X-ORF mutations could be better tools for in vivo studies of PA-X function and IAV pathogenesis.
The key unexpected finding from our study is the link between PA-X and splicing. All other viral host shutoff RNases appear to act at some stage of mRNP loading into the translation apparatus. For example, RNA targeting by the alphaherpesvirus protein vhs is linked to physical interactions with translation initiation factors [bib_ref] Herpes simplex virus virion host shutoff protein is stimulated by translation initiation..., Doepker [/bib_ref] [bib_ref] mRNA decay during herpesvirus infections: interaction between a putative viral nuclease and..., Feng [/bib_ref] [bib_ref] mRNA decay during herpes simplex virus (HSV) infections: protein-protein interactions involving the..., Feng [/bib_ref] and SARS CoV nsp1 only degrades RNAs that are actively translated [bib_ref] A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1..., Kamitani [/bib_ref]. Thus, to our knowledge, there is no other described instance of a host shutoff RNase using splicing as a targeting mechanism. In fact, splicing was reported to protect mRNAs from cleavage by vhs [bib_ref] The Splicing History of an mRNA Affects Its Level of Translation and..., Sadek [/bib_ref]. The connection between splicing and PA-X degradation is evident from the reduced effect of PA-X on intronless mRNAs [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref] , the negative correlation between exon number and degree of degradation by PA-X [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] , and the fact that small changes in the 5 0 splice site can affect the susceptibility to degradation by PA-X [fig_ref] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X [/fig_ref].
These findings begin to shed light on the specificity of PA-X for Pol II transcripts . In cellular transcription, the splicing machinery associates with RNAs through interactions with Pol II, and thus only Pol II transcripts are normally spliced [bib_ref] CTD serine-2 plays a critical role in splicing and termination factor recruitment..., Gu [/bib_ref]. Protein-protein interactions with splicing factors may thus bring PA-X to its Pol II targets. This idea is corroborated by our proteomic analysis, which shows that the PA-X X-ORF interacts with several splicing regulators (PUF60, RBM39, PRPF4) and spliceosome-associated polyadenylation proteins (the CFIm complex proteins NUDT21 and CPSF6) . Furthermore, PA-X activity is in part dependent on the CFIm complex . We speculate that more exons provide more chances for PA-X to be brought to the RNA by these factors, resulting into more efficient turnover of RNAs with more splice sites. Since these proteins do not regulate the processing of all of the mRNAs in the cell to the same extent, PA-X interactions with these proteins could provide an additional mechanism for target discrimination. Further studies will be needed to determine the exact role of these factors.
A targeting strategy based on splicing offers a major benefit to the virus because it provides the ability to easily discriminate between host and viral mRNAs. Viral mRNAs are synthesized by the RdRp, and most of them are not spliced, which renders them ''invisible'' to PA-X. That said, our published results suggest that even the two viral mRNAs that are spliced (nuclear export protein and matrix protein 2 [M2]) are PA-X resistant . However, splicing of viral mRNAs is a fundamentally different process, since the splicing machinery needs to be recruited to the RNAs separately from Pol II [bib_ref] Influenza viruses and mRNA splicing: doing more with less, Dubois [/bib_ref]. It is possible that viral mRNA splicing does not require the CFIm complex or other PA-X-binding partners, because they are auxiliary components of the host RNA processing machinery. The PA-X splicing-based targeting strategy is more efficient at virus versus host discrimination than the translation-based targeting strategy used by herpesviral host shutoff RNases, which leads to the degradation of viral and host mRNAs alike. This is likely because viral translation relies on the same machinery as host translation. While herpesviruses can compensate for the degradation of their own RNAs, this self-sacrifice may not work for a virus such as IAV, which has a shorter replication cycle and a small genome with a limited gene expression program.
Our BioID results suggest that the preference for spliced RNAs may be linked to protein-protein interactions between the PA-X X-ORF and cellular factors. The X-ORF is required for PA-X nuclear localization [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref]. However, enforced nuclear localization of the PA-X RNase domain alone does not fully rescue activity [bib_ref] Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in..., Hayashi [/bib_ref] , suggesting that the X-ORF has additional functions. We identified many X-ORF-interacting proteins with various roles in RNA metabolism in addition to a nuclear import (C-E) NUDT21 and CPSF6 were knocked down by siRNA in A549 cells, using a mixture of 2 siRNAs. Cells were then infected with WT PR8 IAV for 15 h. Infection rates were assessed by staining for IAV proteins and host shutoff by staining for nuclear PABP. (C) Representative immunofluorescence images. Scale bar, 200 mm, indicated as an arrow in the lower left corner. (D and E) change in the fraction of infected cells with nuclear PABP (D) or total cell counts and infected cells (E), relative to control siRNA. Bars are means ± SDs; n R 3. *p < 0.05 and ***p < 0.001. ANOVA followed by Dunnett's multiple comparison test versus control siRNA. See also .
protein (importin 7 [IPO7]). By examining the X-ORF in isolation, we likely excluded indirect interactions via RNA binding of the RNase domain, as well as interactions that are important for PA rather than PA-X function. Among our hits, two nucleolar proteins, NCL and NPM1, were also reported to interact with H5N1 PA-X [bib_ref] Interactomic landscape of PA-X-chicken protein complexes of H5N1 influenza A virus, Li [/bib_ref]. The fact that biotinylated proteins accumulated in the nucleoli also supports the idea that NCL and NPM1, which traffic to nucleoli, come in contact with the BirA*-fused X-ORF and full-length PA-X [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref]. It is unclear whether PA-X interaction with nucleolar proteins is of functional importance, since silencing NCL had little effect on PA-X-mediated host shutoff in infected cells and PA-X is not localized specifically to this compartment [fig_ref] Figure 5: Addition of Introns and Splicing Events Promotes Degradation by PA-X [/fig_ref] ; [bib_ref] Influenza a virus host shutoff disables antiviral stress-induced translation arrest, Khaperskyy [/bib_ref] [bib_ref] Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus..., Khaperskyy [/bib_ref]. NCL has been reported to protect specific RNAs from degradation by viral host shutoff RNases, including PA-X [bib_ref] Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases, Muller [/bib_ref] [bib_ref] A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral..., Muller [/bib_ref]. Therefore, the PA-X-NCL interaction may reflect a different role for NCL in regulating RNA homeostasis during infection. By contrast, we found evidence for the involvement of the CFIm complex (NUDT21 and CPSF6) in host shutoff during infection and PA-X ectopic expression . The findings in infected cells must be interpreted with caution because CPSF6 silencing markedly inhibited viral infection , and S6F). It is unclear whether the reduction in infection rates is connected to PA-X function. While the CFIm complex is more commonly studied for its roles in alternative polyadenylation and mRNA 3 0 processing [bib_ref] Cleavage factor Im (CFIm) as a regulator of alternative polyadenylation, Hardy [/bib_ref] , multiple studies have shown that NUDT21 and CPSF6 are found in purified spliceosome complexes [bib_ref] Large-scale proteomic analysis of the human spliceosome, Rappsilber [/bib_ref] [bib_ref] Comprehensive proteomic analysis of the human spliceosome, Zhou [/bib_ref]. This finding has led to the idea that they may also play a role in the coordination of splicing and 3 0 processing [bib_ref] An active role for splicing in 3 0 -end formation, Martinson [/bib_ref]. In a previous study, we reported that canonical 3 0 end processing may also be linked to PA-X targeting ; the CFIm complex may also explain this connection. Little is known about the function of the CFIm complex, so studying its contribution to PA-X activity and/or IAV infection may advance the understanding of its normal physiological role. Roles for the other candidate PA-Xinteracting proteins remain to be explored; such studies may be hindered if these proteins play PA-X-independent roles in the viral replication cycle or in maintaining general cell viability during infection. For example, we found that silencing RBM39, an alternative splicing regulator, had profound negative effects on cell survival and infection rates . We also wonder whether the association of PA-X with cellular proteins could compromise their normal function. We did not find dramatic changes in host splicing in our dataset (not shown), but others have reported increased intron retention in cells infected with a PR8 chimeric virus bearing a 1918 NS1 protein [bib_ref] Influenza virus infection causes global RNAPII termination defects, Zhao [/bib_ref]. This discrepancy could be due to the way we set up our sequencing pipeline or to the NS1 variant present in the virus. Because these changes were attributed to NS1 activity, we must be cautious in our assessment of PA-X-dependent and PA-X-independent effects in our system.
It is interesting that both PA-X and the well-known influenza host shutoff factor NS1 interact with nuclear mRNA processing machinery to control gene expression [bib_ref] Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of..., Nemeroff [/bib_ref]. Many NS1 variants (but not PR8 NS1) cause host shutoff by bind-ing and inhibiting a component of the 3 0 end RNA processing machinery, CPSF30 [bib_ref] Structural basis for suppression of a host antiviral response by influenza A..., Das [/bib_ref] [bib_ref] Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of..., Nemeroff [/bib_ref]. While this convergence could allow the two proteins to coordinate an attack on the host, studies of engineered and naturally evolved viruses suggest that NS1 and PA-X activities are anti-correlated to prevent cytotoxicity. For example, the original 2009 pandemic H1N1 NS1 does not bind CPSF30, nor does it reduce host gene expression [bib_ref] Inefficient control of host gene expression by the 2009 pandemic H1N1 influenza..., Hale [/bib_ref] , but the more human-adapted NS1 from currently circulating pandemic H1N1 strains does [bib_ref] Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009..., Clark [/bib_ref] [bib_ref] Functional Evolution of the 2009 Pandemic H1N1 Influenza Virus NS1 and PA..., Nogales [/bib_ref]. These H1N1 strains have also accumulated mutations that reduce PA-X activity, suggesting that having two highly active host shutoff proteins may impair viral fitness [bib_ref] Functional Evolution of the 2009 Pandemic H1N1 Influenza Virus NS1 and PA..., Nogales [/bib_ref]. A recent study by the Takimoto lab comparing NS1 and PA-X targeting in a 2009 pandemic strain suggests that NS1 and PA-X have overlapping but not identical targets. NS1 is more clearly directed at downregulation of the innate immune response, whereas PA-X has a broader targeting range. In general, host mRNA processing may be a hub of regulation for influenza because viral mRNAs are generally not processed by host machinery. Also, interactions with host mRNA processing do not directly compromise Pol II activity, which is required for viral replication [bib_ref] Synthesis of influenza virus polypeptides in cells resistant to alpha-amanitin: evidence for..., Lamb [/bib_ref].
Our results affirm the importance of PA-X for the viral replication cycle, as they show that the ability of the virus to regulate host gene expression is severely reduced in the absence of PA-X. Moreover, we have uncovered a unique mechanism of host RNA targeting that can allow PA-X to distinguish not only between host and viral targets but also among cellular targets. For example, the intronless mRNA TAF7, which we examined in [fig_ref] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins... [/fig_ref] , is a component of Pol II pre-initiation complexes. Therefore, PA-X selectivity could have repercussions for the viral replication cycle. Through further elucidation of the PA-X mechanism of action, we will gain important insights into how host shutoff allows the virus to usurp host biosynthetic machinery and expand our knowledge of the link between PA-X and IAV pathogenesis.
# Star+methods
Detailed methods are provided in the online version of this paper and include the following:
[formula] d KEY RESOURCES [/formula]
# Acknowledgments
We thank Albert Tai and the personnel of the Tufts University Core Facility -Genomics Core for help with the RNA-seq. We thank Alejandro Cohen of the Dalhousie Proteomics and Mass Spectrometry Core Facility for support of the BioID proteomics analysis. We thank Andrew Mehle and Richard Webby for constructs, Claire Moore and Andrew Bohm for suggestions and feedback, and members of the Gaglia and McCormick labs for critical reading of the manuscript. This work was supported by a Natalie V. Zucker award from Tufts University ( Firth, A.E., Jagger, B.W., Wise, H.M., Nelson, C.C., Parsawar, K., Wills, N.M., Napthine, S., Taubenberger, J.K., Digard, P., and Atkins, J.F. (2012). Ribosomal frameshifting used in influenza A virus expression occurs within the sequence UCC_UUU_CGU and is in the +1 direction. Open Biol. 2, 120109.
Gaglia, M.M., . A common strategy for host RNA degradation by divergent viruses. J. Virol. 86, 9527-9530. . Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. Nat. Protoc. 9, 2045-2060. Figures 4, S3. For analysis of intronless RNAs, only RNAs that were longer than 300 nt were used, to enrich for mRNAs and long noncoding RNAs and exclude small non-coding RNAs that are transcribed by Pol III or are produced through processing of longer transcripts, like microRNAs and small nuclear and nucleolar RNAs. The length distribution of the remaining intronless RNAs was similar to that of the spliced RNAs. All RNAs were used for the analysis of PA-X downregulation versus length, exon number, and GC content. k-means clustering analysis was carried out using the Cluster 3.0 program (http://bonsai.hgc.jp/$mdehoon/software/cluster/ software.htm). The clustering was done only on RNAs that were detected in all samples tested (6,391 RNAs), because all eight datasets were used to generate the clusters, even though only the mRNA levels for select samples are plotted in [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] for clarity. The classified RNAs listed in Data S1a, S1b, S1c and S2a, S2b, S2c represent these 6,391 RNAs. Because in k-means clustering the number of clusters is user-defined, clustering was attempted with three, four, and five clusters. Four clusters were chosen, because they provided more granularity. For example, they identified a group of RNAs that were only PA-X-dependent in the ectopic expression system. Initializing the program with more than four clusters led to separation of PA-X targets in multiple groups different only by the extent of downregulation, but did not identify other patterns of gene expression. Gene ontology (GO) term analysis was carried out on the DAVID server [bib_ref] Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene..., Huang [/bib_ref] [bib_ref] Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources, Huang [/bib_ref]. The 5 0 splice site quality score was computed using the MaxEntScan::score5ss program using the maximum entropy model [bib_ref] Maximum entropy modeling of short sequence motifs with applications to RNA splicing..., Yeo [/bib_ref]. Other analyses were done using custom scripts in Python2.7. For the ISG analysis [fig_ref] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs [/fig_ref] the list of ISG tested by .
# Star+methods key resources
## Quantification and statistical analysis
For [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] , 5, 7, S4, S6, statistical analysis and plotting were done in GraphPad Prism v7.0d software using the test recommended by the software and indicated in the figure legends. Generally, ANOVA followed by a corrected pairwise test (Tukey's or Dunnett's) was used when more than two samples were analyzed and Student's t test when two samples were compared. For [fig_ref] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host... [/fig_ref] , 2A and 2B, 4, statistical analysis and plotting were done using Python2.7 and the NumPy, SciPy and matplotlib libraries. The Kolmogorov-Smirnoff test was used to compare populations and Spearman's correlation coefficient to analyze the relationship between certain variables and RNA downregulation. Statistical tests used for each panel are noted in and/or Figures.
## Data and software availability
The accession number for the RNA-seq data reported in this paper is GEO: GSE120183 (raw read data and processed data included here as Data S1 and S2).
[fig] Figure 1: PA-X Downregulates Most Cellular RNAs and Is a Major Contributor to Host Shutoff during Influenza A Virus Infection (A) Diagram of mutations in the PR8 PA(DX) virus. Less intense colors indicate lower levels of PA-X. Blue, position of the frameshift. Red, mutated nucleotides in the frameshifting sequence and at PA-X codon 201. (B) HEK293T cells were transfected for 24 h with a b-globin reporter and WT PR8 PA-X (''61'') or variants with the C-terminal X-ORF truncated after the indicated number of amino acids (aa). Levels of b-globin in PA-X transfected cells were measured by RT-qPCR and are plotted relative to vector transfected cells, after normalization to cellular 18S rRNA. Values represent means ± SDs. n = 3. *p < 0.05, and **p< 0.01, ANOVA followed by Dunnett's multiple comparison test versus WT PA-X (61 aa). (C) RNA-seq was carried out on RNA collected 15 h after infection from A549 cells infected with WT PR8 or PR8 PA(DX). The ratio between levels in IAV-infected versus mock-infected cells was computed for each RNA and the distribution of the ratios (log2) is plotted as a frequency histogram. The populations are significantly different (p < 0.001) based on the Kolmogorov-Smirnoff test. The dashed line indicated a ratio of 1 (no change). n R 2. (D) The ratio in RNA levels in WT IAV-infected cells versus mock-infected cells in our study (15 h post-infection, MOI = 1) is plotted against the results from Bercovich-Kinori et al. (2016) (8 h post-infection, MOI = 5 [/fig]
[fig] Figure 2: PA-X Downregulates Most Cellular RNAs in the Absence of Other Viral Proteins RNA and protein samples were collected from control (untransduced) A549 cells or A549 cells expressing doxycycline-inducible PR8 PA-X (WT), PR8 PA-X catalytic mutant (D108A), PR8 PA-X N-terminal endonuclease domain (aa 1-191, ''N term''), or Udorn PA-X 18 h after the addition of doxycycline. (A) RNA-seq was carried out on cells expressing WT or mutant PR8 PA-X (2 clonal lines for each). The ratio between the levels in PA-X-expressing versus control cells was computed for each RNA, and the distribution of the ratios is plotted as a frequency histogram. The dashed line indicates a ratio of 1 (no change). n R 2. (B) The PA-X-dependent changes in RNA levels in infected cells (ratio in PR8 PA(DX) versus WT PR8) are plotted against changes in cells expressing PR8 PA-X versus control cells. p < 0.001, Spearman's test. [/fig]
[fig] Figure 3: k-Means Clustering Reveals Differentially Regulated Groups of RNAs (A-C) Cluster 3 was used to divide cellular RNAs in 4 clusters based on the pattern of fold changes in iPA-X cells (PA-X WT or D108A catalytic mutant versus control) and cells infected with IAV (WT or PA-X-deficient IAV versus mock). Cumulative probability histograms of fold changes for each of the classes are plotted: (A) 2 groups of PA-X targets, (B) PA-X-resistant RNAs, (C) potential PA-X targets that are regulated by other processes during infection. All of the datasets collected were used for clustering, but only select datasets are plotted for simplicity. [/fig]
[fig] Figure 4: RNAs that Are Not Spliced Are Less Sensitive to Regulation by PA-X (A and B) RNA-seq results from Figures 1C and 2A are plotted separately for spliced and intronless RNA as cumulative distribution histograms. (A) Cells overexpressing WT PA-X, clones #1 and #10; (B) cells infected with WT PR8 versus PR8 PA(DX). (C-F) Relative RNA levels in PA-X overexpressing (OE; clone #1) versus control cells are plotted against the number of exons (C and E, log2 scale) or transcript length in kilobases (D and F, log10 scale). (C and D) All RNAs, (E) RNAs with 6 exons, and (F) RNAs 3.5-4.0 kb in length. All of the correlations are statistically significant (p < 0.001, Spearman's test). (G) The number of exons for RNAs identified in the clustering analysis [/fig]
[table] TABLE d: CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell lines d METHOD DETAILS B Plasmids B Cell lines, lentiviral transduction and transfections B Viruses and infections B Preparation of cell lysates containing biotinylated proteins B Neutravidin pull-down B Mass spectrometry sample preparation B Reductive dimethylation and quantitative mass spectrometry B Cell fractionation for RNA analysis B RNA purification, cDNA preparation and qPCR B Intron splicing verification B Co-immunoprecipitation from nuclear lysates B Western blotting and immunofluorescence B RNA-seq B Read alignment and bioinformatic analysis d QUANTIFICATION AND STATISTICAL ANALYSIS d DATA AND SOFTWARE AVAILABILITY SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.03.063. [/table]
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Pharmaceutical and medicinal significance of sulfur (SVI)-Containing motifs for drug discovery: A critical review
a b s t r a c tSulfur (S VI ) based moieties, especially, the sulfonyl or sulfonamide based analogues have showed a variety of pharmacological properties, and its derivatives propose a high degree of structural diversity that has established useful for the finding of new therapeutic agents. The developments of new less toxic, low cost and highly active sulfonamides containing analogues are hot research topics in medicinal chemistry. Currently, more than 150 FDA approved Sulfur (S VI )-based drugs are available in the market, and they are widely used to treat various types of diseases with therapeutic power. This comprehensive review highlights the recent developments of sulfonyl or sulfonamides based compounds in huge range of therapeutic applications such as antimicrobial, anti-inflammatory, antiviral, anticonvulsant, antitubercular, antidiabetic, antileishmanial, carbonic anhydrase, antimalarial, anticancer and other medicinal agents. We believe that, this review article is useful to inspire new ideas for structural design and developments of less toxic and powerful Sulfur (S VI ) based drugs against the numerous death-causing diseases.
# Introduction
The sulfonamide or sulfonyl functional groups have been important motifs in medicinal chemistry since the early discovery of sulfonamide containing antibacterial drugs [bib_ref] Synopsis of some recent tactical application of bioisosteres in drug design, Meanwell [/bib_ref]. The applications of sulfonyl or sulfonamide functional groups in medicinal chemistry cannot be ignored, as it constitutes an important class of drugs used extensively as agricultural and pharmaceutical agents [bib_ref] Recent developments in the synthesis of fused sultams, Majumdar [/bib_ref] [bib_ref] Synthesis of water-soluble, topically effective, intraocular pressure-lowering aromatic/heterocyclic sulfonamides containing cationic or..., Scozzafava [/bib_ref]. The features of S VI -containing species of strong electron withdrawing nature, stability against hydrolysis, resistance to reduction at sulfur, and crisp preference for two-electron processes over radical processes, have already made this group applicable to many productive fields. Sulfonamides as synthetic antifolic agents have been widely used for the anticipation and treat of bacterial infections in biological systems and recently have evoked high favor in biology and medicine due to their wide array of biological activities such as antibacterial , antifungal [bib_ref] Biological activity, design, synthesis and structure activity relationship of some novel derivatives..., Lal [/bib_ref] , antiinflammatory [8e10], antioxidant [bib_ref] Design, synthesis and pharmacological evaluation of (E)-3,4-dihydroxy styryl sulfonamides derivatives as multifunctional..., Xianling [/bib_ref] [bib_ref] Synthesis, antioxidant, enzyme inhibition and DNA binding studies of novel N-benzylated derivatives..., Aadil [/bib_ref] [bib_ref] Sulfonamides as multifunctional agents for Alzheimer's disease, Seema [/bib_ref] [bib_ref] Divergent C-H functionalizations directed by sulfonamide pharmacophores: late-stage diversification as a tool..., Dai [/bib_ref] , antitubercular [bib_ref] New cyrhetrenyl and ferrocenyl sulfonamides: synthesis, characterization, X-ray crystallography, theoretical study and..., Quintana [/bib_ref] [bib_ref] Carbonic anhydrase inhibitors, characterization and inhibition studies of the most active beta-carbonic..., Carta [/bib_ref] , antidiabetic [bib_ref] A 50-year history of new drugs in Japan-the development and progress of..., Ozawa [/bib_ref] [bib_ref] Efficacy of glimepiride in type 2 diabetic patients treated with glibenclamide, Hamaguchi [/bib_ref] , HIV protease inhibitors [bib_ref] Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters, Shen [/bib_ref] [bib_ref] The KLEAN study of fosamprenavir-ritonavir versus lopinavir-ritonavir, each in combination with abacavir-lamivudine,..., Eron [/bib_ref] , antiglaucoma [34e36], antiobesity [bib_ref] Antiobesity carbonic anhydrase inhibitors: a literature and patent review, Scozzafava [/bib_ref] [bib_ref] Novel (4-piperidin-1-yl)-phenyl sulfonamides as potent and selective human b 3 agonists, Hu [/bib_ref] , antiviral [bib_ref] Discovery of HIV-1 protease inhibitors with picomolar affinities incorporating N-aryl oxazolidinone-5-carboxamides as..., Ali [/bib_ref] [bib_ref] Synthesis and antiviral evaluation of acyclic azanucleosides developed from sulfanilamide as a..., Gawin [/bib_ref] , antimalaria [bib_ref] Design and synthesis of new N-(5-Trifluoromethyl)-1H-1,2,4-triazol-3-yl benzenesulfonamides as possible antimalarial prototypes, Boechat [/bib_ref] , MMP inhibitors [bib_ref] Sulphonamides: deserving class as MMP inhibitors?, Jain [/bib_ref] [bib_ref] Role of sulfonamide group in matrix metalloproteinase inhibitors, Cheng [/bib_ref] , non-peptidic vasopressin receptor antagonists [bib_ref] An overview of SR121463, a selective non-peptide vasopressin V2 receptor antagonist, Serradeil-Le [/bib_ref] and translation initiation inhibitors [bib_ref] Novel arylsulfoanilide-oxindole hybrid as an anticancer agent that inhibits translation initiation, Natarajan [/bib_ref] etc. Up to date, more than 150 FDA approved drugs bearing Sulfur (S VI ) motif are available in the market such as celecoxib, meloxicam, piroxicam, sulfasalazine, and so on. The diverse pharmacological activity of S VI in organic molecules makes it a first choice for incorporation by the hybrid approach, which is present in most of the required medicines that are accessible in the market [bib_ref] Recent development of sulfonyl or sulfonamide hybrids as potential anticancer agents: a..., Rakesha [/bib_ref].
Heterocyclic compounds play essential roles in life and biochemical processes [bib_ref] Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory..., Cevher [/bib_ref]. Among them, a huge number of novel sulfonamide derivatives have been reported and tested for both in vivo and in vitro antitumor activities. Some of these highly potent analogues are tested in clinical trials. Hopefully, these may lead to new alternative anticancer drugs avoiding the side effects of the available pharmacological agents. Sulfur (S VI )-containing drugs are still widely used for circumstances of spots and urinary tract infections, and are receiving more renewed interest for the treatment of infections caused by bacterial resistance of other antibiotics [bib_ref] Apricoxib, a novel inhibitor of COX-2, markedly improves standard therapy response in..., Kirane [/bib_ref] [bib_ref] Selection and characterization of HIV-1 showing reduced susceptibility to the non-peptidic protease..., Doyon [/bib_ref]. The excellent biological profile, hydrolytic stability and crystalline nature of sulfonamides have grabbed significant attention from synthetic chemists [bib_ref] Design and synthesis of cyclic sulfonamides and sulfamates as new calcium sensing..., Kiefer [/bib_ref] [bib_ref] Synthesis of radiolabeled biphenylsulfonamide matrix metalloproteinase inhibitors as new potential PET cancer..., Fei [/bib_ref]. These sulfonamide analogues can be traced in a number of well established potential drugs belonging to various types of therapeutic agents. Some of the representative sulfonamides or sulfonyl functional group containing FDA approved drugs are listed in [fig_ref] Table 1: Sulfonamides or sulfonyl group containing FDA approved drugs from 1937 to 2012 [/fig_ref].
In search of more new potent multi-targeted sulfonamide or sulfonyl drugs, many medicinal chemistry scholars focused on sulfonamide nucleus, which has importance in the area of medicinal chemistry, drug development as a core substituent of diverse biological agents [bib_ref] Molecular modeling, synthesis, antibacterial and cytotoxicity evaluation of sulfonamide derivatives of benzimidazole,..., Naaz [/bib_ref]. In order to overcome the resistance and to reduce the adverse effects, continuous efforts are made to synthesize novel multi-targeted bioactive sulfonamide analogues. In this regard, combinations of certain sulfonamides and other drug molecules are being used to develop novel formulations with greater effectiveness as well as less toxicity [bib_ref] Dofetilide, a new class III antiarrhythmic agent, Lenz [/bib_ref].
This present comprehensive review aims to recapitulate the recent biological applications made towards the discovery of novel sulfonyl or sulfonamides functional group containing analogues as potential therapeutic agents and the critical aspects of design and structure-activity relationship (SAR) studies were also briefly explained.
## Biological applications of sulfonyl or sulfonamides functionalities
## Antimicrobial agents
The problem of antibiotic resistance among pathogenic bacteria is as old as antibiotics itself [bib_ref] Resistance to antibiotics: are we in the post-antibiotic era?, Alanis [/bib_ref]. The antibiotic resistance which was accelerated by the use and misuse of antimicrobial drugs has been a major global challenge for public health. Dramatic increase of human pathogenic bacteria was observed from the past decades due to their resistance to one or more antibiotics. A number of infections caused by resistant organisms fail at responding to the conventional treatment and in few cases, the last resort antibiotics have also lost their power [bib_ref] Targeting antibiotic resistance, Chellat [/bib_ref].
In search of some new antibiotics, the sulfonamide functional groups have been fundamental motifs in medicinal chemistry since the early discovery of sulphonamide containing antibacterial drugs [bib_ref] Synopsis of some recent tactical application of bioisosteres in drug design, Meanwell [/bib_ref]. To date, a number of sulfonyl or sulfonamide bearing aromatic heterocycles such as quinazolinones, oxazoles, benzimidazole, thiazole and pyridazine have been successfully developed and employed in clinics with the presence of sulfadiazine, sulfachlorpyridazine, sulfathiazole and sulfisoxazole exhibiting excellent antimicrobial activities [bib_ref] Synthesis of novel sulfanilamide-derived 1,2,3-triazoles and their evaluation for antibacterial and antifungal..., Wang [/bib_ref]. Because of the weak effectiveness and even loss of resistance power of old antibiotics against new and upcoming bacterial pathogens, urgent alternatives were needed to develope novel, less toxic and highly effective antimicrobial agents with distinct structures to fight with emerging antibiotic-resistant bacterial infections. In the first part of this review article, we have focused on sulfonamide analogues as a core substituent of antibacterial agents for drug development. In this regard, the combinations of sulfonamides and other heterocyclic drug molecules are being used to develop novel antibiotic drugs [bib_ref] Molecular modeling, synthesis, antibacterial and cytotoxicity evaluation of sulfonamide derivatives of benzimidazole,..., Naaz [/bib_ref]. Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents are summarized in [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref].
Zhou et al. designed and synthesized a novel series of benzimidazole-derived sulfonamide analogues and evaluated for in vitro antimicrobial activities against different microbial pathogens. Compound 107 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed excellent antibacterial activity against S. aureus with MIC values of 4 mg/mL. The replacement of 4fluorobenzyl group (107) by 2,4-dichlorobenzyl group, 108 showed good antibacterial activity against B. typhi with MIC values 4 mg/mL. Compound 108 showed eight folds higher activity (MIC ¼ 4 mg/mL) than standard Chloromycin against B. typhi [bib_ref] Design, synthesis and antimicrobial evaluation of novel benzimidazole-incorporated sulfonamide analogues, Zhang [/bib_ref].
The above same research group further developed a class of new type of sulphonamide-containing azoles analogues as potential antimicrobial agents. Compound 109 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed excellent antibacterial activity against P. aeruginosa with MIC value of 16 mg/ mL [bib_ref] Synthesis of novel sulfonamide azoles via CeN cleavage of sulfonamides by azole..., Zhang [/bib_ref]. Kamble et al. have reported pyrazole derived sulfonamide analogues as good antibacterial agents. Compound 110 showed potent antibacterial activity against tested bacterial strains S. aureus and S. typhimurium with MIC value of 10 mg/mL each.
Compound 111 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed excellent antibacterial activity against different bacterial pathogens namely B. subtillis and E. coli with MIC value of 10 mg/mL each. To elucidate the structure activity relationship (SAR) of compounds 110 and 111, the presence of electron withdrawing (Br and CF [bib_ref] Synthesis of water-soluble, topically effective, intraocular pressure-lowering aromatic/heterocyclic sulfonamides containing cationic or..., Scozzafava [/bib_ref] groups (EWG) on the sulfonyl attached phenyl ring, increases the bacterial resistance against the tested S. aureus and S. typhimurium strains. But the same moiety with replacement of the -Br functional group, and the inserting of the Cl functional group, compound 111 was found to be highly active against another bacterial strains B. subtillis and E. coli. The lipophilicity as well as nature and position of the substituent present on benzene ring of sulfonamide end affected the antimicrobial activity [bib_ref] Synthesis, anti-inflammatory and antimicrobial evaluation of novel 1-acetyl-3,5-diaryl-4,5-dihydro (1H) pyrazole derivatives bearing..., Keche [/bib_ref]. In 2014, Nasr et al. developed a new type of (continued on next page) sulfonamide containing sulfisoxazole analogues and evaluated for antibacterial activity. Compound 112 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed promising antibacterial activities against most of the tested bacterial strains. Compound 113 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed excellent antibacterial activity against the S. epidermidis, P. vulgaris and K. pneumonia bacterial strains. The analysis of the SAR, revealed that the presence of sulfonamide group with heterocyclic moiety increases the lipophilic characters of the synthesized compounds [bib_ref] Design, synthesis, antimicrobial evaluation and molecular docking studies of some new thiophene,..., Nasr [/bib_ref]. The research group of Padmaja [bib_ref] Synthesis, antimicrobial and antioxidant activities of substituted pyrazoles, isoxazoles, pyrimidine and thioxopyrimidine..., Padmaja [/bib_ref] synthesized heterocycles containing sulfonamides analogues and evaluated for in vitro antimicrobial activities against various microbial pathogens using agar disc diffusion method. Among all the synthesized analogues, isoxazole containing sulfone analog 114 (S. aureus -32 mm, B. subtilis -31 mm, K. pneumoniae-26 mm, P. vulgaris -28 mm in diameter) [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to exhibit the highest inhibitory activity against tested bacterial strains. The presence of EWG (Cl) on phenyl ring of the sulfonyl end and sulfone group infatuated stronger antimicrobial activities compared to the other EDGs. In the continuation of the potent antimicrobial drug developments of sulfone containing heterocyclic derivatives, Lavanya et al. [bib_ref] Synthesis and antimicrobial activity of (1,4-phenylene) bis(arylsulfonylpyrazoles and isoxazoles), Lavanya [/bib_ref] reported 1,4-phenylene) bis (arylsulfonylisoxazoles analogues to have potent antimicrobial properties. Compound 115 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to have the highest antibacterial activity against B. subtilis with zone of inhibition of 38 mm at 100 mg/mL. The elucidating of the SAR indicated that the presence of EWG (Cl) on the phenyl ring of the sulfone end showed maximum antibacterial activity against B. subtilis strain. In another study, a 2-ureidothiophene-3carboxylic acid derivative was synthesized and screened as dual bacterial RNAP and HIV-1 RT inhibitors by Elgaher et al. [bib_ref] Discovery and structure-based optimization of 2-Ureidothiophene-3-carboxylic acids as dual bacterial RNA polymerase..., Elgaher [/bib_ref]. Compound 116 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] displayed more potency against tested S. aureus with high cellular antiretroviral activity. This is probably due to the presence of non-bulky hydrophilic substituents at the ureido side chain for RT inhibition, the hydrophilic and hydrogen bond donor or acceptor substituents at the N-phenyl group are also to improve the inhibitory activity more than 2e3-fold compared with electron-withdrawing groups (EWGs). Novel N-sulfonaminoethyloxime derivatives of dehydroabietic acid were developed by Zhang et al. [bib_ref] The synthesis and antistaphylococcal activity of N-sulfonaminoethyloxime derivatives of dehydroabietic acid, Zhang [/bib_ref] and tested for antibacterial activity against various bacterial pathogens. Among those, compound 119 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] exhibited the superior activity against tested five multidrug-resistances of S. aureus with MIC values between 0.78 and 1.56 mg/mL. The meta-CF 3 phenyl derivative 119 showed the highest activity with MIC of 0.39e0.78 mg/mL against S. aureus Newman. To elucidate the SAR, they demonstrated that the introduce of an electron withdrawing trifluoromethyl group (-CF 3 ) at meta position on the phenyl ring is more beneficial for the increasing antibacterial activity and selectivity compared to other substituents such as chloro, bromo, fluoro, methyl or methoxy groups. Very interestingly, the ortho substituted CF 3 derivative exhibited no in vitro activity against any of the Gram-positive bacterial strains at 50 mg/mL. The tert-butyl and methoxy functional group containing analogues showed decreased antibacterial activity. In addition, the substitution position appeared to have slight influence on the antibacterial activity of electron withdrawing functional group substituted derivatives. Nimbarte and coworkers [bib_ref] Design, synthesis and biological evaluation of 4-(1-(4(sulphanilamide)phenyl)-3-(methyl)-1H-pyrazol-5-yl) dine urea and N-acyl derivatives..., Nimbarte [/bib_ref] developed novel sulfonamide linked piperidine and pyrazole analogues and evaluated for the inhibition of soluble epoxide hydrolysis. Compounds 120 and 121 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] showed the highest inhibitory activity against tested bacterial strains with IC 50 values of 0.220 mM and 0.224 mM, respectively. Zengin and coworkers found a new class of sulfanilamide as potent antimicrobial agents against tested bacterial and fungal strains. Compound 122 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] was found to have the highest antimicrobial activities against B. cereus (MIC value is 33 mm) and E. fecalis (MIC value is 33 mm). The SAR studies revealed that the lipophilicity of the analogues played a crucial role for producing antimicrobial activities. The dimethyl substituted compound 122 had high antimicrobial activity but low lipophilic character [bib_ref] Synthesis of sulfanilamide derivatives and investigation of in vitro inhibitory activities and..., Turkmen [/bib_ref]. Series of sulfonamide containing benzothiazole hybrids were evaluated for in vitro antimicrobial properties against some microbial pathogens. Compounds 123 and 124 and were found to have higher antimicrobial activities compared to the reference Sulfamethoxazole-trimethoprim mixture. The SAR studies revealed that the presence of sulfonamides with an amino group (-NH 2 ) and nitro group (-NO 2 ) at the para position of the phenyl ring showed excellent antimicrobial properties. The replacement of the amino group with nitro group led to the decrease of the antibacterial activity [bib_ref] Synthesis and biological evaluation of sulfonamide thiazole and benzothiazole derivatives as antimicrobial..., Argyropoulou [/bib_ref]. In continuous study of sulphonamide containing benzothiazole analogues as powerful antimicrobial properties, a new class of sulphonamide containing benzothiazole hybrids were synthesized and screened for antimicrobial activity by Patel et al. presence of sulfonamide group at para position of the phenyl group has highly increases the antibacterial properties of all the tested bacterial pathogens. Furthermore, the presence of EWG (Cl) group on the phenyl ring also contributes to increasing the antibacterial activity [bib_ref] Synthesis and antibacterial activity of some heterocyclicderivatives of sulphanilamide, Subudhi [/bib_ref].
## Anti-diabetic activity
Diabetes is one of the most severe diseases rising in the world. According to the estimation data obtained in 2010, around 285 millions of people are suffering from diabetes all over the world and it may increase to 439 million by 2030 [bib_ref] Global estimates of the prevalence of diabetes for, Shaw [/bib_ref] [bib_ref] Global healthcare expenditure on diabetes for, Zhang [/bib_ref]. Change in the blood glucose due to the insulin resistance is observed as the characteristic of being diabetic in 95% of the cases [bib_ref] Type 2 diabetes in the young: the evolving epidemic, Alberti [/bib_ref] which give raise to several more problems like high blood pressure, heart problem, kidney failure, stroke and blindness [bib_ref] Microvascular and macrovascular complications of diabetes, Fowler [/bib_ref]. Therefore, it is highly demanded to develop additional electronic and steric requirements of arylsulfonamidothiazoles with antidiabetic effect [bib_ref] Synthesis of N-(5-chloro-6-(quinolin-3-yloxy) pyridin-3-yl)benzenesulfonamide derivatives as non-TZD peroxisome proliferator-activated receptor g(PPARg) agonist, Bajare [/bib_ref]. [fig_ref] Figure 5: Some representative sulfonyl of sulfonamides as potent anti-diabetic agents [/fig_ref] showed some representative sulfonyl of sulfonamides as potent anti-diabetic agents.
In 2014, Navarrete-V azquez and co-workers designed and developed naphthalene containing sulfonamides as potent antidiabetic agents against 11b-hydroxysteroid dehydrogenase type-1 (11b-HSD1). Among them, compounds 136, 137 and 138 showed promising anti-diabetic activity against 11b-hydroxysteroid dehydrogenase type-1 with % inhibition of 68, 67 and 55 at 10 mM respectively better than the standard drug BVT.14225 (55% inhibition). The SAR studies suggested that both piperidine and pyrrolidine core attached at the amide group were more active compounds to the tested all hybrids [bib_ref] Synthesis of 2-{2-[(a/b-naphthalen-1-ylsulfonyl)amino]-1,3-thiazol-4-yl} acetamides with 11b-hydroxysteroid dehydrogenase inhibition and in combo antidiabetic..., Navarrete-V Azquez [/bib_ref]. A novel class of thiazolidinedione based sulfonamide hybrids were evaluated for antidiabetic activity against the Peroxisome Proliferator Activated Receptor (PPARg) by Naim et al. [bib_ref] Design, synthesis and molecular docking of thiazolidinedione based benzene sulphonamide derivatives containing..., Naim [/bib_ref]. Among these, compound 139 [fig_ref] Figure 6: Sulfonyl of sulfonamides as antidiabetic agents [/fig_ref] was found to be excellent PPAR-g inhibitor of 61.2% with 1.9 folds increase in gene expression. In docking studies, compound 139 displayed good interaction with amino acids Tyr 473, Ser 289, Hie 449, Tyr 327, Arg 288, Met 329 and Leu 228 . This observation indicates that the presence of hydrophobic moiety in 139 is surrounded by hydrophobic amino acids. It is believed that such hydrophobic interactions enhances the ligand receptor complex as well as binding affinity of ligand towards PPARg.
Rathish et al. reported the synthesis and anti-diabetic activity of sulfonamide based pyridazinone derivatives. Compounds 140 and 141 [fig_ref] Figure 8: Sulfonyl of sulfonamides as anti-diabetic agents [/fig_ref] showed excellent anti-diabetic agents with more than 50% reduction in the rise of blood glucose levels. The SAR may be summarized as the introduction of electron withdrawing Cl at para position of phenyl group caused slightly reduction in the activity. On the other hand, the presence of electron releasing functional groups such as methoxy or methyl functional group at phenyl ring slightly caused reduction in the activity. Moreover, the compounds containing less bulky side chains were found to be more favourable for increasing anti-diabetic activity [bib_ref] Synthesis and blood glucose lowering effect of novel pyridazinone substituted benzenesulfonylurea derivatives, Rathish [/bib_ref]. The effect of in vivo antidiabetic activity in non-insulin dependent diabetes mellitus rat of sulfonamide end enhanced the anti-diabetic properties [bib_ref] Synthesis, in vitro and computational studies of protein tyrosine phosphatase 1B inhibition..., Navarrete-Vazquez [/bib_ref]. Recently, how to improve the drug resistance of potent antidiabetic drugs against PTP-1B, has emerged as a key role of insulin signalling target for type 2-diabetes. In 2018, Du and co-workers reported novel PTP-IB inhibitors of a series of ureodo-sulfonamides based analogues. Among these, compounds 146 and 147 showed superior PTP1B inhibitors with IC 50 values of 18.6 nM and 66.2 nM respectively. The SAR suggested that, compound 146 with 2-ethoxy group on B ring was identified to possess 10.9 fold more potent inhibitory activity against the PTP1B enzyme. Compound 147, with the presence of -CONH-(3,4-di-MeO-Ph) group on ring B displayed high potent activity [bib_ref] Discovery of novel high potent and cellular active ADC type PTP1B inhibitors..., Du [/bib_ref].
A series of piperazine-sulfonamide analogues were studied for in vitro a-amylase inhibition activity by Nawaz et al. [bib_ref] Synthesis of piperazine sulfonamide analogs as diabetic-II inhibitors and their molecular docking..., Taha [/bib_ref]. Com- stronger cryptochrome modulator with EC 50 ¼ 0.144 mM. The SAR suggested that, the presence of sulfonamides functional group improved lipophilic efficiency of the potent analog [bib_ref] Carbazole-containing sulfonamides and sulfamides: discovery of cryptochrome modulators as antidiabetic agents, Humphries [/bib_ref]. Recently, Deka and co-workers have prepared a new series of thiazolidinediones hybrids and screened for potent peroxisome proliferatoractivated receptor g (PPARg). Among all the synthesized analogues, compounds 154 and 155 [fig_ref] Figure 9: Sulfonyl of sulfonamides as anti-diabetic agents [/fig_ref] showed maximum PPARg binding affinities (I max ) with 98% and 82% respectively. The SAR revealed that, the introduction of diverse aryl sulfonamides as the polar head group and 1-phenylpiperidine on the tail part highly influenced the PPARg activity. In addition, the presence of electron withdrawing Cl and eCF 3 groups on the phenyl ring of the sulfonamide linker also played a major role for the increases of activity. The presence of electron releasing (OH and eOCH 3 ) groups decreased the activity [bib_ref] Design and synthesis of non-TZD peroxisome proliferatoractivated receptor g(PPARg) modulator, Deka [/bib_ref]. In 2017, Bruning and co-workers designed and synthesized a class of novel 2,4-dichloro-N-(3,5-dichloro-4-(quinolin-3-yloxy) phenyl)benzenesulfonamide analogues for potent PPARg-targeted antidiabetics agents. Compound 156 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] showed the most potent active PPARg inhibitor with EC 50 values is 2 nM. The SAR revealed that, the presence of EWGs (F and Br) on phenyl ring A increased the activity. The sulfonamide moiety and a bromine atom at the para position on the aromatic benzene ring A contributed the potent active PPARg inhibitor [bib_ref] -(quinolin-3-yloxy)phenyl)benzenesulfonamide (INT131) analogs for PPARg-targeted antidiabetics, Frkic [/bib_ref]. Gao and co-workers synthesized a novel series of sulfonamide-1,3,5-triazine-thiazoles derivatives and tested for in vitro inhibitory activity against several DPP enzymes, such as DPP-4, DPP-8 and DPP-9. Compound 157 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to be highly potent against DPP-4 enzyme with IC 50 value of 2.32 nM compared to standard drug alogliptin. The SAR suggested that, compounds containing EWGs had superior inhibitory activity compared to those with EDGs substituent. Furthermore, the presence of additional aromaticity did not influence the activity. Moreover, molecular docking results indicated that, ligand 157 was efficiently docked into the active site of the catalytic triad of Ser 630, Asp 708 and His 740 encompassing both S1 and S2 pocket with CDOCKER interaction energy of 57.80 [bib_ref] Sulfonamide-1,3,5-triazine-thiazoles: discovery of novel class of antidiabetic agent via inhibition of DPP-4, Gao [/bib_ref]. At last, Iqbal and co-workers have developed arylsulfonylspiroimidazolidine-2,4-dione hybrids as potent hypoglycemic and ALR2 agents. Compound 158 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to have the most potent inhibitory activity against ALR2 with an IC 50 value of 0.89 mM. The in vivo hypoglycaemic activity of compound 158 exhibited 72.24% reduction in blood glucose, which was more potent than standard drug glibenclamide (60.92% reduction). The SAR suggested that, the presence of EWG (Cl) on the phenyl ring highly influenced the ALR2 activity. Replacing the halogen atom by methyl or methoxy group led to a reduced activity which was attributable to the lower lipophilicity of these substituents compared to the chlorine atom, and lesser interaction with the active site of aldose reductase. Activity was not really affected when the 2-naphthyl group replaced the para substituted phenyl ring; however, 2-anthraquinyl group was found to be detrimental to the activity. The large size of the 2anthraquinyl group might be responsible for this negative effect [bib_ref] Synthesis, characterization, hypoglycemic and aldose reductase inhibition activity of arylsulfonylspiro, Iqbal [/bib_ref].
## Anti-inflammatory activity
Inflammation is a localised physical condition causing swellness, redness, heat with pain which is mediated by the release of proinflammatory mediators like bradykinin and cytosine increasing the prostaglandin synthesis rate [bib_ref] Cyclooxygenase inhibitory natural products: current status, Jachak [/bib_ref]. Nonsteroidal anti-inflammatory drugs (NSAIDs) existing in two isomeric forms, constitutive form (COX-1) and an inducible form (COX-2) inhibits cyclooxygenases (COX) and thereby inhibiting the biosynthesis of prostaglandins (PGs) [bib_ref] Cyclooxygenase 2 inhibitors: discovery, selectivity and the future, Marnett [/bib_ref] [bib_ref] Cyclooxygenase inhibitors-current status and future prospects, Dannhardt [/bib_ref]. The role of COX-1 enzyme is maintaining the gastric integrity and kidney functioning whereas COX-2 is involved in inflammation and pain [bib_ref] Prostaglandin endoperoxide H synthases-1 and -2, Smith [/bib_ref] [bib_ref] Prostaglandin synthase 2, Herschman [/bib_ref].
The sulfonamide moiety exists as one of the most ubiquitous pharmacophoric functional groups in medicinal chemistry. Sulfonamide group shows a diverse pharmacological activity in the organic molecules and hence it has become a priority while choosing functional group to incorporate in the optimizations by hybrid approach. It was reported earlier that a number of sulfonyl or sulphonamide functional group containing heterocyclic compounds were utilised to demonstrate potential anti-inflammatory activity [99e103]. Moreover, among the highly marketed COX-2 inhibitors that comprise the sulphonamide moiety, SC-558 [bib_ref] Synthesis and in vitro evaluation of new benzenesulfonamides as antileishmanial agents, Borges [/bib_ref] and celecoxib (166) [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] are the major determinant for COX-2 selectivity and in vivo efficacy. Nimesulide (167) [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] is an example of small molecule NSAID S sold in the market today that has the sulfonamide functionality [bib_ref] Mechanism of action of novel anti-inflammatory drugs diflumidone and R-805, Vigdahl [/bib_ref]. Some of them were potential anti-inflammatory analogues as showed in [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref].
A class of novel sulfonamides as potent anti-inflammatory agents were designed and synthesized bearing pyrazolyl derivatives by Bekhit et al. [bib_ref] Synthesis and biological evaluation of some thiazolyl and thiadiazolyl derivatives of 1H-pyrazole..., Bekhit [/bib_ref]. The para-chlorophenyl substituted compound 170 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] emerged as a potent anti-inflammatory agent with protection 77.4% exceeding that of indomethacin. Chowdhury and co-workers reported a family of pyrazole bearing sulfonamides analogues and evaluated for in vitro antiinflammatory activity. Compound 171 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] displayed attractive anti-inflammatory activity compared to the standard antiinflammatory drugs celecoxib and aspirin. SAR studies revealed that the presence of N-methyl-1,2,3,6-tetrahydropyridyl ring significantly increased the bioisosteric effects in the active analogues [bib_ref] Synthesis of new 4-[2-(4-methyl(amino)sulfonylphenyl)-5-trifluoromethyl-2H-pyrazol-3-yl]-1,2,3,6-tetrahydropyridines: a search for novel nitric oxide donor antiinflammatory..., Chowdhury [/bib_ref]. Next, the research group of El-Din et al. developed sulfonamides based hybrids with potent anti-inflammatory activity. Compound 172 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to be most significant candidate, no ulcerogenic effect and with minimal effects on renal function. Novel pyrazole based sulfonamides derivatives were prepared by Küçükgüzel and co-workers and screened for their in vitro anti-inflammatory activity. Among those, compound 173 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] showed promising anti-inflammatory activity [bib_ref] Synthesis and characterization of celecoxib derivatives as possible anti-inflammatory, analgesic, antioxidant, anticancer..., Küçükgüzel [/bib_ref]. (IC 50 ¼ 0.22 mmoL/kg) [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] showed excellent antiinflammatory activity (82% inhibition) and promising analgesic activity. The SAR suggested that, compounds containing 4chlorophenyl pharmacophore exhibited higher activity than other functional substituted analogues (except for benzenesulfonamide azomethine). The effect of the nature of substituent at the 3position of the pyrazole nucleus also played a major role in enhancing the anti-inflammatory activity [bib_ref] Synthesis of novel 1,3,4-trisubstituted pyrazoles as anti-inflammatory and analgesic agents, Ragab [/bib_ref]. Mohammed and Nissan reported novel pyrazole bearing sulphonamide-hydrazones derivatives as potent anti-inflammatory agents. Compound 175 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to be a better antiinflammatory agent than the standard anti-inflammatory drug diclofenac and indomethacine. In addition, molecular docking study revealed that the compound 175 interacted with Tyr 385 and Ser 530 [bib_ref] Synthesis, molecular docking, and biological evaluation of some novel hydrazones and pyrazole..., Mohammed [/bib_ref]. Hassan et al. synthesized a series of benzofuran bearing celecoxib-sulfonamides for the development of novel anti-inflammatory agents. Among those, compound 176 and 177 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] exposed the highest anti-inflammatory activities. Antiinflammatory data revealed that an essential role of compounds 176 and 177 bearing pyridine moiety enhanced the antiinflammatory efficiency in animal models [bib_ref] Celecoxib analogs bearing benzofuran moiety as cyclooxygenase-2 inhibitors: design, synthesis and evaluation..., Hassan [/bib_ref]. Ahmed and coworkers synthesized a new class of curcumin-containing sulfonamides analogues to investigate the activity against antiinflammatory. Compound 178 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was identified as a successful anti-inflammatory agent by 82% inhibition of induced edema which is comparable to standard drug indomethacin (84.4% inhibition). In 2014, Kumar et al. reported an eighteen pyrazolylpyrazolines bearing benzenesulfonamide as potent antiinflammatory agents. Among those, compounds 179 and 180 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] showed excellent anti-inflammatory effects [bib_ref] Benzenesulfonamide bearing pyrazolylpyrazolines: synthesis and evaluation as anti-inflammatory, antimicrobial agents, Kumar [/bib_ref]. Compounds containing sulfonamides based heterocycles have been highlighted for the search of new anti-inflammatory agents.
## Anti-malarial activity
Malaria is a parasitic infection which is spread worldwide mostly affecting and causing serious problems in the tropical and subtropical parts of Asia, Central and South America, Africa and also millions of people are affected in the parts of Middle East [bib_ref] The global fight against HIV/AIDS, tuberculosis, and malaria: current status and future, Vitoria [/bib_ref]. A parasitic species called Plasmodium which is carried by the female of Anopheles mosquito is the cause of this disease which enters into bloodstream of humans by an infected mosquito. The treatment and management of this disease is unreasonably high not only because of medication but also due to low production. The difficulty in controlling malaria lies at growing resistance of malaria parasite to most of the antimalarial drugs used [bib_ref] Antimalarial drug resistance, artemisinin-based combination therapy, and the contribution of modeling to..., Yeung [/bib_ref].
Hence there is a strong need to treat this drug-resistant disease by developing better performing drugs. A continued effort including exploration of potentially bioactive natural product derived compounds is required. Most of the biologically active antimalarial agents contains sulphonamide group [121e123]. The sulfonamide group present in a number of potential anti-malarial analogues were showed in Results from the study indicated that alkyl chain length was critical for antimalarial activity and also the presence of isopropyl groups on the phenyl ring of the sulfonamide end highly enhanced the antimalarial activity [bib_ref] N-(7-Chloroquinolinyl-4-aminoalkyl)arylsulfonamides as antimalarial agents: rationale for the activity with reference to inhibition..., Verma [/bib_ref]. Recently, Oliveira and co-workers reported the potent sulfonamide containing chaclcone hybrids and evaluated for in vitro anti-malarial activity against P. falciparum.
Compound 200 (IC 50 ¼ 2.06 mM) [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to be the best antimalarial agent with good selectivity index [bib_ref] Anti-malarial activity of 4-metoxychalcones: docking studies as falcipain/plasmepsin inhibitors, ADMET and lipophilic..., De Oliveira [/bib_ref]. Muthas and co-workers reported a class of new potent hydroxyethypiperazines bearing benzenesulfonyl hybrids as antimalarial agents. All the synthesized compounds were tested in vitro antimalarial activity against a W2 P. falciparum clone. Among those, compound 201 (IC 50 of 16.9 mM, [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] displayed superior antimalarial activity with IC 50 values of 4.80 mM against W2 P. falciparum clone [bib_ref] Synthesis, biological evaluation, and modeling studies of inhibitors aimed at the malarial..., Muthas [/bib_ref].
Recently, the combination of indoleamides with sulfonyl has been reported as the most active antimalarial agents against Pf3D7 and PfK1 strains. Among those, compounds 202, 203, 204 and 205 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] respectively. SAR revealed that the presence of sulfonyl analogues containing bulky groups such as p-ter-butylphenyl (202 and 204) and 4-chloro-2,5-dimethyl phenyl groups showed the most promising antiplasmodial activity with IC 50 values range between 1.60 and 2.19 mM [bib_ref] Synthesis and antiplasmodial activity of novel indoleamide derivatives bearing sulfonamide and triazole..., Devender [/bib_ref]. Huang and co-workers reported sulphonamide bearing small analogues as potent dual inhibitors of FP-2 and DHFR. Compounds 206 and 207 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] bearing amide and sulphonamide moieties were found to be the most active FP-2 inhibitors. Compound 207 containing thiazole group on amide moiety was most active analogues against FP-2 (IC 50 ¼ 7.0 mM) and DHFR (IC 50 ¼ 6.3 mM). In addition, compound 207 showed reasonable in vivo antimalarial activities compared to standard drug chloroquine diphosphate salt. SAR suggested that the presence of amide, sulphonamide and thiazole groups played a crucial role for enhancing the antimalarial activity [bib_ref] Design and synthesis of small molecular dual inhibitor of falcipain-2 and dihydrofolate..., Huang [/bib_ref]. Caridha and co-workers reported potent thiophene and benzene sulfonamides as antimalarial agents. Among these, bromohydrosulfonylacetamides 208 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to be promising growth inhibition of drug resistant P. falciparum W2 strain as well as low toxicity profiles against mammalian cell lines. Further exploration of 208 with variation in the thiophene and benzene ring substitutions may produce more potent PfCDK inhibitors. Cunico and coworkers have developed hydroxyethylpiperazine analogues and evaluated in vitro anti-malarial agents against a W2 Plasmodium falciparum clone. Compound 209 was found to be the most potent anti-malarial agent with IC 50 value of 4.8 mM against W2 Plasmodium falciparum clone almost as active as standard lopinavir. The SAR revealed that the presence of amine group on the phenyl ring influenced the anti-malarial activity. In addition, the presence of piperazine moiety was also an essential for increases the activity. The sulfonamide functional groups were bridged between the two bioactive analogues [bib_ref] Synthesis, antimalarial evaluation and molecular modeling studies of hydroxyethylpiperazines, potential aspartyl protease..., Cunico [/bib_ref]. (Ab) deposits, oxidative stress, dyshomeostasis of biometals, tauprotein aggregation are considered to be such pathophysiological factors . Unfortunately the medicines for the cure of AD and its progression are not discovered yet. But certain medicines are approved and prescribed for the AD patients for the temporary relief [bib_ref] The significance of the cholinergic system in the brain during aging and..., Schliebs [/bib_ref] [bib_ref] Bioinorganic chemistry of Alzheimer's disease, Kepp [/bib_ref]. In this part of the review article, sulfonamide nucleus is focused as a core substituent of Alzheimers agents for the development of drug [141e144]. Some of the sulfonyl or sulfonamides containing heterocycles represented as potential Alzheimer's agents are summarized in [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref]. Mutahir and co-workers performed novel biphenyl bissulfonamide derivatives as potent acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) agents. Among the tested compounds, compound 217 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to be the most potent activity against AChE (IC 50 2.27 ± 0.01 mM), whereas 218 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] exhibited the highest inhibition for BChE (IC [bib_ref] Apricoxib, a novel inhibitor of COX-2, markedly improves standard therapy response in..., Kirane [/bib_ref] 7.74 ± 0.07 mM). SAR studies revealed that both the 3,30dimethylbiphenyl functionality as well as benzyl moiety on nitrogens played a crucial role for the higher activity of compound 217. The higher activity of 218, bearing n-hexadecanyl moiety on nitrogens, is a similar trend as the case in AChE inhibition which could be attributed to the hydrophobic bulkiness of the n-hexadecanyl group. In addition, molecular docking studies were also performed for the analysis of the binding mode and hydrogen bonding interactions of compound 217 in both cholinesterases enzymes. Ligand binding within the active site of AChE was limited to hydrophobic interactions with Tyr334, Phe331, and Phe330 from anionic sub-site, Tyr70 from acyl pocket as well as Tyr121, Trp279, Asp276 and Phe228 from peripheral anionic site. Biphenyl fragment was engaged in more specific pep and CHep interactions with Tyr334. The arrangement of the most active compound 217 in the active gorge of AChE is shown in [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref]. Ligand binding within the active site of AChE was limited to hydrophobic interactions with Trp84, Phe330, and Phe331 from anionic sub-site, Phe290 from acyl pocket as well as Tyr121, Trp279 and Tyr334 from peripheral anionic site (PAS). Biphenyl fragment was engaged in more specific pep and CH-p interactions with Trp279, Phe331 and Tyr334. Oxygen atoms in sulfonamide groups might create weak H-bonds with hydroxyl group of Tyr70 or unionized form of Asp72. The arrangement of the most active compound 217 in the active gorge of AChE is shown in [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref]. And similar results were observed in case of docking to the active site of BChE. The binding of the tested compound 217 with AChE and BChE was mainly provided due to the presence of hydrophobic interactions. Summing up, it can be assumed that the binding of the tested compounds with AChE and BChE was mainly provided due to the presence of hydrophobic interactions. However, the obtained compounds are interesting starting point for their further development and synthesis of potent cholinesterase inhibitors. Structural modifications leading to increase of number of hydrogen bond donors and acceptors should augment the strength and specificity of binding to enzymes [bib_ref] Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase inhibitors: synthesis, biological evaluation and..., Mutahir [/bib_ref]. Yar et al. reported the novel potent pyridine 2,4,6tricarbohydrazide analogues as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) agents. Compound 219 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] exhibited the most potent activity against tested enzymes such as AChE (IC 50 50.2 mM) and BChE (IC 50 43.8 mM). Overall the compound 219 bearing phenyl group was found to be active against all these tested enzymes [bib_ref] Pyridine sulfonamide as a small key organic molecule for the potential treatment..., Riaz [/bib_ref]. On the other hand, Ulus et al. described acridine-sulfonamide hybrids as potent acetylcholinesterase inhibitor for the treatment of Alzheimer's disease. Compound 220 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] displayed superior activity against AChE with an IC 50 of 0.14 mM [bib_ref] Synthesis of novel acridine-sulfonamide hybrid compounds as acetylcholinesterase inhibitor for the treatment..., Ulus [/bib_ref]. Later, the same research group (Ulus et al.) continued the development of new type of alzheimer's agents in which, sulfonamid bearing tacrine derivatives were synthesized and evaluated for in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities. Compound 221 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to have the highest inhibitory activity on AChE with IC 50 value of 0.009 mM. This value is 220-fold higher than that of galantamine (IC 50 ¼ 2.054 mM). Compound 222 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] displayed the strongest inhibition of BuChE with IC 50 value of 2.250 mM. To elucidate SAR, sulfonamide group present on the para position at the phenyl ring showed good acetylcholinesterase activity (AChE that the presence of electron releasing moiety was crucial for the higher activity of 223 and EWGs inactive against the AChE and BChE enzymes. At the next level of investigation, the authors also performed the molecular docking analysis of potent compound 223 for detailed exploration of its binding pattern within the active sites of AChE and BChE. In addition, docking technique is considered efficient in accurately predicting binding mode of small molecules. The most potent compound 223 was showed detailed exploration of its binding pattern (2D and 3D binding mode of interaction) within the active sites of AChE and BChE. Compound 223 had two hydrogenbond interactions with the peripheral anionic site residue Tyr279 upon binding to AChE [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref]. Additionally, the docked complex of BChE with 223 showed that the hydrophobic patch residue Tyr332 and the catalytic triad residue Gly116 were involved in intra molecular hydrogen bonding [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref]. The docking results showed that 223 were capable of establishing two hydrogen-bond interactions with the peripheral anionic site (PAS) residue Tyr121 upon binding to AChE [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref]. Additionally, the docked complex of BChE with 6j (BChE-6j) showed that the hydrophobic patch residue Tyr128 and the catalytic triad residue Ser198 were involved in intramolecular hydrogen bonding [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] To elucidate the SAR, the presence of 3,4dihydroxy group in the B ring of the chalcone, produced more potent agent than the corresponding 4-hydroxy derivatives. Smaller electron donating groups (CH 3 , OH and NH 2 ) were more favoured than larger species such as OCH [bib_ref] Synthesis of water-soluble, topically effective, intraocular pressure-lowering aromatic/heterocyclic sulfonamides containing cationic or..., Scozzafava [/bib_ref] and EWGs such as NO 2 [bib_ref] Inhibitory evaluation of sulfonamide chalcones on bsecretase and acylcholinesterase, Kang [/bib_ref]. Recently, Wieckowska et al. reported the sulfonamide based piperidine hybrids as potent 5-HT6 receptor antagonist with a cholinesterase inhibitor. Among these, compound 228 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] was found to be the most potent agent against the 5-HT6 receptor (K b ¼ 27 nM), AChE and BuChE (hAChE: IC 50 ¼ 12 nM, hBuChE: IC 50 ¼ 29 nM). In a further drug development program, a novel class of multi-functional ligands were evaluated, in which compound 229 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] the best derivative from the series, represented an excellent starting point for the development of an effective treatment for AD [bib_ref] Novel multitarget-directed ligands for Alzheimer's disease: combining cholinesterase inhibitors and 5-HT 6..., Wię Ckowska [/bib_ref].
## Antileishmanial activity
Disseminated leishmaniasis has become an emerging infectious disease, mostly due to Leishmania braziliensis. L. braziliensis has caused both cutaneous and mucocutaneous leishmaniasis in several Latin American countries [bib_ref] Differential effects of antigens from L. braziliensis isolates from disseminated and cutaneous..., Leopoldo [/bib_ref]. Presently this parasitic disease causes morbidity and mortality, mainly in the developing world [bib_ref] Drug development for neglected diseases: a deficient market and a public-health policy..., Trouiller [/bib_ref]. Toxicity, high costs and development of drug resistance have become obstacles in the prevailing chemotherapic treatment [bib_ref] Combination therapy for visceral leishmaniasis, Van Griensven [/bib_ref]. Sodium stibogluconate (Pentostam ® ) and meglumine antimoniate (Glucantime ® ), the two pentavalent antimonial [Sb(V)] compounds, were first introduced in the 1940s and are being used for all forms of leishmaniasis through parenteral administration [bib_ref] Chemotherapy of leishmaniasis: past, present and future, Mishra [/bib_ref]. Therefore, drugs that are safe, inexpensive and easily available need to be developed immediately. Lead compounds are also now having taken important roles for the future treatment of this disease globally.
Sulfonamides, according to literature have shown versatile antileishmanial activity and have become a structural core in leishmanicidal therapy [157e160]. The sulfonamide group act as a chemical link that allows binding of other potential "active components" such as aromatic and heteroaromatic systems along with the demonstration of antiparasitic activity [161e163].
Marra et al. conducted a study aimed at the preparation and evaluation of potent novel 4-(1H-pyrazol-1-yl)benzenesulfonamide hybrids against the L. infantum and L. amazonensis strains. Compounds 230
(IC 50 ¼ 0.059 mM against L. infantum, IC 50 ¼ 0.070 mM against L. amazonensis) and 231 (IC 50 ¼ 0.065 mM against L. infantum, IC 50 ¼ 0.072 mM against L. amazonensis) [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed most potent activity against the tested L. infantum and L. amazonensis strains. In this case, both compounds 230 and 231 pyrazole baring sulfonamide groups were active for treating infections caused by these two Leishmania strains [bib_ref] 4-(1H-Pyrazol-1-yl)benzenesulfonamide derivatives: identifying new active antileishmanial structures for use against a neglected..., Marra [/bib_ref]. Borges and co-workers reported a new class of pyrazolyl benzenesulfonamide hybrids as potent antileishmanially active candidates against Leishmania amazonensis. Among these, compound 232 (IC 50 value is 6.7 mM) [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] was found to have the most potent activity against Leishmania amazonensis with IC 50 value higher than reference drug ketoconazole [bib_ref] Synthesis and in vitro evaluation of new benzenesulfonamides as antileishmanial agents, Borges [/bib_ref]. Gonz alez-Rosende et al. developed a new series of naphthalene-sulfonamide analogues as potent antileishmanial and trypanocidal inhibitors. Compound 233 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] displayed most potent inhibition on three Leishmania species entitled L. infantum (IC 50 ¼ 23.0 mM), L. amazonensis (IC 50 ¼ 42.9 mM) and T. cruzi (IC 50 ¼ 223.7 mM). In addition, compound 233 was found to be an excellent anti-T. cruzi candidate, and further clinical investigation could be useful in the development of new antichagasic drugs [bib_ref] Gonz alez-Rosende, In Vitro and in Vivo antileishmanial and trypanocidal studies of..., Galiana-Rosell O [/bib_ref]. The group of Palop reported diselenide containing sulfonamide derivatives, which exhibited in vitro leishmanicidal activities against Leishmania infantum intracellular amastigotes and THP-1 cells. Compound 234 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] emerged as the most active compound (IC 50 ¼ 2.8 mM), showing higher activity and much less toxicity against THP-1 cells than reference drug edelfosine. SAR studies, no clear-cut relationship was found but, mutually these results suggested that the sulfonamide scaffold could be a valuable linker for the connection of parent diselenide and para-fluoro phenyl ring attaching both sulfonamide ends [bib_ref] Novel hybrid selenosulfonamides as potent antileishmanial agents, Baquedano [/bib_ref]. In addition, Rodrigues et al. designed and developed chalcone-sulfonamised analogues as potent antileishmanial agents. Compound 235 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] was found to be the best profile against L. braziliensis promastigotes with IC 50 value of 3.5 mM. Moreover, the presence of benzylamino group extensively contributed to this activity. These results revealed the sulfonamide based methoxychalcone hybrids as lead compounds for designing new candidates for leishmaniasis treatment [bib_ref] Synthesis, biological evaluation and SAR of sulphonamide 4-methoxychalcone derivatives with potential antileishmanial..., Andrighetti-Frohner [/bib_ref].
## Tuberculosis activity
Tuberculosis is a highly infectious chronic deadly disease caused by a bacteria called Mycobacterium tuberculosis (MTB). This disease is a threat to the human life affecting lungs primarily (pulmonary TB) apart from other vital organs. Drug-resistant TB (DR-TB), multidrug-resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB) and totally drug resistant TB (TDR) are emerging now a day's which are completely resistant for the action of presently available standard drugs [bib_ref] Recent developments of coumarin-containing derivatives and their anti-tubercular activity, Hu [/bib_ref]. The infection of TB is so high that it has caused deaths of around 1.4 million and 10.4 million clinical cases all over globe as reported in 2015 [bib_ref] Quinoline: A promising antitubercular target, Keri [/bib_ref]. However the treatment of TB with the drugs such as Isoniazid (INH), Ethambutol (EMB0), Rifampicin (RIF) and Pyrazinamide (PZA) is observed to be highly effective for TB. Discovery of Rifampicin (RIF) have helped in obtaining handful of Anti-TB drug compounds to the humans. However, still a number of derivatives are to be explored to stop the activity of bacteria and further spreading of TB. [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed some of the sulfonyl or sulfonamides containing heterocycles as potential TB agents.
Shahul and co-workers reported aminoperidines with benzimidazole derivatives as potent anti-TB agents against Mtb and Mtb DNA gyrase. Compound 241 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] to be further exploited [bib_ref] High-throughput discovery of mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) inhibitors using..., Tan [/bib_ref]. Several sulfonyl-hydrazones were also tested for in vitro anti-TB agents against Mycobacterium tuberculosis-PtpB. Among all the synthesized molecules, compounds 247 (IC 50 ¼ 18 mM), 248 (IC 50 ¼ 21 mM), 249 (IC 50 ¼ 39 mM) and 250 (IC 50 ¼ 41 mM) [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed the most potent PtpB inhibitors.
The SAR suggested that the presence of EWGs (Cl, F and NO 2 ) on the phenyl ring of sulfonamide end enhanced the anti-TB properties [bib_ref] Sulfonyl-hydrazones of cyclic imides derivatives as potent inhibitors of the Mycobacterium tuberculosis..., De Oliveira [/bib_ref]. As the continuous search of new type of potent anti-TB agents, Reddy and co-workers designed and developed new sulfonamide based indole hybrids as potent anti-TB agents. Compound [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] was found to be the best inhibitor against MtbCM. The SAR showed that the indole containing an o-(RSO 2 NH) C 6 H 4 group at C-2 position and a sulfonamide moiety played a key role during its interaction with the active site of CM [bib_ref] A new route to indolesvia in sit-udesilylationeSonogashira strategy: identification of novel small..., Nakhi [/bib_ref]. At last, novel 4-aryl/alkylsulfonylmethylcoumarins hybrids were synthesized and screened for in vitro anti-mycobacterial activity against MTBH 37 Rv. Among these, compound 254 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed excellent anti-TB activity with MIC value of 0.78 mg/mL, eight fold more potent than EMB (MIC: 1.56 mg/mL) and PZA (MIC: 6.25 mg/mL) [bib_ref] Synthesis and in vitro antitubercular activity of 4-aryl/alkylsulfonylmethylcoumarins as inhibitors of Mycobacterium..., Jeyachandran [/bib_ref].
## Antiviral activity
Viruses are infectious agents affecting the life forms. They are responsible for causing various dangerous diseases like human immunodeficiency virus (HIV), hepatitis B and C viruses (HBV and HCV, respectively), severe acute respiratory syndrome (SARS), corona viruses (Middle east respiratory Syndrome, MERS); influenza (seasonal, pandemic), viral haemorrhagic fevers (Ebola), dengue, and chikungunya etc. These diseases have caused adverse impact on human health leading to unexpected illnesses and deaths, troubling day-to-day normal life activities. Viruses are the major cause for the emergence of newer pandemics e.g. H1N1 influenza, Ebola, and Zika virus etc. threatening the public health [bib_ref] Emerging infectious diseases in 2012: 20 years after the institute of medicine..., Morens [/bib_ref].
On the same hand, more than 60 antiviral drugs of diverse chemical classes have been approved by the FDA, mainly for the management of HIV, the hepatitis B and C, herpes and influenza A and B viruses and still many molecules are in various stages of clinical trials. But there is still a pressing need for the development of new drugs acting through several mechanisms and combat the viral resistance as viruses are constantly evolving [bib_ref] HIV drug resistance: problems and perspectives, Pennings [/bib_ref]. However it is always challenging for the medicinal chemists to develop newer drugs understanding unique biological features of viruses and treat the emerging viral disease in one or the other way without harming the host cells [bib_ref] Plant derived antivirals: a potential source of drug development, Mishra [/bib_ref]. [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] showed some of the sulfonyl or sulfonamides potent anti-viral agents.
In recent years, various sulphonamide based isoxazolidines hybrids were synthesized and evaluated for in vitro HIV- [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] was found to be the most potent activity against the single mutants Y181C and Y188L with EC 50 ¼ 0.428 and 0.675 mM, respectively, more potent than reference drug AZT. These results are expected to be helpful in the design of thiophenepyrimidine-based NNRTIs with more potent activity against HIV strains with RT mutations [bib_ref] Discovery of thiophene[3,2-d] pyrimidine derivatives as potent HIV-1 NNRTIs targeting the tolerant..., Kang [/bib_ref]. Zhang et al. explored pyridine-sulfonamides as potent Hepatitis C (HCV) NS4B inhibitor. Compound 267 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] has showed outstanding potency against the HCV 1b replicon, with an EC 50 ¼ 2 nM and a selectivity index of >5000 with respect to cellular GAPDH. The overall profile of compound 267 makes it a good aspirant for future drug development program [bib_ref] Structure-activity relationship (SAR) optimization of 6-(Indol-2-yl)pyridine-3-sulfonamides: identification of potent, selective, and orally..., Zhang [/bib_ref].
Several thiadiazole bearing sulfonamides analogues have also demonstrated promising antiviral activity against tobacco mosaic virus by the half leaf method explored by Yang et al. Compounds 268 (42%) and 269 (42%) [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] showed promising TMV inhibition compared to the reference drug Ningnanmycin (54%). The SAR, structural modification in the sulfonamide moiety has a wide impact on anti-viral activity of the compounds [bib_ref] Synthesis and antiviral activity of 5-(4-Chlorophenyl)-1,3,4-thiadiazole sulfonamides, Chen [/bib_ref]. Hu et al. developed and prepared a new class of chalcone-containing purines and benzenesulfonamide hybrids and tested for antiviral properties against TMV and CMV. Compound 270 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] was found to possess outstanding activity against TMV with the EC 50 value of 51.65 mg/mL, which was better than that of ribavirin (150.45 mg/mL). The SAR analysis showed that introducing EDGs at the 2-position of benzenesulfonamide aromatic rings and low steric hindrance group promoted antiviral properties. These findings indicated that chalcone derivatives were worthy of further research and development as templates for new antiviral agents [bib_ref] Antiviral properties and interaction of novel chalcone derivatives containing a purine and..., Zhou [/bib_ref]. Compound 271 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] was found to have potent (Ki ¼ 0.8 nM, IC 50 ¼ 1.5 mM) antiviral activity. Oral bioavailability of this compound ranged from 42% (rat) to 77% (dog) with t 1/2 ¼ 6 h [bib_ref] Structure-based design of sulfonamide-substituted non-peptidic HIV protease inhibitors, Skulnick [/bib_ref] [bib_ref] Structure-based design of nonpeptidic HIV protease inhibitors: the sulfonamide-substituted cyclooctylpyranones, Skulnick [/bib_ref]. Saturation of the 5,6-double bond in the pyrone ring led to the identification of a compound 272 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] with excellent binding affinity for the HIV protease (Ki values in the 0.05 nM) and excellent antiviral activity in cell culture, with significantly less ED 50 value of 0.95 mM [bib_ref] Cycloalkylpyranones and cycloalkyldihydropyrones as HIV protease inhibitors: exploring the impact of ring..., Romines [/bib_ref]. At last, the continuation of finding new class of potent coumarin-benzimidazole hybrids as potent anti-HCV activity by Hwu et al. Among these, compounds 273 (EC 50 ¼ 10.2 mM) and 274 (EC 50 ¼ 13 mM) [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] displayed excellent antiviral activity against chikungunya virus (CHIKV). The SAR revealed that the extension of the doubly conjugated uracilecoumarins to triply conjugated uracilecoumarinearenes by use of the -SO 2 linker was fundamental to their anti-CHIKV activity. Bezouracil derivatives 273 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] had better selectivity indexes compared to uracil 274 [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref] or thymine [bib_ref] Benzouracil-coumarin-arene conjugates as inhibiting agents for chikungunya virus, Hwu [/bib_ref].
## Carbonic anhydrase inhibition
Carbonic anhydrases (CAs) are a class of metalloenzymes containing zinc as the metal. The roles of these metalloenzymes are the interconversion of carbon dioxide and water to bicarbonate and proton maintaining the acid-base balance in tissues and blood. This enzyme is a multidomain protein containing CA subdomain situated outside the cell. It also possesses high CO 2 hydrase catalytic activity which is inhibited by CA inhibitors belonging to sulphonamide, sulfamate and sulfamide classes of compounds [bib_ref] Biochemical characterization of CA IX: one of the most active carbonic anhydrase..., Hilvo [/bib_ref]. Today around 15 different human CAs are known which are widely distributed in different tissues involving in different physiological process such as cell differentiation and proliferation, pH homeostasis, neurotransmission and pathologies like diuretics, epilepsy, glaucoma, obesity and cancer .
Sulfonamide is considered to be a significant moiety due to its diverse pharmacological activities [bib_ref] Carbonic anhydrase inhibitors and their therapeutic potential, Supuran [/bib_ref] and these have clinical use as carbonic anhydrase inhibitors (CAIs) primarily as diuretics and anti-glaucoma agents. Heterocyclic ring or the aromatic ring containing sulfonamide moieties as zinc binding group as tail approach afford CAIs possessing both high affinity and desired pharmacologic properties and have been already explored in literature [bib_ref] Sulfonamides containing coumarin moieties selectively and potently inhibit carbonic anhydrases II and..., Wang [/bib_ref] [bib_ref] Benzenesulfonamides incorporating bulky aromatic/heterocyclic tails with potent carbonic anhydrase inhibitory activity, Bozdag [/bib_ref].
Very recently, a family of sulfonamide based heterocycles hybrids were designed and biologically evaluated as potent carbonic anhydrase activity against hCA 11 and hCA 1V by Nocentini et al. Compound 275 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to have superior activity with IC 50 values of hCA 11 is 0.4 nM and hCA 1V is 20.5 nM. The SAR revealed that the presence of key functional elements such as pyrazole, isoxazole and sulfonamide functional moiety was beneficial for the enhancing carbonic anhydrase activity [bib_ref] Synthesis and biological evaluation of novel pyrazoline-based aromatic sulfamates with potent carbonic..., Nocentini [/bib_ref]. Khalifah and co-workers designed and developed potent iminothiazolidinone-sulfonamide hybrids and evaluated for their inhibitory effect against four relevant human (h) isoforms of carbonic anydrases (CAs, EC 4.2.1.1) I, II, IV and IX by a stopped-flow CO 2 hydrase assay [bib_ref] The carbon dioxide hydration activity of carbonic anhydrase, Khalifah [/bib_ref]. Compounds 276 and 277 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] showed the most potent active against hCAII with IC 50 values of KIs of 0.41 and 0.46 nM which may be due to the presence of EWGs (Cl and NO 2 ) for highly influencing the strongest inhibitors of hCAII [bib_ref] Synthesis, biological evaluation and computational studies of novel iminothiazolidinone benzenesulfonamides as potent..., Mahmood [/bib_ref].
In 2008, a series benzenesulfonamide linked 1,3,5-triazine hybrids were synthesized in good yield and tested for in vitro carbonic [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to be incorporated with amino, hydrazino, ethylamino, dimethylamino or amino acyl moieties, and showed promising CA activity but incorporation of bulky groups viz., n-propyl, n-butyl, diethylaminoethyl, piperazinylethyl, pyridoxal amine or phenoxy showed least CA activities against hCA I, II and IX inhibitors. Supuran et al. reported sulfonamides linked triazine moieties (279e283) [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] and tested for carbonic anhydrase transmembrane isoforms IX, XII and XIV over cytosolic isoforms I and II. The longer spacer compound (n ¼ 2) has shown more effectiveness as an inhibitor than the intermediate spacer (n ¼ 1), which in turn was more effective than the shorter spacer derivative (n ¼ 0). The short amino alcohol derivative 279 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] has also shown more effective than the bulkier compound 281 [bib_ref] Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I,..., Garaj [/bib_ref]. Mert et al. reported the new class of 5-amino-1,3,4-thiadiazole-2-sulfonamide containing pyrazole hybrids and tested for in vitro inhibitory activity against the isoforms of human cytosolic carbonic anhydrase I and II.
Compounds 284 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] for hCA I (K i ¼ 0.119 mM) and the compound 285 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] for hCA II (K i ¼ 0.084 mM) showed the highest inhibitory activity compared to the rest of the analogues [bib_ref] The synthesis of novel pyrazole-3,4-dicarboxamides bearing 5-amino-1,3,4-thiadiazole-2-sulfonamide moiety with effective inhibitory activity..., Mert [/bib_ref].
## Cannabinoid receptor agonists
Cannabinoid receptors 1 and 2 (CB 1 and CB 2 , respectively) were considered to be the members of the G protein-coupled receptor (GPCR) superfamily in the early of 1990's [bib_ref] Structure of a cannabinoid receptor and functional expression of the cloned cdna, Matsuda [/bib_ref] [bib_ref] Molecular characterization of a peripheral receptor for cannabinoids, Munro [/bib_ref]. Cannabinoid-1 receptor (CB1R) being most abundant neuroregulatory receptors present in the brain, peripheral organs such as adipose tissues, muscle and liver [bib_ref] International union of pharmacology XXVII. Classification of cannabinoid receptors, Howleff [/bib_ref] regulates feeding and appetite [bib_ref] Endocannabinoid signaling in the brain, Wilson [/bib_ref]. Whereas cannabinoid-2 receptor (CB2R) is mostly expressed in the immune system regulating immunity and neurodegeneration [bib_ref] Cultured rat microglial cells synthesize the endocannabinoid 2-arachidonylglycerol, which increases proliferation via..., Carrier [/bib_ref].
Compound 286 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was the best example of sulfonamide group claimed potent cannabinoid receptor agonist. In addition to this finding of new sulfonamides containing cannabinoid receptor drugs, scientists from AstraZeneca have also reported a potent sulphonamide based drug 287 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] acts as both CB 1 /CB 2 dual agonists for the administration of pain [bib_ref] Targeting cannabinoid agonists for inflammatory and neuropathic pain, Cheng [/bib_ref]. Very recently, Watson and co-workers from Pfizer have reported sulfonylbenzimidazole hybrids (288 and 289) [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] as selective CB 2 agonists and as potential analgesic agents devoid of the side effects associated with CB 1 agonists [bib_ref] Optimisation of a novel series of selective CNS penetrant CB 2 agonists, Watson [/bib_ref]. In 2012, Verbist and co-workers identified sulfonylbenzimidazole analogue 290 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] as potent CB 2 -receptor agonists. The compound 290 was found to have no analgesic effect which was demonstrated in pain models. Furthermore, to improve the metabolic stability and solubility, the same group optimized the compound 290 and led to the discovery of relatively polar and peripherally acting CB 2 agonists of compounds 291 and 292 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] [bib_ref] Aerssens, 5-Sulfonyl-benzimidazoles as selective CB 2 agonists-Part 2, Gijsen [/bib_ref]. Greig et al. designed and produced a new class of indole sulfonamides as potent cannabinoid receptor. Compounds 293 and 294 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] displayed outstanding potencies of 4 and 3 nM respectively, and showed good oral exposure and CNS penetration, making them highly versatile tools for investigating the therapeutic potential of allosteric modulation of the cannabinoid system [bib_ref] Development of indole sulfonamides as cannabinoid receptor negative allosteric modulators, Greig [/bib_ref]. The presence of sulfonamides functionalities has proved to be an extremely potent agonists at the hCB 2 receptor, compound 295 (EC 50 ¼ 5.1 nM) and 296 (EC 50 ¼ 7.0 nM) being the most potent hCB 2 receptor agents. These results inspired further development of in vitro profiling of sulfonamides on the hCB 1 receptor and on rat liver microsomes (RLM) [bib_ref] Rapid assessment of a novel series of selective CB 2 agonists using..., Ryckmans [/bib_ref]. Chang et al. developed pyrazole bearing sulfonamide hybrids and evaluated for potent cannabinoid-1 receptor antagonist. Compound 297 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to be most potent cannabinoid-1 receptor antagonist with K i values of 0.3 nM (hCB1R), 21.0 nM (hCB2R) and EC 50 of 3 nM (CB1R). Compound 297 is currently under development for treating obesity and the related metabolic syndrome [bib_ref] -(trifluoromethyl)phenyl)ethynyl)thiophene-2-yl)-1H-pyrazole-3-carboxamide as a novel peripherally restricted cannabinoid-1receptor antagonist with significant weight-loss efficacy..., Chang [/bib_ref].
## Anticonvulsant activity
Epilepsy is a family of neurological disorders caused due to disturbances in the nerve cell activity which is associated with progressively impaired cognition and function, brain damage and other neurological deficits. It has become a common neurological condition affecting 45e100 million people [bib_ref] New anticonvulsant agents, Malawska [/bib_ref]. Fortunately there is availability of antiepileptic drugs [AED's] which allow epileptic patients to maintain a normal and undisturbed life by having satisfied control and total relief of seizers [bib_ref] The epidemiology of epilepsy: the size of the problem, Bell [/bib_ref] [bib_ref] Novel approaches to epilepsy treatment, Sørensen [/bib_ref]. Further improvement in the development of antiepileptic drugs is a requirement for the complete prevention of epilepsy and its progression.
Farag and co-workers developed a set of compounds (298e300) [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] with pharmacophore hybrids and tested for picrotoxin (PIC)-induced convulsions (10 mg/kg, i.p.) in mice. Among them, compound 298 protected all animals better than the reference drug phenobarbital and did not show mortality. Other active compounds 299 and 300 also showed reasonable protections and they decreased the mortality rate up to fifty percent. In continuation, the authors have further modified benzothiazole pharmacophore by introducing sulfonamide group and tested those molecules for their anticonvulsant activity using MES, sc-PTZ seizure tests in swiss albino mice [bib_ref] Synthesis and anticonvulsant activity of sulfonamide derivativeshydrophobic domain, Siddiqui [/bib_ref]. Compounds 301 and 302 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] were found to be the most active in both seizure tests at variant doses and were neurotoxic at the higher doses of 300 mg/kg as similar to standard drug carbamazepine [bib_ref] Synthesis of benzothiazole semicarbazones as novel anticonvulsants-The role of hydrophobic domain, Siddiqui [/bib_ref].
## Anticancer activity
Cancer is been universally known as a disease or a group of diseases causing death. It is found to exist all over the world [bib_ref] Synthesis of potent and selective inhibitors of Candida albicans N-myristoyltransferase based on..., Yamazaki [/bib_ref]. Cancer is meant to be a bunch of cells originated from a single cell due to its uncontrolled growth and rapid proliferation properties [bib_ref] Clinical implications of the p53 tumor-suppressor gene, Harris [/bib_ref]. The problem with the drugs is that it is unable to differentiate between normal and cancerous cell type leading to several serious side effects [bib_ref] Recent advances in the development of dual Topoisomerase I and II inhibitors..., Salerno [/bib_ref]. Development of anticancer theraupetic agents has become a challenge for the medicinal chemists. But a continuous effort is being carried in this area of research to save millions of lives.
In 2017, Nitin and co-workers designed and developed a new class of benzothiazole derived methyl sulfonyl hybrids as potent anticancer agents. Among them, some of the compounds showed superior anticancer activity against human cervical HeLA cell lines. Compounds 303 and 304 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] were extensively inhibiting to the cell growth and GI 50 values were found to be 0.22 and 0.6 mM respectively. The SAR studies revealed that the presence of EWGs (NO 2 ) on the phenyl ring and two sulfonyl groups in the analogues increased the anticancer activity. In addition, the search of novel class of potent sulfonamides bearing hybrids, Ibrahim and coworkers reported isatin-pyrazole benzenesulfonamide derivatives as potent CA inhibitors. Compounds 305 and 306 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] showed . Some of the sulfonyl or sulfonamides containing heterocycles as potential TB agents. good carbonic anhydrases inhibition to the cell lines hCA IX (K i ¼ 15.7 and 7.4 nM, respectively) and hCA XII (K i ¼ 3.7 and 5.4 nM, respectively). The SAR studies suggested that the presence of EWGs (NO 2 ) on the phenyl ring to increased the activity and sulphonamide played a major role in the enhancing the CA activity [bib_ref] Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII, Ibrahim [/bib_ref]. In 2014, Tiangong and co-workers developed compound (307 and 308) [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] , a new class of styrylsulfonyl-methylpyridines hybrids with the anticancer activity against A2780, MCF7 and HCT-116 cell lines by using MTT assay. Compound 307 was found to have the best antitumor activity in a xenograft HCT-116 colon cancer model with GI 50 value of 0.570 mmoL/L. Compound 308 displayed potent antitumor efficacy in the xenograft A2780 with GI 50 value of 0.007 mmoL/L. The SAR studies revealed that compounds containing 2,4,6-trimethoxy and two sulfoonamide group favourably increased the anticancer activity. The presence of EDGs was necessary for to increases the activity and also the presence of pyridine ring in the derivatives increased the oral bioavailability and solubility of the synthesized compounds [bib_ref] Discovery of (E)-3-((styrylsulfonyl)methyl)pyridine and (E)2-((Styrylsulfonyl)methyl)pyridine derivatives as anticancer agents: synthesis, structure-activity relationships,..., Tiangong [/bib_ref]. R. Pingaew et al. [bib_ref] Novel 1,4-naphthoquinone-based sulfonamides: synthesis, QSAR, anticancer and antimalarial studies, Pingaew [/bib_ref] reported a new class of sulfonyl containing thiosemicarbazone and tetrahydroisoquinoline hybrids as potent anticancer agents. Some of the synthesized thiosemicarbazone analogues showed good cytotoxic potency against MOLT-3 cell lines with IC 50 value of 2.13 mg/mL. Compound 309 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to have the best cytotoxic activity against HuCCA-1 and HepG2cells with IC 50 values of 31.00 mg/mL and 10.50 mg/mL respectively. The SAR displayed the presence of EDGs (OMe) and sulfonyl functionalities increased the cytotoxic activity [bib_ref] Synthesis and cytotoxicity of novel N-sulfonyl-1,2,3,4-tetrahydroisoquinoline thiosemicarbazone derivatives, Ratchanok [/bib_ref]. At last, in 2014, Jun and co-workers designed and produced novel 1-sulfonyl indolines analogues in good yields and tested for their antiproliferative activity against various cancer cell lines. Among them, compounds 310 and 311 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] showed good cytotoxicity with IC 50 values in the range of 0.055e0.105 mM and 0.039e0.112 mM, respectively against four human cancer cell lines HCT116, PC3, HepG2 and SK-OV-3. The SAR demonstrated that the presence of EDGs (OMe) significantly increased the activity and the presence of oxazole moiety also influenced the antiproliferative activity [bib_ref] Synthesis and structure-activity relationship of 4-azaheterocycle benzenesulfonamide derivatives as new microtubule-targeting agents, Jun [/bib_ref].
## 5-ht6 receptor
Romero and co-workers reported a new class of thiazolecontaining tryptamine hybrids as potent 5-HT6 receptor agonist. Compound 312 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to have good Ki value of 2.2 nM against 5-HT6 receptor. Compound 312 displayed partial agonistic property in cAMP functional assay with pKi value 6.96 in HEK-293 F cell line. Further in vivo studies indicated that compound 312 effectively improved recognition memory by combined modulation of cholinergic as well as glutamatergic neurotransmission in rats [bib_ref] Efficacy of selective 5-HT6 receptor ligands determined by monitoring 5-HT6 receptor-mediated cAMP..., Romero [/bib_ref]. Hayat et al. found that the benzothiazole-sulfonamide hybrids displayed powerful 5-HT6 receptor antagonists against HeLa cell line. Compounds 313 (IC 50 ¼ 14 mM) and 314 (IC 50 ¼ 3.9 mM) bearing 4-isopropylphenyl and 1-naphthylsulfonamide group at C-6 position of benzothiazole ring, respectively, showed promising inhibition of 5-HT6 receptor antagonists against HeLa cell line. Prio et al. patented various N-phenyl-2,3dihydroimidazol [2,1-b]thiazole-5-sulfonamide derivatives as 5-HT6 receptor ligands. Numerous substitution on phenyl as well as imidazole ring provided suitable ligands and some of them displayed excellent affinity for 5-HT6 receptor. Particularly compounds 315, 316 and 317 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] showed good Ki value of 8.4, 16.9 and 5.4 nM, respectively. At last, Liu et al. explored a number of sulphonamide based benzothiazole hybrids as 5-HT6 receptor agents. All the produced hybrids possessed nanomolar range affinity for 5-HT6 receptor. Among them, compound 318 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] was found to be the best potent 5-HT6 receptor with Ki value of 500 nM. In addition, in vitro and in vivo studies of this compound 318 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] may provide promising leads in future.
## Miscellaneous
Yang et al. designed and developed a class of potent quinolinebased ALDH1A1 inhibitors. The pharmacokinetics (PK) study demonstrated that compounds 319 and 320 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] had realistic drug exposure via po administration and would be suitable for in vivo proof of concept animal studies for a better understanding of the physiological and pathophysiological actions of this enzyme [bib_ref] Discovery of orally bioavailable, quinoline-based aldehyde dehydrogenase 1A1 (ALDH1A1) inhibitors with potent..., Yang [/bib_ref]. Very recently, Iqbal and co-workers found the sulfonamide bearing sultams analogues as potent alkaline phosphatase (bTNAP and bIAP) inhibitors. Among these, compound 321 [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] containing a p-nitro substituent was found to be the best active inhibitor with IC 50 value of 0.11 ± 0.005 mM. This examination highlighted the significance of the presence of EWGs on the para position of phenyl ring for effective bTNAP inhibition. The presence of NO 2 on the para position of phenyl ring 321 was found to be very efficient and selective inhibitor of bTNAP over bIAP. Moreover, the homology built models were then used for the docking studies in order to rationalize the most probable binding interactions of inhibitors with the enzyme. Compound 321 was the most active bTNAP inhibitor, the sulfonamide group found to be oriented towards the Zn 2þ metal ions. [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] displayed the detailed binding site interactions of most active bTNAP inhibitor of compound 321.
Compound 321 was the only compound in which not the sulfonamide group, but the oxygen atom of the p-nitro group was in direct contact (2.1 Å) with the Zn 2þ ion of the active site, since 321 is the most active bTNAP inhibitor, this interaction might be responsible for exceptional inhibitory activity observed for this compound. The presence of a sulfonamide group, as a zinc binding function, is suggested to be the most prominent structural feature in the design of potent AP inhibitors [bib_ref] Facile dimethyl amino group triggered cyclic sulfonamides synthesis and evaluation as alkaline..., Bhatti [/bib_ref]. [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] which only lacked the chloro at 5-position, was observed to be seven-fold decline in the activity. Another compounds 324 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] lacked chloro at position 4 was perceived to be nine times decline in the activity. The SAR revealed that the decrease of chloro substitution from tri-to di-substituted decreased the activity. It is worth displaying in that the synthesized hybrids of biologically active analogues such as oxadiazole ring, sulfone group, hydrazide moiety and aryl rings cordially played their role in exhibiting the activity.
Compound 322 was identified as a lead compound for bglucuronidase inhibitory activity and may be used for further research for finding a powerful inhibitor [bib_ref] Synthesis and in silico studies of novel sulfonamides having oxadiazole ring: as..., Taha [/bib_ref]. Very recently, Iqbal and coworkers developed a class of chalcone-sulfonamide hybrids as potent alkaline phosphatase inhibitors. Among them, compounds 325 and 326 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] showed maximum inhibition of human and rat e5ʹNT with IC 50 values of 0.26 and r5ʹNT with 0.33 mM, respectively. The SAR studies suggested that the presence of EDGs at meta position of phenyl ring displayed greater inhibition. Likewise, the presence of di-substituted bulky electron donating group i.e., methoxy group at 3/4 position showed greater inhibition of~161 fold higher than that of the standard compound sulfamic acid. The most potent inhibitor of 325 and 326 were analyzed in the active pocket of enzyme where its docking poses elaborate the presence of five strong hydrogen bonds with various amino acid residues. Compound 4e also showed strong electrostatic interactions. The two zinc ions within active pocket of h-e5ʹNT showed interaction with nitrogen of sulfonamide moiety by forming a metal acceptor phenomenon [bib_ref] Synthesis, characterization and biological evaluation of novel chalcone sulfonamide hybrids as potent..., Ejaz [/bib_ref]. In 1997, the FDA approved compound Delavirdine (327) [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] was found to be the second NNRTI agent authorized for treatment of HIV-1. Further, crystallographic analysis confirmed that methyl-sulfonamide group at indole ring was essential for enhancing the activity [bib_ref] Unique features in the structure of the complex between HIV-1 reverse transcriptase..., Esnouf [/bib_ref]. Moretto et al. identified a novel pyridothiophene inhibitor of PTP1B with a K i value of 0.370 mM [bib_ref] Bicyclic and tricyclic thiophenes as protein tyrosine phosphatase 1B inhibitors, Moretto [/bib_ref]. The X-ray co-crystal structures of compounds 328 and 329 [fig_ref] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents [/fig_ref] showed one of the sulphonamide oxygens hydrogen bonded to the backbone nitrogen of Gly259 and the other entered into interactions with Arg24 and Arg254 through bridging water molecules [fig_ref] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids [/fig_ref]. These interactions could increase the inhibitor activity up to 25-fold more, but would not bring selectivity over TCPTP. In 2006, Klopfenstein and co-workers developed compound 329 as PTP1B inhibitors with 2-fold selectivity over TCPTP, in which the sulfamic acid moiety picked up hydrogen bonding interactions with Arg24, Arg254, and Gln262 (2F6Z, [fig_ref] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties [/fig_ref] [bib_ref] ,3,4-Tetrahydroisoquinolinyl sulfamic acids as phosphatase PTP1B inhibitors, Klopfenstein [/bib_ref].
With the aim at further investigating the diverse chemical space, the introduction of imidazopyridine ring as a scaffold led to the discovery of two novel series of imidazopyridinylthioacetanilides hybrids. Among these, compounds 330 and 331 (EC 50 ¼ 0.75 mM and 0.21 mM, respectively) [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] were identified as the most potent inhibitors in suppressing HIV-1 replication. These compounds 330 and 331 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] had higher anti-HIV-1 potency compared to reference drug dideoxycytidine [bib_ref] Arylazolyl(azinyl)thioacetanilides. Part 10: design, synthesis and biological evaluation of novel substituted imidazopyridinylthioacetanilides..., Li [/bib_ref]. Later, Kim et al. designed and developed sulfonamide containing hydroxylated chalcones 332 and 333 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] with potential inhibition of trans-sialidase enzyme which was demonstrated by IC 50 values 0.9 and 2.5 mM [bib_ref] Development of new and selective trypanosoma cruzi trans-sialidase inhibitors from sulphonamide chalcones..., Kim [/bib_ref]. In continuous search of potential sulfonamide based chalcone hybrids as potent drugs against some diseases causing pathogens, in 2010, El-Ayache [bib_ref] Novel bisarylsulfonamides and aryl sulfonimides as inactivators of plasminogen activator inhibitor-1 (PAI-1), El-Ayache [/bib_ref] reported polyphenol bearing two polyphenolic moieties separated by a bisarylsulfonamide 334 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] moiety was essential for the activity and the substitution at the benzylic methylene was detrimental to the inhibitory activity. Also, the presence of ortho substituent on the aromatic ring of the benzyl group especially trifluoromethyl group enhanced the Eg5 inhibitory activity [bib_ref] Substituted benzimidazoles: a novel chemotype for small molecule hKSP inhibitors, Lahue [/bib_ref]. Finally, Prachayasittikul and co-workers found triazoles derived sulfonamide analogues as potent aromatase inhibitory activity. Compound 337 [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref] bearing 6,7-dimethoxy substituents on the isoquinoline ring showed the most potent aromatase inhibitory activity (IC 50 ¼ 0.2 mM) without affecting normal cell. The SAR suggested that the lipophilic effect of dimethoxy groups enhanced the activity of compound 337. In addition, molecular docking studies were performed the most active compound 337 and mode of binding interaction of compound 337 was revealed that, the investigated triazoles could closely engage the active site of aromatase through the interactions of hydrophobic, pp stacking and H-bonding. Furthermore, hydrophobic interactions with Arg115 were observed 2D-ligand protein interaction [fig_ref] Figure 4: Sulfonyl of sulfonamides as antimicrobial agents [/fig_ref]. The compound 337 was arranged in such a way to appropriately form hydrogen bonding of the sulfonyl group with the amino group of Ala306 and Ala307 as well as hydrogen bonding of oxycoumarinyl moiety with Ser119. These hydrophobic and hydrogen bonding interactions were suggested to play pertinent roles contributing to the most potent activity of compound 337. Interestingly, isomeric coumarinyl and naphthalenyl triazoles play crucial roles in exerting more potent aromatase inhibitory activity than other tested compounds. This could be attributed to their binding interactions with the aromatase enzyme. The molecular requirements for the most potent triazole inhibitor 337 which contains 6,7-dimethoxy groups, 7-coumaryloxymethyl at position 4 of the triazole ring, and m-substitution of triazole and sulfonyl moieties on the phenyl ring. Such structural features were essential for engaging in hydrophobic, p-p stacking and H-bonding interactions with the aromatase enzyme, particularly, hydrogen bond forming with Arg115 and Ser119 [bib_ref] Synthesis and molecular docking of 1,2,3-triazole-based sulfonamides as aromatase inhibitors, Pingaew [/bib_ref].
# Conclusion
This review updates and summaries the importance of the sulfonyl or sulfonamide based scaffolds (S VI based moieties) in bioactive compounds. As described above, sulfonamide based hybrids have huge range of biological activities such as antimicrobial, anti-diabetic, anti-inflammatory, anti-malarial, anti-tubercular, antiviral, Alzheimer's activity, anti-convulsant, anti-cancer, antitubercular and other activities. Theoretically, sulfonyl or sulphonamide (S VI moieties) based drugs possess the immense potential therapeutic values on diversity of drug targets. The SAR based work will probably continue to play an important role to further optimize the full potential of sulfonamide hybrids. Many of these potential drugs are not yet in clinical trials, but emphasize the exigency in the need for their further derivatization to provide an opportunity for managing therapeutic values more efficiently and with greater efficacy in the future. In addition, these biological agents with promising activity and well-defined mechanisms of action can be considered valuable candidates as prototypes in the design and development of novel and more effective synthetic compounds based potent inhibitors.
[fig] Compound 126: Fig. 4) was found to be antimicrobially active with MIC values in the range of 15.5e31.25 mg/mL. Compounds 125 and 127 (Fig. 4)showed reasonable antimicrobial activity with MIC values in the range of 31.25e62.5 mg/mL. The SAR studies revealed that the presence of strong EWGs such as -F, -Cl, and -NO 2 showed superior antimicrobial activities compared to the EDGs[72]. Various sulfonamide based analogues were synthesized and tested for their in vitro antimicrobial agents by Bhusari et al. Among these, compounds 128e130(Fig. 4)displayed superior antimicrobial agents against the tested microbial pathogens[73]. Subudhi et al. developed a class of novel sulphonamide based analogues and evaluated for their in vitro antimicrobial activities. Compound 131(Fig. 4)showed superior antimicrobial activity against the S. aureus (zone of inhibition 18 mm), E. faecalis (zone of inhibition 23 mm), E. coli (zone of inhibition 18 mm) and P. aeruginosa (zone of inhibition 17 mm). The preliminary SAR study revealed that the [/fig]
[fig] Figure 1: Some of the sulfonyl or sulfonamides containing heterocycles as potential antimicrobial agents. [/fig]
[fig] Figure 2: Some antimicrobial activities of potent sulfonyl or sulfonamides hybrids. [/fig]
[fig] Figure 3: Sulfonyl of sulfonamides with antimicrobial properties. [/fig]
[fig] Figure 5: Some representative sulfonyl of sulfonamides as potent anti-diabetic agents. [/fig]
[fig] Figure 6: Sulfonyl of sulfonamides as antidiabetic agents. [/fig]
[fig] In 2013 ,Figure 7: Ragab et al. prepared and evaluated some novel 1,3Docked pose of compound 139 (turquoise colour) showing hydrogen bond interactions (red dashed lines) with amino acids in the binding site of PPARg. The ligand and amino acids are represented as stick and thin tube model. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) [/fig]
[fig] Figure 8: Sulfonyl of sulfonamides as anti-diabetic agents. Korupolu et al. reported a structural investigation of sulfonamide hydrazones as potent anti-inflammatory agents. Among all the synthesized analogues, compounds 181 and 182 (Fig. 14) showed superior anti-inflammatory activities with IC 50 values of 8.9 and 8.4 mM respectively. Compound 181 was found to be the strongest and the most selective COX-2 inhibitor among the fluorinated derivatives. SAR suggested that the presence of trifluoromethyl group at para position in compound 181 showed good selectivity with COX-2 inhibition activity [115]. Recently, Abdellatif and co-workers reported the synthesis and anti-inflammatory activity study of sulfonamides based imidazolone analogues as selective COX-2 inhibitors. Based on in vitro evaluation, compounds 183 and 184 (Fig. 14) displayed excellent COX-2 potency with IC 50 values of 0.42 and 0.62 mM respectively and the most COX-2 selective indexes as S.I. values of 10.76 and 10.87 respectively. SAR suggested that the presence of EWGs on the phenyl ring influenced the anti-inflammatory activity[116]. [/fig]
[fig] Figure 9: Sulfonyl of sulfonamides as anti-diabetic agents. [/fig]
[fig] 2. 5: Alzheimer's disease (AD) Alzheimer's disease (AD) is a neurodegenerative disorder featured with cognitive dysfunction and memory lapse which accounts for the major dementia cases. According to the present estimation, about 45 million people are going through this disease worldwidely and it may reach up to 131 million by 2050 as per the documentation if left untreated [133e135]. Actual etiology for the AD progression is not known yet but a number of pathophysiology factors are believed to be responsible for the progression of this disease. Deficits of acetylcholine (Ach), inflammation, b-amyloid [/fig]
[fig] 1: replication by Loh et al. Compounds 261 (IC 50 ¼ 93 mM against HIV-I and IC 50 ¼ 91 mM against NL4.3) and 262 (IC 50 ¼ 75 mM against HIV-I [/fig]
[fig] Figure 40: Sulfonyl or sulfonamides with diverse pharmacological properties. [/fig]
[fig] Figure 42: X-ray crystal structures of PTP1B/compound complex (Fig. 42). PTP1B/5 (2B07); (Fig. 43) PTP1B/6 (2F6Z) The residues related to selectivity are labeled with black words and coloured by cyan. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) [/fig]
[fig] Figure 43: X-ray crystal structures of PTP1B/compound complex (Fig. 42). PTP1B/5 (2B07); (Fig. 43) PTP1B/6 (2F6Z) The residues related to selectivity are labeled with black words and coloured by cyan. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) [/fig]
[fig] Figure 44: Sulfonyl or sulfonamides showed diverse biological properties. [/fig]
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Worm tubes as conduits for the electrogenic microbial grid in marine sediments
## This pdf file includes:
Authors note Authors note: C. Reimers' lab has found in continuing work beyond reference (10) Be gave distinct signals demonstrating rapid, nonlocal particle exchange to depths > 10 -12 cm. Particle mixing coefficients (D B ) in the surface 4 cm ranged seasonally from 3.9 -199 cm 2 yr -1 , with the lowest rate during Feb 2010, an annual median of 16 and an average of 42 cm 2 yr -1 .
The lowest rates occurred during winter and spring, and the highest in the fall (data not shown). from the tube (e.g., . |
A case report of bladder and intestinal endometriosis, and the relationship between sex hormone receptor expression and PIK3CA mutation analysis
Background: Extragonadal endometriosis is a rare condition, and its disease manifestation and long-term prognosis have not been elucidated. We report an extragonadal endometriosis case controlled by drug therapy for 14 years with analysis of the sex hormone receptor expression and PIK3CA mutation.Case presentation:The patient was diagnosed with bladder endometriosis at age of 30 years, and underwent bilateral nephrostomy and GnRHa therapy with add-back therapy. The patient was switched to dienogest therapy at age 35 and had hematuria and bloody stools at age 38. PET-CT revealed a 6-cm mass in the bladder with fluorodeoxyglucose accumulation and the diagnosis of endometriosis in the bladder, sigmoid colon, and cecum was confirmed after the biopsy result. The lesion's tubular structures were positive for the estrogen receptor, but only 30% positive for the progesterone receptor, and the H1047R mutation in PIK3CA was found in tubular structures of the bladder lesion. GnRHa therapy caused the tumors to shrink.Conclusion:Decreased progesterone receptor expression and oncogenic mutations may influence the course of less common and rare site endometriosis. Rare site endometriosis often requires long-term hormone therapy, and management should be tailored to the patient's life stage, keeping in mind complications, such as decreased bone density.
# Background
Endometriosis occurs in 10% of sexually active women, 50% of infertile women, and 70% of women suffering from pelvic pain, and requires long-term management. Endometriosis is exacerbated by estrogen and ameliorated by progesterone. Therefore, hormone therapy using gonadotropin releasing hormone agonist (GnRHa) or gonadotropin releasing hormone antagonist, or progesterone therapy using dienogest is recommended as standard therapy. Recent molecular analysis has revealed that deep endometriosis and ovarian endometriosis produce oncogenic mutations in many cases. Endometriosis rarely occurs in extragonadal organs, such as the bladder and intestinal tract, causing organ-specific symptoms. reported that urinary tract endometriosis was present in 52.6% of patients suffering from deep infiltrating endometriosis. In our literature search, no comprehensive reports on this condition were found except several case reports that included rather Open Access *Correspondence: [email protected] Department of Obstetrics and Gynecology, Kindai University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-sayama, Osaka 589-8511, Japan insufficient discussion on the optimal treatment and the related molecular analysis.
We report an extragonadal endometriosis case controlled by drug therapy for 14 years with analysis of the sex hormone receptor expression and PIK3CA mutation. The course and study of this case will aid in understanding this rare condition.
## Case presentation
The studied patient is currently 42-year-old, G0P0. Family history, Nothing noteworthy. Her menstrual period started at age of 13 years. Dysmenorrhea was diagnosed at age of 15 years. At age of 16 years, she had a fever, lower abdominal pain, and fullness of the abdomen. Computed tomography (CT) showed ovarian swelling, and laparotomy was performed. Endometriosis and an infected endometriotic cyst were identified in the abdominal cavity; therefore, ovarian cystectomy and lavage drainage were performed. She visited our hospital at age of 29 years with increased menstrual pain. Low-dose estrogen/progestin (LEP) and non-steroidal anti-inflammatory drugs (NSAIDs) were administered. However, she experienced strong abdominal pains and underwent CT scan that revealed bilateral hydronephrosis. The blood test indicated white blood cells (WBC) 12.8 × 10 3 /uL (reference 3.3-8.6), C-reactive protein (CRP) 20.0 mg/dL (reference 0-0.1), blood urea nitrogen (BUN) 16 mg/dL (reference 8-20), creatinine (CRE) 1.02 mg/dL (reference 0.46-0.79) and urinary occult blood 1 + . The patient was manged by insertion of intravesical catheter and antibiotic. When the renal function improved and inflammation subsided the urinary catheter removed and the patient was discharged. Magnetic resonance imaging (MRI) performed after discharge revealed bladder wall thickening, and bladder endometriosis was diagnosed. At three months, CT scan showed that the hydronephrosis was worsened. A right ureteral stent was placed and the left ureteral stent placement was not possible. The patient showed no improvement in hydronephrosis even after stent implantation, and developed left abdominal pain. She had a fever of 39.0-39.9 °C with WBC 10.4 × 10 3 /uL, CRP 21.5 mg/dL, BUN 28 mg/dL and CRE 3.50 mg/dL. Acute pyelonephritis due to left ureteral obstruction and postrenal renal failure were suspected and a left nephrostomy was performed. However, right hydronephrosis and renal function did not improve, and one month after the left nephrostomy, the right nephrostomy was performed. Later, the renal fistula catheter was regularly replaced. Regarding endometriosis, the GnRHa therapy (leuprorelin acetate 1.88 mg/month) was continued with hormone add-back of conjugated estrogen 0.625 mg every other day until age of 35 years, when the therapy was changed to dienogest 2 mg/day. Her pelvic MRI at age of 36 years revealed only some irregularities of the bladder wall.
At age of 38 years, she had hematuria and bloody stools, and MRI revealed 6 cm-sized mass in the bladder and 1.8 cm-sized mass in the cecum. Positron emission tomography and computed tomography (PET-CT) indicated standardized uptake value (SUV) max of 13.16 in the bladder tumor, and 3.43 in the cecum mass. Malignancy caused by endometriosis was suspected and a cystoscopic guided biopsy was performed that showed the tumor in the bladder was endometriosis . Colonoscopy revealed tumors in the cecum and sigmoid colon which were demonstrated by biopsy to be endometriosis ; endometriosis with tumor formation or polypoid endometriosis was diagnosed. Immunohistochemistry showed that the bladder and intestinal endometriotic lesions were . For progesterone receptor (PGR) expression, the lesions were positive in the interstitial parts, but only 30% in the tubular parts. Using FFPE specimens of lesions in the bladder, cecum, and sigmoid colon, the tubular parts were collected by laser microdissection according to the method described in the previous report, and three hot spots of PIK3CA (E542K, E545K, and H1047R) were examined by droplet digital PCR. The H1047R mutation was found only in the bladder lesion that was the largest.
As treatment, the surgical option was pelvic exenteration that was judged too invasive; therefore, the drug therapy was continued. Based on the history and immunostaining results, the lesion was judged progesteroneresistant but estrogen-dependent; therefore, GnRHa (leuprorelin acetate 1.88 mg) was administered once every 4 weeks for the first 6 months, resulting in a significant reduction of the intravesical mass volume confirmed by pelvic MRI. Later, GnRHa was administered once every 6 weeks, and the total number of administrations to date is 38 GnRHa injections. Bloody stool disappeared, and hematuria rarely appeared. Bone density is gradually decreasing, but no osteoporosis observed.
# Discussion
Bladder endometriosis, causing organ-specific symptoms, is observed in approximately 1% of all endometriosis cases, and the symptoms include dysuria and hematuria. Knabben et al. reported that 68.8% of patients with bladder endometriosis have urinary symptoms. In the present case, bilateral hydronephrosis was observed, probably due to the lower ureter stenosis caused by endometriosis. In urinary tract endometriosis with hydronephrosis, ureteral stent placement is to be used only as adjunct during surgery. Therefore, ureteral stent placement did not improve hydronephrosis, and bilateral nephrostomy was eventually required in our case. In surgery for bladder endometriosis, the lesion should be removed without residue. However, in our case, the bladder lesion is so widespread that surgical urinary tract reconstruction is considered difficult; therefore, bilateral renal fistula is maintained.
There have been multiple case reports showing that PET-CT reveals fluorodeoxyglucose (FDG) accumulation associated with endometriotic lesions, while there is a single report that found no prospective FDG accumulation in 10 endometriosis patients examined preoperatively. In our case, a remarkable increase in intravesical mass was observed during the treatment course, resulting in so-called polypoid endometriosis, and significant accumulation of FDG was observed. To the best of our knowledge, this is the first report of PET-CT findings for polypoid endometriosis, which suggest that PET-CT may not be useful in differentiating tumor-forming polypoid endometriosis from malignant tumors.
In our case, the bladder lesion was exacerbated after switching to dienogest therapy. In the biopsy specimen at that time, PGR expression was observed only in approximately 30% of the endometriotic tubular structures , which might explain their resistance to the progesterone preparation. In endometriosis, progesterone resistance can be caused by molecular mechanisms, such as gene polymorphisms, altered microRNA expression, and epigenetic modifications of PGR. In our case, tubular structures with and without PGR expression were found distinctively adjacent to each other, that may suggest the methylation of the PGR promoter. On the other hand, ER expression found in 100% of the tubular structures may explain the successful GnRHa therapy. Thus, in endometriosis examining the expression of the sex hormone receptor by biopsy is considered useful for selecting the optimal hormone therapy.
Sequencing analysis of genes in deep endometriosis and endometriotic cyst revealed that oncogenic mutations frequently occur in endometriosis. Endometriotic lesions containing genetic mutations found at multiple distant sites were reported to be identical clones. Similarly, in our case, the endometriosis lesions in the bladder and intestinal tract are considered clones; however, the mechanism of this phenomenon comparable to cancer metastasis is unknown. To the best of our knowledge, this is the first report of gene mutation analysis in endometriosis observed in so-called extragonadal organs namely the bladder and intestine. The oncogenic mutation of PIK3CA was only detected in the bladder lesion, probably due to its lesion size and marked FDG uptake. It should be noted that oncogenic mutation frequently found in endometriosis does not immediately indicate susceptibility to carcinogenesis, and that the malignant transformation of bladder and ureteral endometriosis is extremely rare. There is a review report that driver mutations in endometriosis are not necessarily synonymous with malignancy or precancerous lesions, and in light of this, the PIK3CA mutation observed in this case may be a passenger mutation rather than a driver mutation.
GnRHa therapy for more than 6 months accompanies bone density decrease. Our patient has been on GnRHa therapy for 11 years, except for 3 years on dienogest. The add-back therapy with estrogen performed during the first 6 years and every 6 weeks during the recent 5 years may help reduce the side effects of GnRHa. Currently, her bone density level is in the normal range, but close continued monitoring required. |
Characterization of immune features and immunotherapy response in subtypes of hepatocellular carcinoma based on mitophagy
Mitophagy is suggested to be involved in tumor initiation and development; however, mitophagy heterogeneity in hepatocellular carcinoma (HCC) and its association with immune status and prognosis remain unclear. Differentially expressed genes (DEGs) were identified using expression profiles acquired from The Cancer Genome Atlas (TCGA). Mitophagy-related subtypes were identified using the ConsensusClusterPlus software. The differences in prognosis, clinical characteristics, and immune status, including immune cell infiltration, immune function, immune-checkpoint gene expression, and response to immunotherapy, were compared between subtypes. A mitophagy-related gene signature was constructed by applying least absolute shrinkage and selection operator regression to the TCGA cohort. The International Cancer Genome Consortium cohort and the cohort from Peking Union Medical College Hospital were utilized for validation. Carbonyl cyanide m-chlorophenylhydrazone was used to induce mitophagy in HCC cell lines to obtain our own mitophagy signature. Real-time polymerase chain reaction was used for the experimental validation of the expression of model genes. Two mitophagy-related subtypes with distinct prognoses, clinical characteristics, immune states, and biological function patterns were identified based on the mitophagy-related DEGs. The subtype that showed higher mitophagy-related DEG expression had worse survival outcomes, suppressed immune function, higher immune-checkpoint gene expression, and a better response to immunotherapy, indicating that this subpopulation in HCC may benefit from immune-checkpoint blockade therapy and other immunotherapies. A risk model consisting of nine mitophagy-related genes was constructed and its performance was confirmed in two validation cohorts. The risk score was an independent risk factor even when age, sex, and tumor Frontiers in Immunology (2022) Characterization of immune features and immunotherapy response in subtypes of hepatocellular carcinoma based on mitophagy. Front. Immunol. 13:966167. stage were considered. Our study identified two distinct mitophagy subtypes and built a mitophagy signature, uncovering mitophagy heterogeneity in HCC and its association with immune status and prognosis. These findings shed light on the treatment of HCC, especially with immunotherapy.
# Introduction
Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer cases, which is ranked the fifth in cancerrelated death [bib_ref] Hepatocellular carcinoma in non-alcoholic fatty liver disease-a review of an emerging challenge..., Geh [/bib_ref]. Despite much progress in the diagnosis and treatment of HCC, the prognosis of patients with HCC remains poor, with a median survival time of 9 months (3). For patients with HCC at early stage, curative treatments such as radiofrequency ablation and liver section can achieve a 40%-70% 5-year survival rate; and palliative treatments such as transarterial chemoembolization has been shown to improve median OS of intermediate stage HCC to approximately 20 months. For HCC at advanced stage or terminal stage, survival outcomes are still unsatisfactory even with the help of molecular therapy. Immunotherapies, such as immunecheckpoint blockade (ICB), have shown strong antitumor activity and lead to a substantial prolonged survival for advanced HCC, whereas only a subset of patients can benefit from these therapies [bib_ref] Advances in immunotherapy for hepatocellular carcinoma, Sangro [/bib_ref]. Therefore, there is an urgent need to explore the underlying molecular mechanisms of HCC and provide new targets and strategies for treatment.
Major breakthroughs in a mechanism called mitophagy have recently gained considerable attention [bib_ref] Molecular mechanisms and physiological functions of mitophagy, Onishi [/bib_ref]. Mitophagy, also known as mitochondrial autophagy, eliminates denatured or damaged mitochondria, preventing the accumulation of mitochondrial DNA mutations and maintaining mitochondrial quality [bib_ref] Mechanisms of mitophagy, Youle [/bib_ref]. Hence, mitophagy plays a vital role in regulating energy metabolism and removing excessive cytotoxic reactive oxygen species [bib_ref] ROS and the DNA damage response in cancer, Srinivas [/bib_ref]. Mitophagy plays a dual role in the development of cancer by suppressing tumors at an early stage and promoting tumors at an advanced stage [bib_ref] Eiyama A, Okamoto K. PINK1/Parkin-mediated mitophagy in mammalian cells, Xu [/bib_ref]. The ubiquitindependent PINK1/Parkin pathway is the most common mitophagy cascade, and some core genes within this pathway, such as PINK1 and PARK2, can predict prognosis in patients with papillary renal cell cancer (10, 11). However, the role of mitophagy-related genes (MRGs) in HCC is not fully understood. Several studies have reported heterogeneity in autophagy in other types of cancer [bib_ref] Identification of a five autophagy subtype-related gene expression pattern for improving the..., Zhang [/bib_ref] [bib_ref] Autophagic heterogeneity in gastric adenocarcinoma, Yoon [/bib_ref]. As a specific form of autophagy, the heterogeneity of mitophagy likely influences the development and prognosis of HCC. Research focused on mitophagy may help to concretize problems. Therefore, we aimed to investigate the role of MRGs and mitophagy-related subtypes in HCC, focusing on the association with immune status and response to immunotherapy, as there has been massive interest in immunotherapy for HCC [bib_ref] Immunotherapy for hepatocellular carcinoma: Current and future, Johnston [/bib_ref].
A flowchart of the study design is shown in. In this study, we first screened differentially expressed MRGs (DEMs) between tumor and normal tissues of patients with HCC. Based on the expression profile of the DEMs, we classified the patients into two subtypes and explored their prognoses, clinical characteristics, immune states, and drug sensitivities. Subsequently, based on the MRG signature, a prognostic model was constructed and validated in two cohorts. Moreover, we explored the differences in biological functions between these subtypes and risk groups. Cell experiment and qPCR were performed to validate our results. Our findings are helpful in accurately characterizing HCC and providing personalized treatment for patients.
# Materials and methods
## Data acquisition
We obtained two gene sets from public databases by searching the keyword "mitophagy": one is Mitophagy-animal pathway (Entry: hsa04137), which contains 72 genes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (https://www.kegg.jp/kegg/pathway.html), the other one is REACTOME-MITOPHAGY gene sets (source: R-HSA-5205647), which contains 29 genes from the C2:CP : REACTOME in Molecular Signatures Database with Gene Set Enrichment Analysis (GSEA) (http://www.gsea-msigdb.org). Since 20 genes overlapped in two gene sets, we eventually acquired a total of 81 MRGs for subsequent analyses. The RNA-seq and clinical information of HCC samples were downloaded from the Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/), which included 50 normal samples and 374 cancer samples. We also acquired two HCC cohort datasets for validation: one was downloaded from the International Cancer Genome Consortium (ICGC) database (https://dcc.icgc.org/), which included 243 cancer samples; the other was collected from Peking Union Medical College Hospital (PUMCH) and included 20 patients with HCC (Supplementary . The cohort from our center was approved by the Ethics Committee of PUMCH and CAMS (Chinese Academy of Medical Sciences) & PUMC (Peking Union Medical College), and written informed consent was obtained from all patients.
## Identification and analysis of dems
DEMs between tumor tissues and normal tissues in TCGA were screened using the "limma" R package (15) based on the following criteria: |log 2 fold change| > 0.5 and false discovery rate < 0.05. The protein-protein interaction network of DEMs was obtained from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org), and the interaction between core genes was visualized using Cytoscape software (version 3.8.2) (16).
# Consensus cluster analysis
We used the ConsensusClusterPlus software (17) of R to perform unsupervised consensus clustering of TCGA dataset based on the expression of DEMs. The optimal cluster number k was determined by evaluating the delta area, consensus cumulative distribution function, and consensus matrix. Principal component analysis was used to verify the results of the cluster analysis. The correlation between clusters and clinical variables was tested using Chi-square test.
# Immune status analysis
To explore the impact of mitophagy on patient immune status, two mitophagy-related subtypes were compared in terms of differences in infiltrating immune cells, immune function, immune-checkpoint gene expression levels, and response to immunotherapy. We quantified the relative abundance of immune cell types and the activity of immune function in each sample using single sample GSEA algorithms through the R package "GSVA" [bib_ref] GSVA: gene set variation analysis for microarray and RNA-seq data, Hänzelmann [/bib_ref]. The expression levels of immunecheckpoint genes can reflect the response to ICB treatment; thus, the following well-known immune-checkpoint genes were chosen for expression level evaluation in each subtype: CTLA4, CXCL9, CD8A, TBX2, PDCD1, LAG3, HAVCR2, IFNG, TNF, and CD274. Moreover, Tumor Immune Dysfunction and Exclusion (TIDE) scores [bib_ref] Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response, Jiang [/bib_ref] of each HCC sample were calculated online (http://tide.dfci.harvard.edu/) and compared between subtypes to verify the differences in response to immunotherapy. Study flowchart. DEMs, Differentially expressed mitophagy-related genes; ICB, immune-checkepoint blockade.
# Drug sensitivity analysis
To discover potential drugs for patients with different mitophagy-related subtypes of HCC, we evaluated their responses to various antitumor drugs using the "pRRophetic" R package [bib_ref] pRRophetic: an r package for prediction of clinical chemotherapeutic response from tumor..., Geeleher [/bib_ref] , which is based on the Genomics of Drug Sensitivity in Cancer database.
## Construction and validation of risk model
First, we performed univariate Cox regression analysis on MRGs to screen for survival-related prognostic genes in the TCGA cohort. We then obtained genes for model construction by intersecting prognostic genes with DEMs, followed by least absolute shrinkage and selection operator (LASSO) regression using the "glmnet" R package [bib_ref] Regularization paths for generalized linear models via coordinate descent, Friedman [/bib_ref] to form the final gene signature for the risk model. The risk score was formulated as follows:
risk score ¼ b1 ñ gene1 expression + b2 ñ gene2 expression + … + bn ñ gene expression where b represents the coefficient value of each gene. Patients in the training and validation cohorts were divided into high-and low-risk groups based on the median risk scores. The receiver operating characteristic (ROC) curves in each cohort were plotted using the R package "timeROC" [bib_ref] Estimating and comparing time-dependent areas under receiver operating characteristic curves for censored..., Blanche [/bib_ref] , and the time-dependent area under the curve values were measured to evaluate the performance of the model. Univariate and multivariate Cox regression analyses were used to evaluate whether the risk score was an independent predictor.
# Functional analysis
KEGG and Gene Ontology (GO) enrichment analyses were utilized for functional annotation of DEMs and differentially expressed genes (DEGs) between subtypes using the "ClusterProfiler" R package [bib_ref] clusterProfiler: An r package for comparing biological themes among gene clusters, Yu [/bib_ref]. Significant GO terms and pathways were selected with a p-value cutoff of < 0.01. The biological functions enriched in the high-and low-risk groups of the TCGA cohort were derived by GSEA of KEGG pathways. MHCC97H was from the Liver Cancer Institute (Zhongshan Hospital, Fudan University, China). SNU398 was from ATCC (Manassas, VA, USA). Huh7 and MHCC97H were cultured in Dulbecco's modified Eagle medium, supplemented with 10% fetal bovine serum and 1% antibiotics in 5% CO 2 at 37°C. SNU398 was routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics in 5% CO 2 at 37°C. Then three HCC cell lines were treated with 10mM carbonyl cyanide m-chlorophenylhydrazone (CCCP, selleck,China), which was used to induce mitophagy, for 24 hours [bib_ref] Metformin inhibits hepatocellular carcinoma development by inducing apoptosis and pyroptosis through regulating..., Yapryntseva [/bib_ref]. RNA sequencing for Huh 7, MHCC97H, SNU398 cells treated with or without CCCP by Beijing Auwigene Tech, Ltd (Beijing, China) using the Illumina second-generation high-throughput PE150 sequencing platform (Illumina, Inc., CA, United States). Between cell lines treated with and without CCCP, top 100 differentially expressed genes ranked by |log 2 fold change| were considered as mitophagy signature for validation.
## Real-time polymerase chain reaction
Total RNA was extracted from liver tumors and peritumoral tissues using TRIzol (Invitrogen, Thermo Fisher, Waltham, MA, USA), following the manufacturer's instructions. RNA (1 μg) was reverse transcribed using the Hifair ® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen Biotechnology, Shanghai, China) in a 20 ml reaction. After 20fold dilution, 4 ml of cDNA was used as a template in a 20 ml realtime polymerase chain reaction (PCR). For real-time PCR, amplification was performed for 40 cycles using BlasTaq ™ 2X qPCR MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada). Primers were designed on exon junctions to prevent co-amplification of genomic cDNA; the sequences are presented in the Supplementary .
## Statistical analyses
All statistical analyses were performed using R (version 4.0), and relevant packages were applied for processing and visualization. The Wilcoxon test was used to compare differences in continuous variables. The overall survival was evaluated using the Kaplan-Meier analysis, and the survival curve was plotted using the R package "survminer" (http://cran. r-project.org/). The log-rank test was used to examine the differences between subtypes or groups. If not specifically stated, bilateral p < 0.05 was considered statistically significant.
# Results
## Profile and functional annotation of dems between tumor and normal samples
We collected 81 MRGs from the database gene sets, and 51 DEMs were identified between tumor and normal samples from the TCGA cohort. In order to verify DEMs acquired from one public database, we performed RNA sequencing on 8 pairs of HCC samples and peritumoral tissues, and the expression matrix can be found in . Of 42 DEMs identified in our own samples, 35 DEMs overlapped with 51 DEMs from TCGA cohort, and 39 of 40 DEMs identified in ICGC cohort overlapped with those in TCGA cohort, indicating the reliability of DEMs identified in TCGA cohort. A heatmap of the 51 DEMs is shown in . Fifty of the DEMs were upregulated in tumors, primarily including genes involved in the PINK1/Parkin pathway (PINK1, PARK2, ATG family, and TOMM family) and receptor-mediated mitophagy (FUNDC1, PGAM5, and ULK1).
In addition, oncogenes such as TP53, KRAS, and HRAS were also upregulated, as these genes may be related to hypoxic stress. Only one gene, JUN, was downregulated. We then performed protein-protein interaction analysis to determine the interactions between DEMs and the core network, as shown in ; the TOMM family, MFN1, VDAC1, PARK2, and RPS27A were hub genes, and oncogenes such as HRAS, KRAS, and TP53 participated in the core network of mitophagy. The KEGG analysis in showed that, besides mitophagy and autophagy, these DEMs were also enriched in the PD-1/PD-L1 checkpoint pathway, which is related to the response to ICB treatment of HCC. Pathways involving hepatitis B and apoptosis were also enriched, and they were shown to be involved in tumorigenesis and the development of HCC (26, 27).
## Subpopulation of hcc on account of expression pattern of dems
Consensus clustering was applied to identify HCC subtypes based on the expression levels of the DEMs acquired from the previous step. We determined the k value as 2, at which point the relative change in area under the cumulative distribution function reached an approximate maximum and the consensus matrix showed a clear boundary simultaneously . Therefore, two clearly distributed subtypes were classified; these were denoted as cluster 1 (containing 211 samples) and cluster 2 (containing 163 samples). To further verify the clustering result, principal component analysis was performed, and the principal component distribution was in accordance with the consensus matrix, ensuring the stability of consensus clustering . The DEM expression and clinical features of each sample grouped by cluster are shown in a heat map . Cluster 2 generally had higher DEM expression than cluster 1. Tumor stage and grade were found to be correlated with subtype. Cluster 2 had a higher proportion of tumors with advanced stage and high grade. Moreover, the Kaplan-Meier survival analysis showed that the two subtypes had significant differences in OS . Cluster 2 tended to have worse outcomes than cluster 1 (p = 2.618e-04), with 5-year survival rates of 37.6% and 56.0%, respectively. The difference in prognosis between the two clusters was in accordance with their differences in tumor stage and grade. These findings confirm the existence of mitophagy heterogeneity in HCC and its impact on the development and prognosis of HCC.
## Characterization of immune status and drug sensitivity affected by mitophagy heterogeneity in hcc
To compare the immune characteristics of the two mitophagy-related subtypes, we first estimated immunocyte infiltration and immune function using single sample GSEA algorithms. As shown in , compared with cluster 1, cluster 2 showed higher infiltration of activated dendritic cells (aDCs), immature dendritic cells (iDCs), macrophages, follicular helper T cell (Tfh), T helper 2 cell (Th2) and regulatory T cells (Treg). Regarding immune function in , cytolytic activity and type II interferon response were increased in cluster 1, while cluster 2 had strong antigen-presenting cell (APC) co-stimulation and major histocompatibility complex (MHC) class I reactions. To explore the effect of mitophagy on the response to ICB treatment, we compared the expression levels of immunecheckpoint genes in each subtype. As shown in , all immune-checkpoint genes were consistently overexpressed in cluster 2, indicating that cluster 2 tended to be more sensitive to ICB treatment. Furthermore, we calculated the TIDE score of every sample and the scores were significantly lower in cluster 2 than in cluster 1, further verifying that patients in cluster 2 may be more likely to benefit from immunotherapy . In contrast, with higher TIDE scores, cluster 1 was more likely to achieve tumor immune escape and exhibit a lower response rate to ICB treatment.
We also evaluated the drug sensitivity of each subtype to identify potential chemotherapeutic drugs. Lower IC50 values indicate higher sensitivity. As shown in , compared with cluster 2, cluster 1 was more sensitive to AKT inhibitor III, epidermal growth factor receptor inhibitors such as erlotinib, gefitinib, and lapatinib, and vascular endothelial growth factor receptor inhibitors such as axitinib and sunitinib. Conversely, cluster 2 had a higher response rate to AZD8055 (mTOR inhibitor), bleomycin, cisplatin, etoposide, sorafenib, and methotrexate.
## Functional annotation of degs between subtypes
To reveal the differences in biological functions between the two subtypes, we conducted GO and KEGG enrichment analysis on the DEGs between the two subtypes with a cutoff of |log 2 fold change|> 1 and false discovery rate < 0.05. A total of 260 DEGs met the criteria, and the results showed that complement and coagulation cascades, the peroxisome proliferators-activated receptors signaling pathway, bile secretion, chemical carcinogenesis, and drug and compound metabolism were significantly enriched in the KEGG pathway analysis . The results of GO enrichment analysis are shown in . The comparison of immune status between mitophagy-related subtypes. Box plots showing the differences of infiltrating immunocyte abundance (A), immune reaction activity (B), expression of immune-checkpoint genes (C) and violin plots showing Tumor Immune dysfunction and Exclusion (TIDE) score (D). The asterisks represent the p value (*p < 0.05; **p < 0.01; ***p < 0.001). ns, not significant.
## Constructing prognosis model of mrgs
Defining the TCGA dataset as a training cohort, we performed univariate Cox regression analysis on 81 MRGs, 35 of which were significantly associated with the OS of patients with HCC (p < 0.05). After intersection with 51 DEMs, we obtained 24 genes for model construction . All 24 genes were risk genes with a hazard ratio of > 1 . The LASSO regression model was then utilized, and nine genes were screened to build the prognostic risk model . The risk score was calculated using the corresponding coefficients and gene expression. Finally, the risk score model was formulated as follows: Additionally, we created a Sankey diagram to show the connection among mitophagy subtypes, risk scores, and survival .
## Validation of model efficiency
To verify the performance of the risk model, we performed Kaplan-Meier survival analysis in the training and validation cohorts. The survival curves showed that improved survival rates of low-risk patients continued for nearly 7 years in the TCGA training cohort (p = 9.707e-04), and this advantage existed in the ICGC validation cohort (p = 1.749e-04) . In addition, we used an HCC cohort (n=20) registered in our center to validate the risk model, and the difference in OS was still significant (p = 3.924e-02) . Regarding model accuracy, the 1-year, 3-year, and 5-year AUC of the model for OS was 0.781, 0.690, and 0.650, respectively, in the TCGA training cohort , and 0.709, 0.749, and 0.716, respectively, in the ICGC validation cohort . The AUC of the model in PUMCH cohort was still satisfactory A B D E C Construction of a LASSO regression model and correlation between subtypes and risk groups. . We ranked the risk scores of patients with HCC in all cohorts from low to high, and the survival status and time of each patient were plotted according to the risk score . The plot revealed that high-risk patients generally had poorer survival rates than low-risk patients.
To determine whether the risk score is an independent risk factor for the prognosis of patients with HCC, we performed univariate Cox regression on the risk score and clinical variables . The results showed that only the stage and risk scores were significantly associated with OS (p < 0.001). Next, these variables were included in the multivariate Cox regression analysis. After correction for other confounding factors, including age, sex, stage, and grade, the risk score was still significantly associated with OS, implying that the risk score was an independent risk factor (p < 0.001) .
## Functional enrichment analysis based on the risk score
We performed GSEA on the TCGA cohort, and the most significantly enriched KEGG pathways are shown in. The cell cycle, mTOR signaling pathway, NOTCH signaling pathway, endocytosis, and pathways in cancer were enriched in the high-risk group. Primary bile acid biosynthesis, drug metabolism, cytochrome P450, fatty acid metabolism, glycine serine and threonine metabolism, and linoleic acid metabolism pathways were enriched in the low-risk group.
## Validation of mitophagy heterogeneity through cell experiment
In order to validate mitophagy-related subtypes obtained using public mitophagy gene sets, we acquired mitophagy signature through inducing mitophagy in HCC cell lines for same analyses in TCGA HCC dataset. The expression matrix of HCC cell lines before and after mitophagy induction was demonstrated in Supplementary Table 1 and the expression levels of mitophagy signature genes were applied for clustering of TCGA HCC dataset. As shown in, tumor grade and stage were still correlated with clusters. And cluster 2 had significantly worse survival outcome than cluster 1 (p = 0.006). Similar to results in , cluster 2 had higher infiltration of aDCs, iDCs, macrophages, Tfh, Th2, and Treg than cluster 1. In terms of immune function, type II interferon response was still suppressed in cluster 2, while checkpoint, APC co-stimulation, APC co-inhibition, HLA, MHC class I, and proinflammation exhibited higher levels in cluster 2, which confirmed the association between mitophagy and immune status in HCC. In addition, regarding response to ICB treatment, all immune-checkpoint genes except CXCL9 were significantly overexpressed in cluster 2, and TIDE scores remained lower in cluster 2 than in cluster 1, indicating that patients in cluster 2 tended to benefit from ICB treatment. These findings following cell experiment further validated our results from using public MRGs.
## Validation of expression of model genes in tissue
To verify the reliability of the results acquired from the public database, we further validated the expression levels of the nine genes consisting of the mitophagy signature in five pairs of HCC tissues and peritumoral tissues. As shown in, the tumor expressed significantly higher mRNA levels of all genes (ATG9A, ATG12, HRAS, MFN1, NRAS, PGAM5, SQSTM1, TOMM22, and TOMM5) than peritumoral tissue, which was consistent with the public database analysis. Gene-set enrichment analysis identifying KEGG pathways enriched in the high-and low-risk group. KEGG, Kyoto Encyclopedia of Gene and Genome.
# Discussion
Emerging immunotherapy, especially ICB treatment, has become an effective and promising option to treat HCC (28). However, only a portion of patients respond to immunotherapy; thus, it is important to determine which groups of patients can benefit from immunotherapy, facilitating the progress of personalized treatment. Recently, mitophagy has attracted the attention of researchers as a potential therapeutic target for cancer. Hence, this study aimed to investigate mitophagy heterogeneity in HCC and its association with immune status, identify two mitophagy subtypes with distinct clinical and immune characteristics, and offer more detailed insights into immunotherapy or combination therapy for HCC.
HCC has been confirmed to exhibit high molecular heterogeneity [bib_ref] Tumour evolution in hepatocellular carcinoma, Craig [/bib_ref]. We identified two mitophagy subtypes in TCGA HCC samples based on the expression levels of DEMs, showing that these two subtypes with different mitophagy patterns were characterized by significantly different tumor stages and prognoses. This verified that mitophagy heterogeneity is associated with HCC development and has prognostic value in HCC, although the underlying mechanisms are still not well understood. Mitophagy appears to be tumor-promoting or tumor-suppressive, depending on the tumor type and intrinsic stage [bib_ref] Mitophagy in tumorigenesis and metastasis, Poole [/bib_ref]. For instance, PARK2, which encodes a core mitophagy protein, Parkin, was found to be inactivated in colon and lung cancer [bib_ref] Somatic mutations of the parkinson's disease-associated gene PARK2 in glioblastoma and other..., Veeriah [/bib_ref]. Parkin-null mice are susceptible to spontaneous HCC (32). In contrast, upregulation of the mitochondrial inner membrane protein STOML2 can amplify PINK1/Parkin-mediated mitophagy and facilitate the migration and invasion of HCC cells, thus promoting HCC growth and metastasis [bib_ref] STOML2 potentiates metastasis of hepatocellular carcinoma by promoting PINK1-mediated mitophagy and regulates..., Zheng [/bib_ref]. Previous studies have also found that hyperactivated mitophagy can induce sorafenib resistance in HCC under hypoxic stress [bib_ref] Mitophagy promotes sorafenib resistance through hypoxia-inducible ATAD3A dependent axis, Wu [/bib_ref]. Therefore, the dual role of mitophagy may be involved in HCC heterogeneity. In our study, nearly all DEMs were upregulated in HCC samples compared with normal tissues, and cluster 2 had generally higher expression levels of DEMs but worse prognosis than cluster 1. Based on the above evidence, cluster 2 is likely to be characterized by higher mitophagy activity, which results in a more advanced tumor and worse survival outcome. Furthermore, regulation of various mitophagy pathways, such as PINK1/Parkin-mediated mitophagy and BNIP3/BNIP3L/ FUNDC1-mediated mitophagy, may also be involved in HCC heterogeneity, which warrants further study. To determine whether mitophagy heterogeneity has an impact on the tumor immune microenvironment, we evaluated the differences in immune characteristics between the two subtypes. Immune cell infiltration is closely related to clinical outcomes, and immune cells can serve as an immunotherapy target [bib_ref] Targeting immune cells in the tumor microenvironment of HCC: New opportunities and..., Hao [/bib_ref]. Our single sample GSEA results indicated that cluster 2 had a higher abundance of regulatory T cells and macrophages, which are considered to be HCC promoting [bib_ref] The role of tumor-associated macrophages in primary hepatocellular carcinoma and its related..., Deng [/bib_ref]. Moreover, cluster 2 was characterized by higher expression levels of immune-checkpoint genes. Overexpression of immune-checkpoint genes can suppress the antitumor immune response so that tumor cells can easily evade immune surveillance. These findings explain the poor survival outcomes of cluster 2.
ICB therapy can restore dysfunctional immune system and has achieved remarkable results in cancer treatment. ICB agents against programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have been approved for HCC by the FDA [bib_ref] The current landscape of immune checkpoint blockade in hepatocellular carcinoma: A review, Pinter [/bib_ref]. However, the limited response rate makes it important to screen patients who are sensitive to ICB therapy. Cluster 2 showed higher expression levels of immune checkpoint genes, indicating a better response to ICB treatment. The TIDE algorithm is believed to perform better than the expression level of immune check-points in predicting the survival outcome of cancer patients treated with ICB agents [bib_ref] Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response, Jiang [/bib_ref]. Corresponding to the prediction based on immune-checkpoint expression, the TIDE results revealed that cluster 2 was more likely to respond to ICB treatment. Therefore, ICB treatment may help reverse the poor prognosis of cluster 2. Taken together, mitophagy heterogeneity in HCC may influence immune status and can predict the response rate to ICB agents, revealing the association between mitophagy and immunity. This result enhanced our understanding of the heterogeneity of HCC, promoting personalized therapy in clinical practice and inspiring immunotherapy development in scientific research and trials. Furthermore, our study explored potential drugs for subpopulations with different mitophagy patterns, providing ideas for synergistic combination of ICB agents and targeted therapies. Systemic therapy in HCC should be explored to improve clinical efficacy [bib_ref] Combinational immunotherapy for hepatocellular carcinoma: Radiotherapy, immune checkpoint blockade and beyond, Lee [/bib_ref].
To more precisely predict the prognosis of patients with HCC, we constructed a risk model based on a mitophagy signature. Notably, all nine genes were risk factors for HCC. Of these genes, ATG9A and ATG12 are core regulators of autophagy (39). MFN1, also known as mitofusin-1, was analyzed both in vivo and in vitro and its effects on HCC metastasis were revealed [bib_ref] MFN1-dependent alteration of mitochondrial dynamics drives hepatocellular carcinoma metastasis by glucose metabolic..., Zhang [/bib_ref]. PGAM5 is an atypical m i t o c ho n d r i a l s e r i n e / t h r e o n i n e p h o s p h a t a s e t h a t dephosphorylates FUNDC1 to activate mitophagy. Previous studies have reported that depleting PGAM5 inhibits tumor development and enhances the 5-fluorouracil sensitivity of HCC cells [bib_ref] High PGAM5 expression induces chemoresistance by enhancing bcl-xL-mediated anti-apoptotic signaling and predicts..., Cheng [/bib_ref] [bib_ref] Receptor-mediated mitophagy in yeast and mammalian systems, Liu [/bib_ref]. The TOMM complex (translocase of the outer mitochondrial membrane) imports nearly all mitochondrial proteins from the cytoplasm into the mitochondria, and TOMM22 functions as a central receptor [bib_ref] Expression of the bcl-2 family genes and complexes involved in the mitochondrial..., Kravic [/bib_ref]. Although no significant increase in the expression of TOMM genes was observed in prostate cancer compared to normal tissues, our results demonstrated that this protein was elevated in HCC and may be a good candidate biomarker; this requires validation (44). Through ROC analysis in different cohorts, we found that our risk model showed better efficacy in predicting prognosis compared to models constructed based on other gene signatures, such as pyroptosis in HCC [bib_ref] Identification of a pyroptosis-related gene signature for predicting overall survival and response..., Zheng [/bib_ref]. Remarkably, the performance of the model was consistently stable and even better in the validation cohort of ICGC and our own cohort than in the training cohort, which verifies the robustness of our risk model. Functional analyses revealed that various metabolic pathways were enriched in the mitophagy subgroups and risk groups. Metabolic reprogramming is a hallmark of tumor growth and progression [bib_ref] Metabolic reprogramming for cancer cells and their microenvironment: Beyond the warburg effect, Sun [/bib_ref]. Mitophagy plays a critical role in the metabolic adaptation of cancer cells so that these cells can survive under stress factors produced in the tumor microenvironment, and these adaptions are closely related to the acquisition of metastatic potential and chemoresistance (47). Therefore, some metabolic regulators or pathways related to mitophagy may serve as new therapeutic targets for cancer. Additionally, metabolic pathway regulation can affect immune cell function and fate, leading to a connection to the immune microenvironment [bib_ref] Metabolic pathways in immune cell activation and quiescence, Pearce [/bib_ref]. This crosstalk between metabolic reprogramming and the immune microenvironment adds further layers to the search for novel therapeutic strategies, regardless of forthcoming challenges. Combining existing evidence and our results, we hypothesize that a metabolismmitophagy-immunity network exists in HCC, which needs to be explored and validated in future studies.
Nevertheless, our study has several limitations. First, our study focused on the expression of genes, lacking multi-omics data, such as copy number variations and DNA methylation. Second, the study was conducted retrospectively based on data from a public database rather than using a prospective cohort. Furthermore, HCC cell lines and our own HCC cohort used for validation had limited sample sizes, though the results are still reliable. Finally, the mechanisms underlying mitophagy, metabolism, and immunity in tumors warrant further study.
# Conclusion
In summary, we identified two prognostically and clinically relevant mitophagy subtypes in HCC. These two subtypes differed in multiple aspects, including immune characteristics, responses to immunotherapy, and biological functions. We also constructed a mitophagy-related risk model that exhibited stable efficiency and performed better than models based on other signatures. The expression of these model genes was subsequently validated using laboratory results. These findings suggest mitophagy as a potential treatment target and shed new light on the strategy of immunotherapy in HCC.
# Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
# Ethics statement
The studies involving human participants were reviewed and approved by ethics committee of PUMCH and CAMS (Chinese Academy of Medical Sciences) & PUMC (Peking Union Medical College). The patients/participants provided their written informed consent to participate in this study. |
Race and Genetics in Congenital Heart Disease: Application of iPSCs, Omics, and Machine Learning Technologies
Congenital heart disease (CHD) is a multifaceted cardiovascular anomaly that occurs when there are structural abnormalities in the heart before birth. Although various risk factors are known to influence the development of this disease, a full comprehension of the etiology and treatment for different patient populations remains elusive. For instance, racial minorities are disproportionally affected by this disease and typically have worse prognosis, possibly due to environmental and genetic disparities. Although research into CHD has highlighted a wide range of causal factors, the reasons for these differences seen in different patient populations are not fully known. Cardiovascular disease modeling using induced pluripotent stem cells (iPSCs) is a novel approach for investigating possible genetic variants in CHD that may be race specific, making it a valuable tool to help solve the mystery of higher incidence and mortality rates among minorities. Herein, we first review the prevalence, risk factors, and genetics of CHD and then discuss the use of iPSCs, omics, and machine learning technologies to investigate the etiology of CHD and its connection to racial disparities. We also explore the translational potential of iPSC-based disease modeling combined with genome editing and high throughput drug screening platforms.
# Introduction
CHD is the most frequently occurring birth defect, and despite technological advances in healthcare, children afflicted with CHD continue to face significant morbidity and mortality [bib_ref] Congenital heart disease in the general population: changing prevalence and age distribution, Marelli [/bib_ref]. Moreover, it remains largely unknown why certain populations (e.g., racial minorities) are disproportionally affected by this disease. Herein, we first review the prevalence, risk factors, and genetics of CHD. We then discuss the applications of iPSCs, omics, and machine learning technologies to better understand disease mechanisms especially in connection to racial disparities.
## Congenital heart disease: definition, classifications and prevalence
Cardiac malformations present at birth make up a relevant component of pediatric cardiovascular disease that constitutes a significant percentage of clinically significant birth defects, occurring in about 4 to 50 per 1,000 live births [bib_ref] Genetic basis for congenital heart defects: current knowledge: a scientific statement from..., Pierpont [/bib_ref]. CHD is defined as a gross structural abnormality of the heart or intrathoracic great vessels arising before birth with potential functional significance [bib_ref] The incidence of congenital heart disease, Hoffman [/bib_ref]. It is the most frequently diagnosed congenital defect among newborns and has consistently been a primary cause of morbidity and mortality among those affected [bib_ref] The developmental genetics of congenital heart disease, Bruneau [/bib_ref]. It is estimated that 4 to 10 live-born infants per 1,000 are diagnosed with CHD in the first year of life [bib_ref] Congenital heart disease: incidence and inheritance, Hoffman [/bib_ref] [bib_ref] Report of the task force on children and youth, Moller [/bib_ref]. Additionally, 1 in 4 infants with critical CHD require surgery in the first year of life [bib_ref] Cost-effectiveness of routine screening for critical congenital heart disease in US newborns, Peterson [/bib_ref].
CHD can be classified either as simple, moderate, or complex based on survival, prognosis, and frequency of complications. Simple CHD typically does not require extensive surgery to repair. Common instances of simple CHD defects include isolated congenital valve disease (e.g., bicuspid aortic valve), mild pulmonary stenosis, and minor atrial septal defect (ASD) or ventricular septal defect (VSD) [bib_ref] Simple congenital heart disease: a complex challenge for public health, Buratto [/bib_ref] [bib_ref] Prevalence of congenital heart disease, Hoffman [/bib_ref]. Moderate CHD requires expert care to repair to achieve better prognosis/survival compared to complex CHD [bib_ref] The incidence of congenital heart disease, Hoffman [/bib_ref]. Examples of moderate CHD include coarctation of the aorta, Ebstein anomaly, and more complex ASD/VSD [bib_ref] The incidence of congenital heart disease, Hoffman [/bib_ref]. Complex CHD typically presents early with profound hypoxemia and/or hemodynamic complications that generally require early interventions. Examples of frequently occurring complex CHD include complex tetralogy of Fallot (ToF), transposition of great arteries, and hypoplastic left heart syndrome (HLHS). ToF is a common cardiac defect that accounts for 5.4% of all CHDs and about 60% of conotruncal defects, excluding transposition of great arteries [bib_ref] Congenital heart disease and genetic syndromes: specific correlation between cardiac phenotype and..., Marino [/bib_ref] [bib_ref] Genetic and environmental risk factors of major cardiovascular malformations: the Baltimore-Washington infant..., Ferencz [/bib_ref] [bib_ref] Non-cardiac malformations in individuals with outflow tract defects of the heart: The..., Lurie [/bib_ref]. It is characterized by right ventricular outflow tract obstruction, VSD, overriding aorta, and right ventricular hypertrophy. HLHS patients present with abnormally severe underdevelopment of the left ventricle, mitral valve, and aorta [bib_ref] Intrinsic endocardial defects contribute to hypoplastic left heart syndrome, Miao [/bib_ref]. Without treatment, most subjects with complex CHD will die in the first year of life, with very few reaching adulthood [bib_ref] Prevalence of congenital heart disease, Hoffman [/bib_ref] [bib_ref] Natural history of transposition of the great arteries: anatomy and birth and..., Liebman [/bib_ref].
## Risk factors of congenital heart disease
There are several risk factors contributing to CHD, including genetic/familial contributors as well as environmental/nongenetic factors promoting CHD development [bib_ref] Congenital heart disease: current knowledge about causes and inheritance, Blue [/bib_ref]. Prenatal maternal conditions or exposures associated with an increased risk for CHD may be further categorized into modifiable vs. non-modifiable. Examples of modifiable risk factors include maternal dietary deficiency, substance abuse, obesity/diabetes, and air pollution. Examples of non-modifiable risk factors include maternal rheumatologic disorders, genetics, medications, metabolic disorders, and infections (e.g., rubella) .
## Modifiable risk factors
Studies have shown that daily folic acid deficiency during the pre-and peri-conception periods can be a CHD risk factor [bib_ref] Congenital heart disease: current knowledge about causes and inheritance, Blue [/bib_ref]. Moderate maternal vitamin D deficiency has also been shown to significantly increase the risk of offspring developing CHD [bib_ref] Environmental risk factors for congenital heart disease, Kalisch-Smith [/bib_ref] [bib_ref] A compromised maternal vitamin D status is associated with congenital heart defects..., Koster [/bib_ref]. Additionally, more recent studies show that embryonic hypoxic exposure, caused by maternal smoking, increases the risk of CHD [bib_ref] Environmental risk factors for congenital heart disease, Kalisch-Smith [/bib_ref] [bib_ref] Maternal cigarette smoking and congenital heart defects, Correa [/bib_ref] [bib_ref] Risk of congenital heart defects in the offspring of smoking mothers: a..., Sullivan [/bib_ref]. Furthermore, maternal alcohol consumption, which can lead to the development of fetal alcohol syndrome disorder (FASD), is a commonly known risk factor for CHD. Heart defects such as VSD, ASD, and conotruncal defects occur quite frequently in FASD, with an estimated 67% of cases reported with CHD [bib_ref] Environmental risk factors for congenital heart disease, Kalisch-Smith [/bib_ref] [bib_ref] Congenital heart defects and fetal alcohol spectrum disorders, Burd [/bib_ref] [bib_ref] Prenatal alcohol exposure and congenital heart defects: a meta-analysis, Yang [/bib_ref]. Gestational diabetes is one of the most influential risk factors for CHD [bib_ref] Epidemiology of gestational diabetes mellitus and its association with Type (2) diabetes, Ben-Haroush [/bib_ref] , increasing the risk by 3-to 5-fold [bib_ref] Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease, Basu [/bib_ref]. Poorly controlled maternal diabetes creates an unfavorable environment for embryonic development, exposing the fetus to elevated blood glucose levels, which is a major teratogen for diabetic embryopathy [bib_ref] Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease, Basu [/bib_ref]. Maternal diabetes induces cardiac malformation before the seventh week of gestation [bib_ref] Noninherited risk factors and congenital cardiovascular defects: current knowledge: a scientific statement..., Jenkins [/bib_ref] with frequent cardiovascular malformations such as laterality and looping defects, including transposition of the great vessels, ASDs, VSDs, and HLHS [bib_ref] Genetic and environmental risk factors of major cardiovascular malformations: the Baltimore-Washington infant..., Ferencz [/bib_ref] [bib_ref] Noninherited risk factors and congenital cardiovascular defects: current knowledge: a scientific statement..., Jenkins [/bib_ref] [bib_ref] Diabetes mellitus during pregnancy and the risks for specific birth defects: a..., Becerra [/bib_ref]. Finally, multiple studies have suggested a link between air pollutants exposed during first trimester and the risk of VSD, ASD, and PDA. In 2013, Agay-Shay et al. evaluated the connection between maternal exposure to air pollution and congenital heart defects, finding an association between at least six separate cardiac anomalies with exposure to one of six major pollutants [bib_ref] Air pollution and congenital heart defects, Agay-Shay [/bib_ref] [bib_ref] Association between maternal exposure to ambient air pollution and congenital heart disease:..., Dadvand [/bib_ref] [bib_ref] Ambient air pollution and risk of congenital anomalies in England, Dolk [/bib_ref] [bib_ref] Relation between ambient air quality and selected birth defects, seven county study, Gilboa [/bib_ref] [bib_ref] Ambient air pollution and birth defects in, Hansen [/bib_ref] [bib_ref] Pless-Mulloli T. Maternal exposure to ambient air pollutants and risk of congenital..., Rankin [/bib_ref] [bib_ref] Ambient air pollution and risk of birth defects in Southern California, Ritz [/bib_ref] [bib_ref] Ambient air pollution and cardiovascular malformations in, Strickland [/bib_ref].
## Non-modifiable risk factors
Maternal exposure to pesticides and some therapeutic agents such as anti-seizure medications, thalidomide, and indomethacin tocolysis have been known to contribute to CHD development [bib_ref] Noninherited risk factors and congenital cardiovascular defects: current knowledge: a scientific statement..., Jenkins [/bib_ref] [bib_ref] Genetic and environmental risk factors in congenital heart disease functionally converge in..., Lage [/bib_ref]. Additionally, potential confounding effects have been observed in studies evaluating the effect of maternal antidepressant use, specifically selective serotonin reuptake inhibitors (SSRIs) [bib_ref] Congenital heart disease: current knowledge about causes and inheritance, Blue [/bib_ref]. Several studies also suggest that maternal viral infection in early pregnancy, such as rubella or cytomegalovirus, is associated with significantly increased risk of CHD. Maternal comorbidities such as rheumatologic disorders (e.g., systemic lupus erythematous) and metabolic disorders (e.g., phenylketonuria) have also been shown to increase the risk of CHD.
Moreover, accumulating data suggest racial and ethnic disparities in multiple key outcome measures for patients with CHD [bib_ref] Racial and ethnic disparities in postoperative mortality following congenital heart surgery, Oster [/bib_ref]. Multiple studies have confirmed that when compared with their Caucasian counterparts, the incidence of all CHD in black infants was ∼50% higher and also attributed to more severe and complex CHD types [bib_ref] Ethnic and socioeconomic variation in incidence of congenital heart defects, Knowles [/bib_ref]. Additionally, substantial data indicate that infant and child mortality from CHD is consistently higher among blacks when compared to Caucasians, which has been a reoccurring trend [bib_ref] Mortality resulting from congenital heart disease among children and adults in the..., Gilboa [/bib_ref]. In a 2003-2006 analysis of neonatal mortality due to CHD, the Centers for Disease Control and Prevention found that black infants had 20% higher mortality than Caucasian infants [bib_ref] Racial and ethnic disparities in postoperative mortality following congenital heart surgery, Oster [/bib_ref] [bib_ref] Racial differences by gestational age in neonatal deaths attributable to congenital heart..., Petrini [/bib_ref]. The precise reason for this disproportional rate of CHD mortality among blacks and Hispanics remains unknown, but may be correlated FIGURE 1 | Risk factors of congenital heart disease. Modifiable risk factors associated with congenital heart disease include diet, obesity, substance abuse, and air pollution, whereas non-modifiable risk factors include rheumatologic disorders, genetic variants, specific medications, and infection. Each factor influences the development of this disease throughout pregnancy, effectively comprising the etiology of CHD and irreversibly altering the morphology of the fetal heart.
to underlying genetic variations specific to minority genomes, amongst other potential causes.
## Genetics of chd
Multiple clinical, epidemiological, and embryological studies have acknowledged the significance of genetic factors in CHD etiology [bib_ref] Congenital heart disease and genetic syndromes: specific correlation between cardiac phenotype and..., Marino [/bib_ref]. Although a growing number of genetic contributors for CHD have been discovered, the etiology for the majority of CHDs remains unknown [bib_ref] Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease, Basu [/bib_ref]. Recent progress made possible by new molecular biology methods has identified some of the genes responsible for CHDs [bib_ref] Congenital heart disease and genetic syndromes: specific correlation between cardiac phenotype and..., Marino [/bib_ref]. Furthermore, previous studies positing a multifactorial inheritance hypothesis for the etiology of CHDs (i.e., via combined effects of multiple genes interacting with environmental and/or stochastic factors) may be further investigated using modern techniques [bib_ref] Congenital heart disease and genetic syndromes: specific correlation between cardiac phenotype and..., Marino [/bib_ref] [bib_ref] Multifactorial inheritance hypothesis for the etiology of congenital heart diseases: the genetic-environmental..., Nora [/bib_ref] [bib_ref] The evolution of specific genetic and environmental counseling in congenital heart diseases, Nora [/bib_ref].
The genetic etiologies of CHD are heterogeneous and include chromosomal as well as Mendelian factors [bib_ref] Risk factors in congenital heart disease, Stoll [/bib_ref]. Previously CHD was primarily understood in the context of chromosomal abnormalities. Chromosomal anomalies are responsible for about 8-10% of presenting cases of CHD [bib_ref] Congenital heart disease: current knowledge about causes and inheritance, Blue [/bib_ref] [bib_ref] Inheritance of congenital heart disease, Roos-Hesselink [/bib_ref]. Chromosomal aneuploidy was the first identified genetic cause of syndromic CHD, accounting for a significant proportion of CHD [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref]. Chromosome anomalies also represent the most prevalent association, being diagnosed more frequently in patients with CHD than control subjects [bib_ref] Congenital heart disease and genetic syndromes: specific correlation between cardiac phenotype and..., Marino [/bib_ref]. Investigation into the genetic component for CHD was initiated based on their recurrence in families and by studies showing a correlation between CHD and inherited microdeletion syndromes [bib_ref] The developmental genetics of congenital heart disease, Bruneau [/bib_ref]. Previous research found that chromosomal aneuploidy is a genetic cause of syndromic CHD responsible for a large proportion of CHD [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref]. The most common chromosomal aneuploidy causing CHD is Down syndrome due to trisomy of chromosome 21 and partial trisomy 21 (i.e., translocation, mosaicism) [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref]. It is estimated that 50% of the patients with trisomy 21 (T21) and Turner syndrome (TS) have CHD [bib_ref] Congenital heart disease and cardiac procedural outcomes in patients with trisomy 21..., Morales-Demori [/bib_ref]. Similarly, other syndromic diseases such as Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), the most common micro-deletion disorder in humans, are characterized by craniofacial, parathyroid, and thymic defects as well as cardiac outflow tract malformations [bib_ref] The congenital heart disease genetic network study: cohort description, Hoang [/bib_ref].
Over the past decade, remarkable advances in genetic sequencing technologies have enabled more rapid discovery of new genes contributing to CHD that allowed healthcare researchers to better understand the genetic basis of CHD [bib_ref] Genetics of congenital heart disease: the glass half empty, Fahed [/bib_ref] [bib_ref] Genetic basis for congenital heart disease: revisited: a scientific statement from the..., Pierpont [/bib_ref]. For instance, next-generation sequencing (NGS) enables quick analysis of large amounts of genetic information [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref] [bib_ref] Disease gene identification strategies for exome sequencing, Gilissen [/bib_ref] [bib_ref] Genetics of congenital heart disease: the contribution of the noncoding regulatory genome, Postma [/bib_ref]. Bioinformatic analysis is vital to processing and analyzing the resultant biological data. Single-nucleotide polymorphisms (SNPs) represent changes in single nucleotides found in coding or non-coding regions of the genome [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref]. So far, several SNPs have been identified in the development and progression of CHD [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref]. For example, a study conducted on 114 CHD patients in 2001 by Junker et al. reported that MTHFR 677TT genotype was associated with CHDs such as pulmonary valve stenosis, coarctation of the aorta, HLHS, and aortic valve stenosis [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref] [bib_ref] Infant methylenetetrahydrofolate reductase 677TT genotype is a risk factor for congenital heart..., Junker [/bib_ref]. De novo mutations represent another genetic variant arising during embryogenesis that are known to influence CHD. In 2020, Homsy et al. used exome sequencing to identify an excess of protein-damaging de novo mutations in genes highly expressed in the developing heart [bib_ref] De novo mutations in congenital heart disease with neurodevelopmental and other congenital..., Homsy [/bib_ref].
Another useful advance is the identification of copy number variations (CNVs). CNVs of DNA sequences are sub-chromosomal changes resulting in a large deletion or amplification of DNA segments due to inappropriate recombination that leads to alterations in genes [bib_ref] Genetics of congenital heart disease: past and present, Muntean [/bib_ref]. CNVs are important because subtle variations in the number of copies of genes can significantly affect the development of cardiovascular disease [bib_ref] Diversity of human copy number variation and multicopy genes, Sudmant [/bib_ref]. CNVs of various sizes can be identified by cytogenetic techniques such as comparative genomic hybridization (CGH) or multiplex ligation-dependent amplification (MLPA) [bib_ref] Diversity of human copy number variation and multicopy genes, Sudmant [/bib_ref] , which can be used for detecting CNV in patients with isolated CHD [bib_ref] Screening of congenital heart disease patients using multiplex ligationdependent probe amplification: early..., Sørensen [/bib_ref].
## Genetic link to racial and ethnic disparity
The term "race, " in its traditional genetic conceptualization, is often determined through patterns of human variation reflected from our evolutionary history to serve as a definitive measure [bib_ref] Disparities in infant mortality: what's genetics got to do with it?, David [/bib_ref]. Individuals belonging to different "races" are assumed to differ at the genome level [bib_ref] Disparities in infant mortality: what's genetics got to do with it?, David [/bib_ref]. The assumption of "race" is an essential prerequisite when considering differences and commonalities among patients or groups of patients [bib_ref] Disparities in infant mortality: what's genetics got to do with it?, David [/bib_ref]. Genetic variations that determine phenotypic differences among individuals may also influence disease development, including cardiovascular disease states.
While studies have suggested that racial and ethnic differences may influence the prevalence and outcomes of CHD, the precise genetic and/or environmental causation is not well-established [bib_ref] White-black differences in cardiovascular malformations in infancy and socioeconomic factors, Correa-Villaseñor [/bib_ref] [bib_ref] Racial and temporal variations in the prevalence of heart defects, Botto [/bib_ref]. It is possible that certain variants or CHD-susceptible genetic loci are more prevalent in certain ethnic or racial groups [bib_ref] White-black differences in cardiovascular malformations in infancy and socioeconomic factors, Correa-Villaseñor [/bib_ref] [bib_ref] Racial and temporal variations in the prevalence of heart defects, Botto [/bib_ref]. However, the temporal and racial variations in CHD occurrence remain poorly understood, highlighting the necessity to study further its genetic and environmental determinants [bib_ref] Racial and temporal variations in the prevalence of heart defects, Botto [/bib_ref]. Additionally, racial and ethnic variations that influence CHD development may also be attributed to socioeconomic differences, cultural factors, lifestyle variation, and other factors that indicate environmental exposures [bib_ref] Racial and temporal variations in the prevalence of heart defects, Botto [/bib_ref]. Resultant disparities in maternal health can impact the fetal environment and lead to birth defects, which may be partly explained by epigenetic mechanisms such as posttranslational modifications of histones, DNA methylation, and non-coding RNAs [bib_ref] Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease, Basu [/bib_ref] [bib_ref] Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental..., Jaenisch [/bib_ref] [bib_ref] Epigenetic factors and cardiac development, Van Weerd [/bib_ref]. In a recent study, Basu et al. reported an epigenetic mechanism underlying the gene-environment interaction between Notch1 haploinsufficiency and maternal diabetes mellitus that leads to CHD [bib_ref] Epigenetic mechanisms underlying maternal diabetes-associated risk of congenital heart disease, Basu [/bib_ref].
## Use of ipscs to model chd
A compelling new paradigm in cardiovascular disease modeling is the use of induced pluripotent stem cells (iPSCs) and their differentiated cardiovascular cells to establish in vitro models of human physiology [bib_ref] Will iPSCcardiomyocytes revolutionize the discovery of drugs for heart disease?, Bruyneel [/bib_ref]. First introduced in 2007, human iPSCs have revolutionized biomedical research by improving disease modeling and interrogation of drug response and toxicity, as well as generating a variety of cell types for therapeutic transplantation, amongst other advances [bib_ref] Patient-specific stem cells and cardiovascular drug discovery, Mordwinkin [/bib_ref] [bib_ref] Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Takahashi [/bib_ref]. Patientspecific iPSCs, especially in combination with advanced NGS technologies, are crucial in accelerating the investigation of molecular mechanisms for cardiovascular disorders while also helping to identify novel therapeutic targets for these diseases [bib_ref] Patient-specific stem cells and cardiovascular drug discovery, Mordwinkin [/bib_ref]. A significant advantage is that iPSCs can be differentiated into various cell types such as cardiomyocytes (iPSC-CMs), endothelial cells (iPSC-ECs), and cardiac fibroblasts (iPSC-CFs), making it possible to study human genetics and proteins in their native cellular context [bib_ref] Will iPSCcardiomyocytes revolutionize the discovery of drugs for heart disease?, Bruyneel [/bib_ref].
Another benefit of using iPSCs derived from adult somatic cells is that they can be tailored to the unique individual genetics of patients. This generates novel insights into the molecular mechanisms of heart disease that may allow clinicians to deliver patient-specific pharmacological, genetic and cellular therapies in the future [bib_ref] Human stem cells for modeling heart disease and for drug discovery, Matsa [/bib_ref]. In 2020, Miao et al. utilized scRNA-seq analysis with patient-specific iPSC-ECs and human fetal heart tissue to reveal endocardial functional defects and aberrant endocardiummyocardium crosstalk in HLHS [bib_ref] Intrinsic endocardial defects contribute to hypoplastic left heart syndrome, Miao [/bib_ref]. This innovative study focused on mutations in a transcription factor (ETS) and a chromatin remodeler (CHD7), identifying a large, downstream gene network that was differentially expressed in control vs. HLHS endocardial cells [bib_ref] Intrinsic endocardial defects contribute to hypoplastic left heart syndrome, Miao [/bib_ref]. Another study by Paige et al. observed a drastic impairment in contractility of HLHS iPSC-CMs in addition to associated changes in gene expression that significantly overlapped prior studies of human heart failure (67). Using iPSC-CMs they discovered 3 gene sets that were identified as molecular coordinators in heart failure (i.e., local, pathway, and central coordinators) [bib_ref] Patient-specific induced pluripotent stem cells implicate intrinsic impaired contractility in hypoplastic left..., Paige [/bib_ref]. Finally, Kitani et al. provided genome-wide transcriptomic profiles of iPSC-CMs that were established from 5 patients with single ventricle disease (SVD) (including 1 HLHS, 2 tricuspid atresia, 1 double-outlet right ventricle, 1 double-inlet left ventricle) vs. five patients with nonsyndromic ToF [bib_ref] RNA sequencing analysis of induced pluripotent stem cell-derived cardiomyocytes from congenital heart..., Kitani [/bib_ref]. They discovered that both SVD iPSC-CMs and ToF iPSC-CMs express unique transcriptomes compared with non-CHD iPSC-CMs. These and other studies provide growing evidence for the effectiveness of utilizing iPSCs to model disease states and develop novel therapeutic regimens [fig_ref] TABLE 1 |: iPSC-based studies to model CHD [/fig_ref].
## Modeling racial influence of chd using ipscs
Patient-specific iPSCs help make it possible to analyze causal relationships in specific variants identified via SNP or CNV that are most prevalent among minorities (i.e., blacks and Hispanics). In 2016, Tomita-Mitchell et al. performed NGS on a multigenerational family with a high prevalence of CHD, identifying a rare variant in the α-myosin heavy chain (MYH6) gene [bib_ref] Impact of MYH6 variants in hypoplastic left heart syndrome, Tomita-Mitchell [/bib_ref]. Additionally, Glessner et al. identified a loss-offunction mutation in ETS1 in patients with a hypoplastic left ventricle and other features found in Jacobsen syndrome [bib_ref] Intrinsic endocardial defects contribute to hypoplastic left heart syndrome, Miao [/bib_ref] [bib_ref] Increased frequency of de novo copy number variants in congenital heart disease..., Glessner [/bib_ref]. Future advances should be directed toward identifying specific genetic variants that may be disproportionately affecting blacks and Hispanic cohorts, consequently influencing the severity of CHD. Traditionally, minorities are not as well-represented in clinical trials or drug development compared to their Caucasian counterparts, a gap that may be addressed at least partially by using iPSCs to help identify drug or therapeutic applications effective in these populations. Various studies have shown the value of iPSCs in elucidating the molecular and cellular mechanisms of cardiac arrhythmias in disease states while providing a robust platform for the development novel drugs for clinical therapy [bib_ref] Human stem cells for modeling heart disease and for drug discovery, Matsa [/bib_ref]. However, to date, little research has made the connection between racial disparities among patients with CHD that disproportionally affect minorities, and the full utility of iPSCs to address possible underlying genetic causal variants remains to be explored.
## Advancing technologies
Using iPSCs to recapitulate a clinically relevant readout in a high throughput assay, researchers are now able to leverage chemical and functional genomics (e.g., siRNA and CRISPR screening) to better understand and interpret disease mechanisms for identifying novel therapeutic targets [bib_ref] Will iPSCcardiomyocytes revolutionize the discovery of drugs for heart disease?, Bruyneel [/bib_ref]. The applications of these advanced technologies will expand rapidly in the future to improve our ability to understand and ultimately treat CHD [bib_ref] Will iPSCcardiomyocytes revolutionize the discovery of drugs for heart disease?, Bruyneel [/bib_ref]. Elucidating the complex pattern of functional interactions between genomic variation and environmental exposure that regulate essential biological systems during heart development may help us better understand the correlation between CHD mortality and racial disparities. The ultimate goal is to create treatment platforms that will help reduce disproportionate mortalities among minorities [bib_ref] Genetic and environmental risk factors in congenital heart disease functionally converge in..., Lage [/bib_ref].
Additionally, advances in genome-editing technologies have enabled biomedical researchers to precisely edit or introduce mutations in disease-causing genes to analyze the relative contribution of a single mutation on the severity of the disease phenotype [bib_ref] Will iPSCcardiomyocytes revolutionize the discovery of drugs for heart disease?, Bruyneel [/bib_ref]. The utilization of genome-edited iPSC lines whose cardiovascular disease-associated mutations/variants are engineered into the same genetic background by endonuclease [i.e., zinc finger nucleases (ZFN), transcription activatorlike effector nuclease (TALEN)] or palindromic repeat [i.e., clustered regularly interspaced short palindromic repeats (CRISPR)] is instrumental for generating libraries of disease-specific cardiomyocytes for drug testing and disease modeling [bib_ref] Human stem cells for modeling heart disease and for drug discovery, Matsa [/bib_ref] [bib_ref] Genome editing of human embryonic stem cells and induced pluripotent stem cells..., Wang [/bib_ref].
Another emerging technology that has the potential to enhance the management and understanding of CHD is machine learning. Future management and treatment of CHD will rely on increased understanding of the underlying mechanism of CHD, more precise early detection, and enhanced management strategies. Machine learning is an increasingly popular method to unearth underlying patterns in large datasets and in turn convert latent patterns into opportunities for early and precise predictions. Machine learning has already been utilized with CHD datasets to achieve early CHD diagnosis and to identify risk factors and optimal management strategies with great success. Using over 44,000 patient medical records for 10,019 patients, Diller et al. developed a deep learning model to predict primary clinical diagnosis, disease complexity, and optimal treatment regimen (69). In the future, CHD patients may be triaged at an early stage in an evidencebased manner and treatment strategies can be streamlined through in silico predictions. Further, machine learning is being used to identify and validate causes of CHD on a population level, in one instance elucidating the relationship between air pollutants and increased fetal CHD risk in pregnant women [bib_ref] Single-cell transcriptomic analysis of cardiac differentiation from human PSCs reveals HOPX-dependent cardiomyocyte..., Friedman [/bib_ref].
In addition to modeling the clinical progression of CHD, machine learning offers the potential, when combined with current CHD modeling platforms such as iPSCs and omicstechnology, to unearth underlying genetic mechanisms of CHD. In particular, machine learning has been instrumental in analyzing many recently created cardiovascular single cell RNA sequencing (scRNA-seq) datasets [bib_ref] Single-cell RNA sequencing in cardiovascular development, disease and medicine, Paik [/bib_ref]. scRNA-seq are large datasets that benefit from the capability of machine learning models to learn from the datasets to identify inherent patterns and structure. Two classes of unsupervised machine learning models, dimensionality reduction and clustering techniques, have formed the basis of scRNA-seq analysis and visualization [bib_ref] Challenges in unsupervised clustering of single-cell RNA-seq data, Kiselev [/bib_ref]. The resolution of sc-RNAseq datasets combined with the data analysis power of machine learning have led to countless discoveries in CHD: scRNA-seq datasets have been used to create cell atlases of cardiac development [bib_ref] Transcriptomic profiling maps anatomically patterned subpopulations among single embryonic cardiac cells, Li [/bib_ref] , perform lineage reconstruction to delineate coronary artery development (75), and uncover mechanisms that regulate emergence and segregation of early cardiac lineages that form the heart (76). Specifically, when applied to iPSCs, machine learning has been used to identify key regulators in cardiac development from scRNA-seq of iPSC-CM differentiation [bib_ref] Intrinsic endocardial defects contribute to hypoplastic left heart syndrome, Miao [/bib_ref] [bib_ref] Single-cell transcriptomic analysis of cardiac differentiation from human PSCs reveals HOPX-dependent cardiomyocyte..., Friedman [/bib_ref] ; and mechanisms FIGURE 2 | Applications of genomics and patient-specific iPSCs to reveal race-related genetic contribution to congenital heart disease. In combination with next generation sequencing and genome editing technology, iPSC-based disease modeling could be utilized to identify genetic variants that exist disproportionally within racial minority communities, thus providing a valuable tool for developing novel therapeutic treatment options to help those who have offspring suffering from CHD. of hypoplastic left heart syndrome from scRNA-seq of iPSCendothelial cells [bib_ref] Intrinsic endocardial defects contribute to hypoplastic left heart syndrome, Miao [/bib_ref].
# Conclusion
In summary, by utilizing advances in genomics and genetic technologies in combination with iPSCs, an innovative platform can be developed to address racial disparities in mortality rates among patients with CHD . By identifying novel genetic variants that are specific to minorities disproportionally afflicted with this disease, novel cardiovascular disease modeling systems can be designed to improve the manner in which CHD has been treated. By using the aforementioned gene editing technology as a therapeutic option, this could ultimately make it possible to identify the causes of racial disparities and find effective treatments to reduce them.
# Author contributions
MM, J-WR, and JW contributed in drafting the manuscript. AZ, GL, and AR editing the manuscript. All authors contributed to the article and approved the submitted version.
# Funding
This work was supported by National Institutes of Health Grants K08 HL148540 (J-WR), F30 HL156478 (AZ), and R01 HL126527, R01 HL130020, R01 HL146690, and American Heart Association 17MERIT3361009 (JW). Due to space limitation, we apologize in advance for not being able to include all references on this topic.
[table] TABLE 1 |: iPSC-based studies to model CHD. [/table]
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Achieving remission or low disease activity is associated with better outcomes in patients with systemic lupus erythematosus: a systematic literature review
# Introduction
A treat-to-target (T2T) strategy has been proposed for several chronic diseases in order to improve the affected patients' treatment, and thus, their outcome; in systemic lupus erythematosus (SLE), however, a uniform definition of treatment goals is lacking.
The ideal goal is remission, which was defined in 2015 and modified in 2021 by the DORIS (Definition Of Remission In SLE) group as the absence of clinical disease activity (Clinical Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)=0 and Physician Global Assessment (PGA) <0.5), with no or minimal intake of glucocorticoids (prednisone daily dose not higher than 5 mg/ day) and/or immunosuppressive drugs on stable maintenance dose. 1 2 However, some modifications of this definition have been reported in the literature.
Nevertheless, as remission state is not achieved frequently, [bib_ref] Prevalence of remission and its effect on damage and quality of life..., Mok [/bib_ref] [bib_ref] Remission of systematic lupus erythematosus, Drenkard [/bib_ref] [bib_ref] Analysis of complete remission in systemic lupus erythematosus patients over a 32-year..., Medina-Quiñones [/bib_ref] low disease activity (LDA) has been proposed as an alternative target. To this end, there are several
## Key messages
What is already known about this subject? ► Remission and low disease activity (LDA) have been reported as potential targets in the systemic lupus erythematosus (SLE) treatment.
What does this study add? ► Remission and LDA (regardless of the definitions used) are associated with better outcomes.
How might this impact on clinical practice or future developments? ► Remission and LDA should be considered as the target for the management of patients with SLE. ► However, it is important to have a uniform definition of both.
## Lupus science & medicine
definitions about LDA in the literature; for example, the Asia Pacific Lupus Consortium (APLC) has introduced the lupus low disease activity state (LLDAS): SLEDAI ≤4, which allows a low level of disease activity, without activity in major organ systems or new disease activity, PGA ≤1, prednisone daily dose not higher than 7.5 mg/day and/ or immunosuppressive drugs on maintenance dose. [bib_ref] Definition and initial validation of a lupus low disease activity state (LLDAS), Franklyn [/bib_ref] The Toronto Lupus Cohort investigators have proposed using the term low disease activity (LDA by Toronto Lupus Cohort): SLEDAI (excluding serology)≤2, without prednisone and immunosuppressive drugs. [bib_ref] Defining low disease activity in systemic lupus erythematosus, Polachek [/bib_ref] All these definitions allow the use of antimalarials. The probability of patients achieving these states seems to vary according to a number of factors including race/ethnicity, in particular African ancestry, age at diagnosis, [bib_ref] Predictors of remission and low disease activity state in systemic lupus Epidemiology..., Ugarte-Gil [/bib_ref] previous disease activity, [bib_ref] Remission in systemic lupus erythematosus: durable remission is rare, Wilhelm [/bib_ref] major organ involvement and treatment. [bib_ref] Remission in systemic lupus erythematosus: durable remission is rare, Wilhelm [/bib_ref] [bib_ref] Predictors of predominant lupus low disease activity state (LLDAS-50), Babaoglu [/bib_ref] [bib_ref] Predictors of remission and low disease activity state in systemic lupus Epidemiology..., Ugarte-Gil [/bib_ref] Furthermore, the clinical impact of achieving such states in several clinical outcomes has been examined. [bib_ref] New therapeutic strategies in systemic lupus erythematosus management, Gatto [/bib_ref] The outcome most frequently evaluated has been organ damage accrual; in fact, in several cohorts, remission and/or LDA have been found to prevent damage, but the exact definitions used for these states have not been uniform. [bib_ref] Prevalence of remission and its effect on damage and quality of life..., Mok [/bib_ref] One of the main challenges is to validate whether all these definitions are indeed predictive of outcomes such as organ damage, death, recurrent flares, number of hospitalisations and quality of life (QoL), and which of them would be the better option. Therefore, our aim was to perform a systematic review of the current literature to assess the impact of the existing definitions of remission/ LDA on relevant outcomes of patients with SLE.
# Methods
## Search strategies
A systematic review according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement, Moher [/bib_ref] was carried out. The protocol was registered with PROSPERO (CRD42020162724).
We used the electronic databases PubMed (1946-week 2, April 2021), Cochrane library (1985-week 2, week 2, April 2021) and EMBASE (1974-week 2, April 2021) were searched. We used the Medical Subject Heading (MeSH) terms and Key words in all possible combinations using Boolean operators with the following search strategy: 'systemic lupus erythematosus', 'lupus', 'SLE', 'remission', 'low disease activity status', 'low lupus disease activity status', 'minimal disease activity'. References of all included full-text articles were handsearched in order to find additional references from the articles that seem to be relevant for the review. Details of the full search strategy are listed in online supplemental table 1.
These articles were downloaded into EndNote software (V.9.3.2); duplicates were deleted. Two independent teams examined each selected article and performed data extraction independently (MFU-G and CR-S or CM-P and GP-E). In case of disagreement, a third investigator was consulted. Discrepancies were resolved by consensus. The literature review team also made every effort to identify multiple publications from a single cohort.
## Criteria for the selection of studies
We included both observational studies (case-control, cross-sectional or cohort) and clinical trials on adults or children with SLE in LDA (using a validated definition) or remission (as defined by available criteria) and reporting different disease outcomes in the follow-up (mortality, damage, flare, health-related QoL (HRQoL), risk of cardiovascular disease, hospitalisations and direct healthcare cost). A minimum sample size of 100 patients was required for an article to be included. Patients needed to have similar duration of follow-up in studies that reported flare rates (using a validated definition) as percentages; alternatively, reported flares per person-years was used in cases where patients had unequal follow-up duration. Damage data, as assessed by the validated instrument (the Systemic Lupus International Collaborating Clinics/ American College of Rheumatology Damage Index (SDI)), were considered.
Studies published only as abstracts were excluded. Articles written in English or Spanish were included. Case reports, case series, editorials, comments, letters and reviews were excluded.
## Data extraction
Two reviewers independently screened all articles and applied the eligibility criteria to identify appropriate Epidemiology and outcomes studies for inclusion; the selected articles were then abstracted, also independently, using a predetermined form. Information was collected on the study characteristics (study design, country, sample size), the number of participants, gender, age, major clinical variables (damage), definition of LDA/remission used, flare rates or flares per person-years, HRQoL scores, HRQoL instruments, hospitalisation rates, mortality rates, direct healthcare cost, definitions of cardiovascular disease and rates or risk of cardiovascular disease. If the same article reported more than one definition of the states or more than one outcome, all of them were included in the respective analyses.
## Epidemiology and outcomes
Evaluation of the quality of the studies The quality of the studies identified was assessed using the Newcastle-Ottawa Scale (NOS) for cohort and casecontrol studies a tool specifically developed to assess the quality of observational studies. [bib_ref] Both prolonged remission and lupus low disease activity state are associated with..., Tsang-A-Sjoe [/bib_ref] The scoring system covers three major domains: selection of cohorts or cases and controls (maximum four points), comparability of selected groups (maximum two points) and ascertainment of either the exposure or the outcome of interest (maximum three points): the resulting score ranges from 0 to 9; a higher score represents a better methodological quality. While there is no validated cut-off value to discern between studies of good or poor quality, studies with a score of ≥7 were arbitrarily defined as being of high quality.Strategy for analysis synthesis Due to the diversity of remission and LDA definitions, outcomes, heterogeneity of the results and of the different statistical tests performed in the selected articles, a metaanalysis was felt not to be feasible for most of the outcome variables; therefore, the studies selected were summarised using a narrative synthesis approach. A description and rationale were provided for grouping studies for synthesis (eg, according to outcomes type). Established metrics were used to measure the direction and magnitude effect of association between remission/LDA and outcomes (eg, OR, risk ratio (RR), HR, among others) when they were available. Summary tables and structured narrative were employed to descriptively summarise and compare each included study and to examine the heterogeneity across studies. [bib_ref] Synthesis without meta-analysis (swim) in systematic reviews: reporting guideline, Campbell [/bib_ref]
# Results
## Study selection and characteristics of studies included
Our search identified 7497 articles, of which 31 studies met the inclusion criteria. 3-5 7 11 14-18 The study selection process and reasons for exclusion are shown in [fig_ref] Figure 1: PRISMA flowchart. [/fig_ref]. Four studies were cross sectional, 27 were longitudinal, 12 (38.7%) were from Europe, 10 (32.3%) from Asia and Australia, 5 (16.1%) from Latin America and 4 (12.9%) from the USA and Canada. The large majority of studies were of high quality according to NOS [fig_ref] Table 1: Characteristics of the articles included in this systematic review [/fig_ref].
## Remission and lda rates
The rates of remission and LDA varied depending on both the definition used and the population studied.
Remission was more frequent in European populations being as high as 88.1% in one study, but it was as low as 3.5% when the definition excluded patients under treatment and a duration of the remission of at least 7 years. LDA was also more frequent in European populations; however, the rate depended on the definition used; as expected, the less stringent the definition, the more frequently this outcome was achieved. These data are depicted in online supplemental table 2.
## Mortality
Six studies including 3933 patients evaluated mortality as an outcome, two evaluated the impact of remission and LDA on mortality, two only LDA, one only remission and one compared remission and LDA. Among the four studies reporting the impact of LDA on mortality, two of them reported a reduction on mortality (HR 0.3% and 1.4% in those in LDA and 6.9% in those active) and two did not, although the trend was similar (HR 0.30 and 0.81, p: not significant). Among the three studies evaluating the impact of remission (compared with those not on remission) on mortality; two of them reported a reduction on mortality (HR 0.08% and 5% in those in remission and 17.7% in those not in remission), whereas the other did not (HR 0.56, p value not significant). In another report, remission was not statistically different from LDA in terms of the mortality rate. These data are depicted in table 2.
## Damage accrual
Sixteen studies including 8288 patients evaluated damage accrual. In the majority of studies, both remission and LDA prevented damage accrual when compared with patients who did not attain these states (risk measures between 0.04 and 0.95 for remission and between 0.07 and 0.90 for LDA, depending on the definition). In most of the studies, LDA also included those patients who were on remission; however, depending on the definition used, there could be a difference between those in remission and those in LDA, being better to be on remission. These data are depicted in tables 3 and 4.
## Flare
Five studies including 3033 patients evaluated longitudinally the occurrence of flares after achieving these states. Remission and LDA reduced the probability of flares in all studies included, regardless of the definition used (HR between 0.26 and 0.70 for remission and between 0.41 and 0.74 for LDA); however, the longer the duration of the state, the lower the risk. Only one study compare remission versus LDA and it did not find a statistically significant difference. These data are depicted in table 5.
## Epidemiology and outcomes
Health-related quality of life (HRQoL) Ten manuscripts including 4480 patients evaluated HRQoL. Remission and LDA were associated with a better HRQoL being this impact more consistent on the physical components of HRQoL, and less so on the mental components of HRQoL. These data are depicted in tables 6 and 7.
Other outcomes Three manuscripts including 802 patients evaluated other outcomes. Being on remission and LDA was associated with a lower hospitalisation rate; LDA was associated with lower medical cost and prolonged remission with lower cardiovascular risk. These data are depicted in table 8.
# Discussion
Our systematic literature search showed that being in remission or LDA, regardless of the definitions used, was associated with better outcomes in patients with SLE, the most commonly reported outcomes being lower damage accrual, fewer flares and a better HRQoL. The association with a lower mortality rate was less consistently reported.
In terms of mortality, LDA was associated with lower mortality in two studies, one from the Toronto Lupus Cohort, 7 which had a more stringent definition of LDA (SLEDAI ≤2 without treatment) and the other from Norway 40 (which allowed a SLEDAI ≤4, excluding new activity and major organ activity, and allowing prednisone ≤7.5 mg/day and immunosuppressive drugs on maintenance dose); similarly, remission was associated with lower mortality in a study from Mexico 4 and in one from the UK. [bib_ref] Analysis of complete remission in systemic lupus erythematosus patients over a 32-year..., Medina-Quiñones [/bib_ref] However, in the GLADEL 14 and the LUMINA 20 cohorts, the association between remission and LDA and mortality was not statistically significant, although the trend was in the protective direction. This lack of association between achieving these outcomes and mortality could be due to a relatively short follow-up time in these cohorts. The Toronto Lupus Cohort compared remission and LDA and found no statistically significant difference between the two states in terms of mortality. [bib_ref] Defining low disease activity in systemic lupus erythematosus, Polachek [/bib_ref] Remission was associated with a lower risk of damage accrual in several cohorts from Asia, Europe, North America (USA-Canada) and Latin America 3 7 14-16 ; however, the minimum time on remission needed to prevent damage accrual has yet to be determined. According to the Padua cohort, being in remission for less than 1 year was not protective against damage, [bib_ref] The effect of different durations of remission on damage accrual: results from..., Zen [/bib_ref] whereas according to the Hopkins cohort, being in remission even less than 25% of the follow-up time prevented the accrual of damage. 1818 According to the GLADEL cohort, being in remission prevented not only the accrual of any damage but also the accrual of severe damage (an increase in the SDI of at least 3 points) and from non-glucocorticoid (GC)-related damage and severe damage. [bib_ref] Remission and low disease activity status (LDAS) protect lupus patients from damage..., Ugarte-Gil [/bib_ref] Additionally, the longer the duration of remission, the lower the probability of damage accrual. [bib_ref] The effect of different durations of remission on damage accrual: results from..., Zen [/bib_ref] ; however, in the Padua cohort, being on LDA for less than 1 year did not prevent the accrual of damage. [bib_ref] Lupus low disease activity state is associated with a decrease in damage..., Zen [/bib_ref] In the Hopkins cohort, being in LDA for less than 25% of the follow-up did not prevent the accrual of damage. [bib_ref] Comparison of remission and lupus low disease activity state in damage prevention..., Petri [/bib_ref] Being in LDA prevented also severe damage accrual, non-GC and GC-related damage [bib_ref] Remission and low disease activity status (LDAS) protect lupus patients from damage..., Ugarte-Gil [/bib_ref] ; furthermore, the longer the duration of LDAS, the less the damage accrued. [bib_ref] Remission and low disease activity state (LDAS) are protective of intermediate and..., Alarcón [/bib_ref] In the Toronto cohort, being on remission and LDA (SLEDAI ≤2 without treatment) did not differ in terms of the risk of damage accrual 7 ; however, in the Padua cohort, being in remission was associated with a lower risk of damage that being on LLDAS (which allowed a SLEDAI ≤4, excluding new activity and major organ activity, and allowing prednisone ≤7.5 mg/day and immunosuppressive drugs on maintenance dose). [bib_ref] Lupus low disease activity state is associated with a decrease in damage..., Zen [/bib_ref] Probably, the difference in the definitions used in both cohorts could explain these results. Consistent with these results, prolonged remission was associated with a lower probability of cardiovascular events. [bib_ref] Prolonged remission is associated with a reduced risk of cardiovascular disease in..., Fasano [/bib_ref] Being in remission or LDA reduced the risk of any flares, being those mild-moderate or severe. Only in the Toronto cohort remission and LDA (SLEDAI ≤2 without treatment) were compared, but no differences were found. [bib_ref] Defining low disease activity in systemic lupus erythematosus, Polachek [/bib_ref] Patient perspective is important in defining the optimal treatment target. In previous reports, the association between disease activity and HRQoL has been low or absent. [bib_ref] Factors affecting quality of life in patients with systemic lupus erythematosus: important..., Elera-Fitzcarrald [/bib_ref] Notably, remission and LDA have been found to be associated with a better HRQoL in cross-sectional and longitudinal studies regardless of whether generic or lupus-specific measures were used. These associations were more consistently reported in the physical than in the mental domains, probably because the mental domains are affected also by comorbid conditions such as depression, fibromyalgia and anxiety. It has been suggested that specific measures may ascertain better QoL dimensions specific to patients with SLE. [bib_ref] Factors affecting quality of life in patients with systemic lupus erythematosus: important..., Elera-Fitzcarrald [/bib_ref] Finally, remission and LDA could reduce hospitalisation rate; this has been reported in the Peruvian Almenara Lupus cohort [bib_ref] Remission and low disease activity state prevent hospitalizations in systemic lupus erythematosus..., Reátegui-Sokolova [/bib_ref] ; LDA could also reduce annual medical cost as reported in a study from an Australian cohort. [bib_ref] Lupus low disease activity state and reduced direct health care costs in..., Yeo [/bib_ref] It is important to point out that this information needs to be confirmed in other populations.
Taking together, being on remission or on LDA, regardless of the definitions used, is associated with better outcomes, including mortality, damage, flares, HRQoL, hospitalisation and cost. It is important, however, to point out that a uniform definition of both states is desirable in order to make these results comparable. The current definition of remission, as proposed by the DORIS group, takes into account two physician disease activity measures (clinical SLEDAI=0 and PGA<0.5) as well as treatment (prednisone daily dose not higher than 5 mg/day and/ or immunosuppressive drugs on maintenance dose),and, even not all the studies used this definition, the large majority used 2015 or 2021 DORIS definitions 1 2 or a very similar definition. LDA should be different enough from remission in order to define a group of patients
## Lupus science & medicine
with a better prognosis than those with active disease, but, not as good as the prognosis of those on remission; in this context, the definition proposed by APLC is a good option as it allows a higher level of disease activity (SLEDAI ≤4 and PGA≤1), excludes activity in major organs and new activity, and also allows a higher dose of prednisone (7.5 mg/day) and keeping the immunosuppressive drugs on maintenance dose. [bib_ref] Definition and initial validation of a lupus low disease activity state (LLDAS), Franklyn [/bib_ref] Additionally, in the KORNET cohort from Korea, LLDAS, but not LDA (SLEDAI ≤2 without treatment) or MDA (minimal disease activity) were predictive of good outcomes. [bib_ref] Comparison of three different definitions of low disease activity in patients with..., Kang [/bib_ref] However, more information is needed in order to determine if being on remission is better than being on LDA. About the duration of these states, it seems that achieving these states even for a short period of time is associated with better outcomes, but the longer the patient remains on these states, the better the outcomes will be. These analyses have some limitations; first, as the studies included used different definitions for remission and LDA, a meta-analysis could not be performed. Second, the duration of follow-up in some studies reviewed was not long enough for the assessment of mortality. Third, there are only a few studies for some of the outcomes assessed; this precludes us from making stronger conclusions.
The main strength of this report is the inclusion of several different populations from across the world and several outcomes, allowing us to evaluate the real impact of remission and LDA in the prognosis of patients with SLE.
In conclusion, being in remission or LDA (regardless of the definition) is associated with improved outcomes in patients with SLE. These results reinforce the relevance of these outcomes for the management of patients with SLE.
In order to facilitate the implementation of a T2T strategy in SLE, it is important to have an uniform definition of remission 1 and LDA. Twitter Manuel Francisco Ugarte-Gil @mugartegil and Guillermo J. Pons-Estel @ gponsestel Contributors All authors were involved in drafting or revising this article critically for important intellectual content, and all authors approved the final version to be published. MFU-G has full access to all of the data from the study and takes responsibility for their integrity and the accuracy of the analyses performed.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
[fig] Figure 1: PRISMA flowchart. [/fig]
[table] Table 1: Characteristics of the articles included in this systematic review [/table]
[table] Table 2: Impact of remission and LDA on mortality* *If an article included more than one definition, a row per definition is included. AM, antimalarials; IS, immunosuppressive drug; LDA, low disease activity; LDAS, low disease activity status; NR, not reported; PDN, prednisone; PGA, Physician Global Assessment; RR, risk ratio; SLAM, Systemic Lupus Activity Measure; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index. [/table]
[table] Table 3: Impact of remission on damage [/table]
[table] Table 6: Impact of remission on HRQoL* [/table]
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The search for, and chemistry and mechanism of, neurotrophic natural products
Neurotrophic factors, now termed neurotrophins, which belong to a class of polypeptidyl agents, have been shown to potentially be beneficial for the treatment of neurodegenerative diseases such as Alzheimer's disease, because endogenous neurotrophic factors (NGF, BDNF, NT3, NT4) have been recognized to play critical roles in the promotion of neurogenesis, differentiation, and neuroprotection throughout the development of the central nervous system. However, high-molecular weight proteins are unable to cross the blood-brain barrier and are easily decomposed by peptidase under physiological conditions. To address this issue, small molecules that can mimic the functions of neurotrophic factors would be promising alternatives for the treatment of neurodegenerative disease. We have continued to search for natural products having typical neurotrophic properties, which can cause neurogenesis, enhance neurite outgrowth, and protect neuronal death using three cellular systems (PC12, rat cortical neurons, and MEB5 cells). In this review, we summarize the neurotrophic activities and synthesis of dimeric isocuparane-type sesquiterpenes from the liverwort, Mastigophora diclados, the mechanism of neurotrophic neolignans, magnolol, honokiol and their sesquiterpene derivatives, and introduce unique neurotrophin-mimic natural products, including seco-prezizaane-type sesquiterpenes from the Illicium species, vibsane-type diterpenes from Viburnum awabuki, and miscellaneous natural products with neurotrophic effects discovered by us.Graphic abstractNeuronal stem cells Neurons Death of neuronsNeurotrophic compounds O O O O H MeO O O O OH O O OH OH OHPublisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
# Introduction
In recent years, the percentage of elderly people has increased. In Japan, the population ratio of people aged more than 65 years is estimated to reach 29.1% by 2020 and further increase to 38.5% by 2050. In a superaged society, people wish for healthy longevity and are eager for a fulfilling welfare society. On the other hand, with age, we suffer from various diseases, such as cardiovascular diseases, cancers, and dementia, and thus, it is essential to not only explore the etiology of these diseases but also develop therapeutic drugs and preventive methods.
In particular, the number of elderly individuals who suffer from senile dementia has increased through this superaged society. Senile dementias are regarded as neurodegenerative diseases, which are categorized as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic sclerosis, and are characterized by nervous system dysfunction resulting from progressive neuronal degeneration [bib_ref] Neurodegeneration: a question of balance, Thompson [/bib_ref]. In Japan, the elderly population with neurodegenerative diseases will increase to 8,300,000 by 2030 unless suitable medical treatments are not realized. AD is the most prevalent form of dementia, accounting for 50-56% of cases at autopsy and in clinical settings, and AD combined with intracerebral vascular diseases accounts for another 13-17% of cases.
The principle risk for AD is age. The incidence of AD doubles for every 5 years of age, but AD is not necessarily the outcome of aging [bib_ref] Alzheimer's disease, Querfurth [/bib_ref]. The brain regions involved in learning and memory processes are reduced in size in AD patients as a result of degeneration of synapses and death of neurons [bib_ref] Pathways towards and away from Alzheimer's disease, Mattson [/bib_ref]. It has been more than 15 years since it was first proposed that AD might be caused by deposition of amyloid β-peptide (Aβ) in plaques in the brain [bib_ref] The amyloid hypothesis of Alzheimer's disease: progress and problems on the road..., Hardy [/bib_ref]. Accumulation of Aβ in the brain triggers the remaining AD pathogenesis, including the formation of neurofibrillary tangles containing tau protein, causing the degeneration of neurons and resulting in AD. Although tremendous efforts have been made according to the amyloid hypothesis, new drugs for the treatment of AD have not been successfully developed [bib_ref] Alzheimer's drugs show progress, Reardon [/bib_ref] [bib_ref] Clinical trials of disease-modifying therapies for neurodegenerative diseases: the challenge and the..., Lang [/bib_ref]. This is presumably because the underlying pathogenesis of AD still remains to be explored [bib_ref] Enhancing central nervous system repair-the challenges, Xu [/bib_ref].
It is well known that following neuronal injury, adult neurons have an intrinsic ability and dynamic repair mechanism within the central nervous system to regenerate and produce neuronal cells and restore neuronal networks, although this capacity is limited and the regions that are able to regenerate neurons are restricted. From this perspective, we initiated our research project to discover small molecule natural products that have the potential to act as neurotrophins to enhance neurogenesis, promote neurite outgrowth, and protect the death of neurons. In this review, we will introduce our own research program on the basis of neurotrophic properties and then highlight neurotrophic natural products, in particular, focusing on the chemistry and biological profiles of our discovered active compounds.
Neurotrophins and the screening system to search for neurotrophin mimetics Neurotrophins (neurotrophic factors) have been shown to be potentially beneficial in the treatment of neurodegenerative diseases such as AD, Parkinson's disease (PD) and Huntington's disease (HD) because endogenous neurotrophic factors have been recognized to play critical roles in the promotion of neurogenesis, differentiation, and neuroprotection throughout the development of the central nervous system [bib_ref] Neurotrophins and proneurotrophins: focus on synaptic activity and plasticity in the brain, Gibon [/bib_ref]. In mammals, the known neurotrophins are nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) [bib_ref] Neurotrophins and proneurotrophins: focus on synaptic activity and plasticity in the brain, Gibon [/bib_ref]. These neurotrophins bind selectively to their tyrosine kinase receptors TrkA, TrkB and TrkC, and all of them bind non-selectively to the neurotrophin receptor p75, resulting in activation of neuronal signal transduction related to the broad spectrum of biological activities exerted by neurotrophins [bib_ref] TRK receptors: roles in neuronal signal transduction, Huang [/bib_ref] [bib_ref] The neurotrophin receptor p75 NTR : novel functions and implications for disease..., Dechant [/bib_ref]. Therapeutic uses of NFs by intracranial injections, transplantation of cells secreting NFs, or gene therapy have shown promising results in animal models of neuronal degeneration as well as in clinical trials [bib_ref] Nerve growth factor treatment for Alzheimer's diseases: the experience of the first..., Hefti [/bib_ref] [bib_ref] A phase 1 clinical trial of nerve growth factor gene therapy for..., Tuszynski [/bib_ref] [bib_ref] Potential therapeutic uses of BDNF in neurological and psychiatric disorders, Nagahara [/bib_ref]. However, as NFs are high-molecular weight proteins, they have been unable to cross the blood barrier and are easily decomposed by peptidase under physiological conditions. To address this issue, small molecules that can mimic the functions of neurotrophic factors would be promising alternatives for the treatment of neurodegenerative disease [bib_ref] Neurotrophic factor mimetics, Swain [/bib_ref] [bib_ref] Neurotrophic natural products: chemistry and biology, Xu [/bib_ref].
Our protocol of searching for small molecules with neurotrophic properties is how to discovery NT mimicking compounds as well as to implicate active compounds in the key physiological functions of NTs: differentiation (neurogenesis), development (neurite outgrowth promotion) and survival (protection of neuronal death) of neurons [bib_ref] Search for neurotrophin-mimic natural products for prevention and treatment of neurodegenerative disease, Kubo [/bib_ref]. We applied three cells to the assay system; rat pheochromocytoma PC12 cells [bib_ref] Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which..., Greene [/bib_ref] , primary cultured rat cortical neurons [bib_ref] Effects of recombinant human basic fibroblast growth factor and its modified protein..., Abe [/bib_ref] and mouse multipotent neural precursor cells (MEB5) [bib_ref] Establishment of an epidermal growth factor-dependent, multipotent neural precursor cell line, Nakagaito [/bib_ref]. Both PC12 cells and NGF-mediated PC12 cells are used as the primary screening to identify active candidates. PC12 cells generate and extend neurites in response to NGF though the direct activation of the TrkA receptor or enhancing the intercellular NT signal pathway to induce neuritogenesis; whereas, NGF-mediated PC12 cells can extend the length of neurites to show neurite outgrowth through various mechanisms. Withdrawal of NGF from the culture medium causes the death of PC12 cells.
This method is used to screen for protection against neuronal death. Primary cultured cortical neurons are used for the second screening to confirm neurite outgrowth promotion or protection of neuronal death under different culture conditions. Finally, MEB5 cell lines have been used to ascertain whether active compounds have the potential to induce the differentiation of stem cells into neurons. In this review, our chemical and biological studies on natural products with neurotrophic activity are compiled [bib_ref] Search for novel neurotrophic factor-like substances in natural products, Fukuyama [/bib_ref] [bib_ref] Search for constituents with neurotrophic factor-potentiating activity from the medicinal plants of..., Li [/bib_ref] [bib_ref] Nonpeptide neurotrophic agents useful in the treatment neurodegenerative diseases such as Alzheimer's..., Akagi [/bib_ref].
## Mastigophorenes: isocuparane-type sesquiterpene dimers from the liverwort
## Mastigophora diclados
The liverworts elaborate a wide variety of terpenoids and lipophilic aromatic substances, which have been very often found to show different types of biological activity [bib_ref] Bryophytes: bio-and chemical diversity, bioactivity and chemosystematics, Asakawa [/bib_ref]. M. diclados (Brid.) Nees is a rather primitive liverwort and is commonly found in tropical Asiatic areas. Our independent study [bib_ref] Mastigophorenes: novel dimeric isocuparane-type sesquiterpenoids from the liverwort Mastigophora diclados, Fukuyama [/bib_ref] [bib_ref] Novel neurotrophic isocuparane-type sesquiterpene dimers, mastigophorenes A, B, C and D, isolated..., Fukuyama [/bib_ref] on the ether extract of M. diclados collected in Boruneo resulted in the isolation of four unique dimeric isocuparanes, mastigophorenes A (1), B (2), C (3), and D (4), together with their monomer unit, herbertenediol (5) [bib_ref] Structures of ent-herbertane sesquiterpenoids displaying antifungal properties from the liverwort Herberta adunca, Matsuo [/bib_ref]. Mastigophorenes A (1), B (2), and D (4) were found to exhibit interesting neurotrophic properties at concentrations ranging from 0.1 to 10 μM, which could enhance neurite-sprouting and network formation in primary cell cultures derived from embryonic rat cerebral hemispheres [bib_ref] Mitotic neuroblasts in dissociated cell cultures from embryonic rat cerebral hemispheres express..., Asou [/bib_ref]. On the other hand, mastigophorene C (3) and the monomeric compound, herbertenediol [bib_ref] Pathways towards and away from Alzheimer's disease, Mattson [/bib_ref] , suppressed the neurite outgrowth under the same conditions.
The dimeric compounds 1-4 could be derived from herbertenediol (5), a cometabolite in the plant, by phenolic oxidation. Compounds 1-4 are presumably biosynthesized via the phenoxy radical products formed from the one-electron oxidation of 5, and then the formed radicals are subsequently converted into radical A or benzyl radical B which would give rise to a quinone methide via one more oxidation along with the loss of a proton radical. Homocoupling between two radicals A would lead to mastigophorenes A (1) and B (2) followed by aromatization; whereas, mastigophorene D (4) could be produced from the direct coupling between two benzyl radicals B. An alternative heterocoupling between [bib_ref] First synthesis of mastigophorenes A and B, by biomimetic oxidative coupling of..., Bringmann [/bib_ref]. However, the direct oxidation of herbertenediol (5) with (tert-BuO) 2 failed to yield dimers 1 and 2, resulting in a complex mixture, and thus protecting the 1-hydroxy group of 5 was essential for successful oxidative coupling. We applied horseradish peroxidase (HRP)-catalyzed oxidative phenolic coupling to 5, which resulted in the direct formation of 1 (10%) and 2 (18%) with recovery of 5 (72%) [bib_ref] Total syntheses of neuroprotective mastigophorenes A and B, Fukuyama [/bib_ref] [bib_ref] Total synthesis of herbertenediol, an isocuparane sesquiterpene isolated from liverworts, Fukuyama [/bib_ref]. With a large amount of synthesized 1 and 2 in hand, the neurotrophic properties of 1 and 2 were evaluated in detail in primary cultured fetal rat cortical neurons. A neurite outgrowth assay was performed using 18-day-old fetal rat cortical neurons in serum-free DMEM supplemented with B-27. Morphological evaluation, as shown in , indicated that mastigophorenes A (1) and B (2) not only promoted significant neurite outgrowth but also formed a network of neurons at 0.1 and 1 μM. A neuronal survival assay was carried out using the same neuronal cultures in serum-free DMEM supplemented with N-2, and the neuronal viability was assessed by the WST-8 assay. As summarized in [fig_ref] Figure 4: Neuroprotective activity of mastigophorenes A [/fig_ref] , compounds 1 and 2 maintained neuronal survival at 0.1 and 1.0 μM, but lost their survival effect at 10 μM. These results suggest that mastigophorenes A (1) and B (2) can protect neurons from being insulted by toxic substances such as oxygen free radicals [bib_ref] Total syntheses of neuroprotective mastigophorenes A and B, Fukuyama [/bib_ref].
In addition, it should be noted that diasteroselective syntheses of mastigophorenes A (1) and B (2) with an atropenantioselective construction of the biaryl axis have been achieved by Bringmann [bib_ref] Nondynamic and dynamic kinetic resolution of lactones with stereogentic centers and axes:..., Bringmann [/bib_ref] , Meyers [bib_ref] Total syntheses of (-)-herbertenediol, (-)-mastigophorene A, and (-)-mastigophorene B. Combined utility of..., Degnan [/bib_ref] , and Feringa [bib_ref] Palladium-catalyzed, tert-butyllithium-mediated dimerization of aryl halides and its application in the atropselective..., Buter [/bib_ref].
## Magnolol, honokiol and sesquiterpene-neolignans from magnolia bark
The bark of the Magnolia tree, Magnolia obovata Thumb. and M. officinalis Rhed. have been used in traditional herbal medicines in China, Korea and Japan. Magnolia bark is an important ingredient in Hange-kobokuto and Sai-boku-to preparations for the treatment of gastrointestinal disorders, anxiety and allergic diseases [fig_ref] Figure 5: Neolignans 6-8 and sesquiterpene-neolignans 9-15 from Magnolia obovata 1 3 [/fig_ref]. Moreover, other reported beneficial effects of Magnolia bark include anticancer, anti-inflammatory, antiplatelet and antioxidant activities [bib_ref] Therapeutic applications of compounds in the Magnolia family, Lee [/bib_ref].
Magnolol [bib_ref] The amyloid hypothesis of Alzheimer's disease: progress and problems on the road..., Hardy [/bib_ref] and honokiol (7), biphenyl neolignans, are the main constituents of Magnolia bark and have been reported to have a variety of biological properties such as antioxidative, antitumor, antidepressant, antidiabetic, antiinflammatory, neuroprotective, and antimicrobial activities [bib_ref] Advances on semisynthesis. Total synthesis, and structure-activity relationships of honokiol and magnolol..., Yang [/bib_ref] [bib_ref] Safety and toxicology of magnolol and honokiol, Sarrica [/bib_ref]. In addition to their biological properties, magnolol [bib_ref] The amyloid hypothesis of Alzheimer's disease: progress and problems on the road..., Hardy [/bib_ref] and honokiol (7) were found to have neurotrophic activity in primary cultured rat cortical neurons at concentrations ranging from 0.1 to 10 μM, but obovatol (8) had no activity even at 10 μM [bib_ref] Neurotrophic activity of honokiol on the cultures of fetal rat cortical neurons, Fukuyama [/bib_ref] [bib_ref] Neurotrophic sesquiterpene-neoligans from Magnolia obovata: structure and neurotrophic activity, Fukuyama [/bib_ref]. Further studies on the minor components resulted in the isolation of various novel sesquiterpenes linked to biphenyl-or biphenylether-type neolignans termed sesquiterpene-neolignan, eudesmagnolol (9), eudeshonokiols A (11) and B (10), eudesobovatols A (13) and B [bib_ref] TRK receptors: roles in neuronal signal transduction, Huang [/bib_ref] , clovanemagnolol [bib_ref] Nerve growth factor treatment for Alzheimer's diseases: the experience of the first..., Hefti [/bib_ref] , and caryolanemagnolol [bib_ref] A phase 1 clinical trial of nerve growth factor gene therapy for..., Tuszynski [/bib_ref] [bib_ref] Novel neurotrophic sesquiterpene-neolignans from Magnolia obovata, Fukuyama [/bib_ref] [bib_ref] Structure of clovanemagnolol, a novel neurotrophic sesquiterpene-neolignan from Magnolia ovovata, Fukuyama [/bib_ref] [bib_ref] Structure of eudesmagnolol and eudeshonokiol, novel sesquiteprene-neolignans isolated from Magnolia obovata, Fukuyama [/bib_ref]. Among them, clovanemagnolol [bib_ref] Nerve growth factor treatment for Alzheimer's diseases: the experience of the first..., Hefti [/bib_ref] and caryolanemagnolol [bib_ref] A phase 1 clinical trial of nerve growth factor gene therapy for..., Tuszynski [/bib_ref] could not only accelerate neurite outgrowth but also activate choline acetyltransferase activity (ChAT) at the concentration of 0.01 μM [bib_ref] Neurotrophic sesquiterpene-neoligans from Magnolia obovata: structure and neurotrophic activity, Fukuyama [/bib_ref].
Clovanemagnolol [bib_ref] Nerve growth factor treatment for Alzheimer's diseases: the experience of the first..., Hefti [/bib_ref] and caryolanemagnolol [bib_ref] A phase 1 clinical trial of nerve growth factor gene therapy for..., Tuszynski [/bib_ref] are most likely to be converted from caryophyllene β-oxide and caryophyllene α-oxide, respectively, according to Barton's results [bib_ref] Neurotrophic sesquiterpene-neoligans from Magnolia obovata: structure and neurotrophic activity, Fukuyama [/bib_ref] [bib_ref] Sesquiterpenpoids. Part III. The stereochemistry of caryophyllene, Aebi [/bib_ref]. The proposed biosynthetic pathway as shown in Scheme 2 is initiated by the oxidation of (-)-caryophyllene, providing caryophyllene β-oxide or caryophyllene α-oxide. Acidc activation of both epoxides leads to an intramolecular attack of the exocyclic alkene, generating the diastereomeric tricyclic cation intermediates A or B. Cation A is trapped by magnolol (6), directly forming clovanemagnolol [bib_ref] Nerve growth factor treatment for Alzheimer's diseases: the experience of the first..., Hefti [/bib_ref] , whereas cation B is trapped by 6, giving rise to caryolanemagnolol [bib_ref] A phase 1 clinical trial of nerve growth factor gene therapy for..., Tuszynski [/bib_ref]. Siegel et al. synthesized 14 and 15 in two steps according to the postulated biosynthetic pathways [bib_ref] Biomimetic syntheses of the neurotrophic natural products caryolanemagnolol and clovanemagnolol, Cheng [/bib_ref] [bib_ref] Neuronal growth promoting sesquiterpene-neolignans; syntheses and biological studies, Cheng [/bib_ref]. Synthesized 14 and 15 were confirmed to significantly promote neuronal growth at 0.01 μM in the primary cultured embryonic cortical neurons, similar to the neurotrophic effects of natural products [bib_ref] Hippocampal and cortical neuronal growth mediated by the small molecule natural product..., Khaing [/bib_ref] [bib_ref] Neuronal growth promoting sesquiterpene-neolignans; syntheses and biological studies, Cheng [/bib_ref].
These results suggest that the lipophilic derivatives of simple biphenyl neolignans, magnolol (6) and honokiol [bib_ref] Alzheimer's drugs show progress, Reardon [/bib_ref] can enhance neurotrophic activity. Comparing the neurotrophic properties between 6 and 7, 7 was found to be more potent than 6 [bib_ref] Neurotrophic activity of honokiol on the cultures of fetal rat cortical neurons, Fukuyama [/bib_ref]. For further developments of more effective derivative, honokiol (7) was synthesized in 21% yield over 14 steps by utilizing a Pd-catalyzed Suzuki-Miyaura reaction [bib_ref] Efficient synthesis and structure-activity relationship of honokiol, a neurotrophic biphenyl-type neolignan, Esumi [/bib_ref]. In addition, the structure-activity relationship between neurite outgrowth-promoting activity and the O-methylated and/or the hydrogenated analogs 7a-7f, as summarized in , was examined in the primary cultured rat cortical neurons.
As a result, honokiol (7), 2-O-methylhonokiol (7a), and 3′-dihydroallylhonokiol (7d) had striking effects on the morphology of cortical neurons as shown in [fig_ref] Figure 7: Morphology of cultured rat cortical neurons demonstrated with anti-MAP2 immunochemical staining [/fig_ref]. Analogs 7b, 7c and 7f showed no enhancement of neurite extension. As shown in , quantitative analysis of the longest neurite length affected by each compound at the concentrations of 0.1 and 1 μM indicated that 7a and 7d have the potential to enhance neurite extension in cultured rat cortical neurons with a potency that is as high as that of 7; whereas, analogs 7b, 7c, and 7f showed diminished neurotrophic efficiency. Thus, these results suggest that with various substituents at the 3′-allyl position of 7 and evaluated their neurotrophic effects in neurite outgrowth of differentiated Neuro2a cells after treatment with NGF but could not find new analogs that were more potent than 7 [bib_ref] Synthesis and neurite growth evaluation of new analogues of honokiol, a neolignan..., Kumar [/bib_ref]. Recently, we successfully synthesized honokiol (7) in 74% yield over five steps [bib_ref] Efficient synthesis of neurotrophic honokiol using Szuzuki-Miyaura reactions, Harada [/bib_ref] , thereby a large amount of honokiol is now available for assessing its in vitro/in vivo biological activities and preparing a variety of derivatives. Next, after preparing several compounds and assessing their neurotrophic activity, we found two fluorescently labeled derivatives, 7-MCM (7-methoxycoumarin-3-carbonyl) (7g) and NBD (7-nitrobenzyl-2-oxa-1,3-diazolyl) (7h) that were suitable to be probe molecules to identify the intra/intercellular targets of 7. Rat cortical neurons were incubated with 5 μM 7h, 7g, and 7MCME for 1 h, and then their distributions in cortical neurons were monitored by fluorescence imaging under a microscope. As shown in [fig_ref] Figure 9: Distribution of fluorescent derivatives in primary cultured rat cortical neurons [/fig_ref] , the apparent fluorescence was observed in intercellular regions, but the fluorescent molecule itself, 7MCME, showed no fluorescence in any of the neurons. Taking a closer look at the fluorescent images, it is interesting to note the fluorescent vesicles assembled at the neck and/or branch of the dendrites in each cell body. These results suggest that honokiol could be taken up into cells and interact with specific targets, which would be associated with neurite outgrowth [bib_ref] Neurotrophic activity and mechanism of honokiol and magnolol, Zhai [/bib_ref].
Pharmacological studies of honokiol (6) and magnolol [bib_ref] Alzheimer's drugs show progress, Reardon [/bib_ref] have revealed that their effects on central nerves, such as depressant, muscle-relaxant, and anxiolytic effects, are mainly ascribed to their actions on GABA A receptors [bib_ref] Honokiol and Magnolol selectively interact with GABA A receptor subtypes in vitro, Ai [/bib_ref]. In our search for neurotrophic compounds, 6 and 7 were identified as neurotrophic compounds that upregulate the activity of choline acetyltransferase in neuronal culture [bib_ref] Neurotrophic sesquiterpene-neoligans from Magnolia obovata: structure and neurotrophic activity, Fukuyama [/bib_ref]. Furthermore, we found that 6 and 7 could promote neurite outgrowth and neuronal survival under serum-free conditions in cultured rat cortical neurons [bib_ref] Neurotrophic activity of honokiol on the cultures of fetal rat cortical neurons, Fukuyama [/bib_ref]. Additionally, it should be noted that magnolol could prevent the decrease in age-dependent neuronal loss in the hippocampus of senescence-accelerated mice (SAMP1) [bib_ref] Neurotrophic effect of magnolol in the hippocampal CA1 region of senescence-accelerated mice..., Matsui [/bib_ref]. The intriguing effects of honokiol and magnolol prompted us to investigate the mechanisms underlying their neurotrophic actions using the cultured neurons. In general, neurotrophins such as NGF, BDNF, and NT-3 bind to the extracellular domain of the tyrosine kinase receptors TrkA, TrkB, and TrkC, respectively, and thereby activate the respective tyrosine kinase in the intracellular domain. When target signal proteins bind to tyrosine kinases, they are phosphorylated to adopt active forms, and then transfer signals to their downstream. Among the NT-activated signaling molecules, Ca 2+ , MAPK (ERK), and Akt, are indispensable for transferring intracellular signals to nuclei [bib_ref] Small molecule Trk receptor agonists and other neurotrophic factor mimetics, Pollack [/bib_ref]. First, we examined the intracellular Ca 2+ response in primary cultured rat cortical neurons and human neuroblastoma SH-SY5Y cells by fluo-3 fluorescence imaging analysis. Magnolol and honokiol increased cytoplasmic free Ca 2+ with a characteristic lag phase. The cytoplasmic free Ca 2+ increase was independent of extracellular Ca 2+ but dependent on the activation of phospholipase C and inositol 1,4,5-triphosphate (IP 3 ) receptors, indicating an increase in cytoplasmic free Ca 2+ through a phospholipase C-mediated pathway. Thus, 6 and 7 cause the release of Ca 2+ from intracellular stores, resulting in an increase in cytoplasmic Ca 2+ [bib_ref] Honokiol and magnolol induce Ca 2+ mobilization in rat cortical neurons and..., Zhai [/bib_ref]. Regarding the effects of 6 and 7 on extracellular signal-regulated kinase (ERK1/2) and Akt, honokiol-induced neurite outgrowth in the cultured rat cortical neurons was significantly reduced by PD98059, a mitogen-activated protein kinase inhibitor, but not by LY294002, a phosphoinositide 3-kinase inhibitor. Honokiol also enhanced the phosphorylation of ERK1/2 in a dose-dependent manner; whereas, the effect of 7 on Akt phosphorylation was characterized by transient enhancement of 10 min and lasting inhibition after 30 min. The phosphorylation of ERK1/2 enhanced by 7 was inhibited by PD98059 as well as KN93, a Ca 2+ /calmodulin-dependent kinase II (CaMKII) inhibitor. Moreover, the products of the phosphoinositide-specific C (PLC)-derived inositol 1,4,5-triphosphate (IP 3 ) and 1,2-diacylglycerol (DAG) Scheme 2 The proposed biosynthesis of clovanemagnolol [bib_ref] Nerve growth factor treatment for Alzheimer's diseases: the experience of the first..., Hefti [/bib_ref] and caryolanemagnolol [bib_ref] A phase 1 clinical trial of nerve growth factor gene therapy for..., Tuszynski [/bib_ref] Fig. [bib_ref] The amyloid hypothesis of Alzheimer's disease: progress and problems on the road..., Hardy [/bib_ref] Structure of honokiol (7) and its analogs 7a-7f, and florescent derivatives 7g-7h were measured after honokiol treatment. Taking these results with our previous findings, as depicted in , signal transduction from PLC, IP 3 , Ca 2+ , and CaMKII to ERK1/2 has been proposed for a mechanism involved in honokiol-induced neurite outgrowth [bib_ref] Honokiol-induced neurite outgrowth promotion depends on activation of extracellular signal-regulated kinases (ERK1/2), Zhai [/bib_ref].
Furthermore, magnolol and honokiol were shown to be able to prevent age-related learning and memory impairment and cholinergic deficits in senescence-accelerated mice (SAMP8) [bib_ref] Magnolol and honokiol prevent learning and memory impairment and cholinergic deficit in..., Matsui [/bib_ref]. Magnolol and honokiol were orally administered to 2-month-old SAMP8 mice once a day for 14 days. The SAMP8 mice showed significant impairment of learning and memory at 4 and 6 months of age. This age-related learning and memory impairment was prevented by pretreatment with either 6 (10 mg/kg) or 7 (1 mg/kg). In addition, 6 and 7 prevented age-related cholinergic defects and enhanced phosphorylation of Akt, a member of the prosurvival pathway, in the forebrain at 2 months of age [fig_ref] Figure 11: Prenylated C 6 -C 3 compounds and neolignans from Illicium species Scheme... [/fig_ref].
Recently, it was reported that 6 and 7 showed antidepressant-like effects on unpredictable chronic mild stress (UCMS)-treated rats by enhancing BDNF expression and serotonergic activity in the hippocampus [bib_ref] Antidepressant-like effect of magnolol on BDNF up-regulation and serotonergic system activity in..., Li [/bib_ref] [bib_ref] Honokiol exerts antidepressant effects in rats exposed to chronic unpredictable mild stress..., Wang [/bib_ref]. Matsui et al. reported that 6 significantly improved depressive behavior in olfactory bulbectomized (OBX) mice in the tail suspension test, significantly enhanced hippocampal neurogenesis and increased phosphorylation of Akt and cyclic AMP-responsive element-binding protein (CREB) [bib_ref] Magnolol eanhances hippocampal neurogenesis and exerts antidepressant-like effects in olfactory bulbectomized mice, Matsui [/bib_ref]. These data demonstrate that magnolol (6) has antidepressant-like effects on behaviors and actions by enhancing hippocampal neurogenesis and neurotrophin-related intracellular signaling in mice.
Magnolol (6) and honokiol (7) are well known to have potent antioxidant effects [bib_ref] Antiperoxidative activity of neolignans from Magnolia obovata, Haraguchi [/bib_ref]. Oral administration of 6 to C57BL/6N mice after 1-methyl-4-phenyl-1,2,3,6-terahydropyridinium (MPTP) treatment, an in vivo model of Parkinson's model, almost completely prevented MPTP-induced lipid peroxidation in the stratum, suggesting that 6 has protective effects on the onset of cognitive impairments via an antioxidative mechanism. This is also consistent with the increasing lipid hydroperoxide level in the brain of SAMP8 at 2 months of age, which may be a cause of the age-related impairments and degeneration seen in the brain [bib_ref] Brain lipid hydroperoxide level increases in senescenceaccelerated mice at an early age, Yasui [/bib_ref].
## Neurotrophic compounds from illicium species
The genus Illicium, belonging to the family Illiciaceae, consists of approximately 40 species around the world. This genus is mainly distributed in the eastern North America, Mexico, the West Indies and eastern Asia. The fruits of the Quantitative analysis of anti-MAP2 immunochemically stained processes affected by honokiol (7) and its analogs 7a-7f. In each group, the average lengths of the primary processes were determined from 100 neurons selected in random fields. **p < 0.01; ***p < 0.001 compared with control. Data presented here are derived from one of the two repeated experiments with similar results 7g 7h 7MCME Illicium species are distinctive star-shaped follicles that emit a characteristic refreshing flavor. In particular, the fruits of I. vernum Hook, Chinese star anise, are the source of economically important product derived from this genus, which is widely used as a spice for flavoring food and beverages. Hence, essential oils have been the primary subject of chemical research on Illicium species, and the presence of volatile phenols has been reported as constituents of various parts of all Illicium species studied so far. The chemical constituents of the Illicium species are classified into three groups; sesquiterpenes, prenylated C 6 -C 3 compounds, and triphenylneolignan (sesquineolignan). Some of these compounds are not only unique in their architectural structure but also exert intriguing bioactive effects on the neuronal system [bib_ref] Chemical constituents of the genus Illicium, Fukuyama [/bib_ref] [bib_ref] Chemistry and neurotrophic activity of seco-prezizaane-and anislatone-type sesquiterpenes from Illicium species, Fukuyama [/bib_ref]. We reported the isolation and structure of tricycloillicinone (16) [bib_ref] Tricycloillicinone, a novel prenylated C 6 -C 3 compound increasing choline acetyltransferase..., Fukuyama [/bib_ref] and (2R)-12-chloro-2,3-dihydroillicinone E (18) [bib_ref] New chlorine-containing prenylated C 6 -C 3 compounds increasing choline acetyltransferase (ChAT)..., Fukuyama [/bib_ref] from the woods of I. tashiroi. Compounds 16 and 18 were found to increase choline acetyltransferase (ChAT) activity by 143% and 228% at 30 μM in cultured P10 rat septal neurons, respectively. Lately, both compounds were shown to promote neurite outgrowth in NGF-mediated PC12 cells and primary cultured rat cortical neurons at concentrations as high as 50, 100 μM. On the other hand, compounds 19-21, without a chlorine atom, did not have neurotrophic activity [bib_ref] Prenylated C 6 -C 3 compounds related to illicinone E from Illicium..., Fukuyama [/bib_ref]. Another bicyclic nonaromatic phenylpropanoid, bicycloillicinone asarone acetal [bib_ref] Neurotrophic factor mimetics, Swain [/bib_ref] , was isolated from the same plant source as 16 and was found to enhance ChAT activity at 30 μM, which catalyzes the synthesis of acetylcholine from its precursor [bib_ref] Bicycloillicinone asarone acetal: a novel prenylated C 6 -C 3 compound increasing..., Fukuyama [/bib_ref]. Acidic hydrolysis of 17 led to its core aldehyde, bicycloillicinone 17a, and cathechol portion 17b (Scheme 3).
The syntheses of 16 and the core structure 17a were achieved by Danishefsky's group [bib_ref] A fully synthetic route to the neurotrophic illicinones: syntheses of tricycloillicinone and..., Pettus [/bib_ref]. Cholinesterase inhibitors such as donepezil and tacrine, which are capable of increasing neurotransmitter acetylcholine levels by inhibiting acethylcholinesterase (AChE) activity, are now in use for the treatment of AD [bib_ref] Cholinesterase inhibitors for Alzheimer's disease therapy: from tacrine to further applications, Giacobini [/bib_ref]. Thus, compounds 16-18, which can induce ChAT, an enzyme responsible for the biosynthesis of acetylcholine, should contribute to increased acetylcholine levels and support the survival of cholinergic neurons. As prenylated phenylpropanoids with neurotrophic activity, illicinin A (22) and compound 23 were isolated from I. anisatum [bib_ref] Isolation, synthesis, and neurite outgrowth-promoting activity of illicinin A from the flowers..., Takaoka [/bib_ref]. Compounds 22 and 23 were found to significantly promote neurite outgrowth at concentrations ranging from 0.1 to 10 μM in primary cultured rat cortical neurons. Illicinin A (22) and its derivatives 22a-22c were synthesized for structure-activity relationship studies by applying Pd-catalyzed Stille reactions and then were assessed for the neurite length of rat cortical neurons at 1 μM. As a result, compound 22c showed reduced activity, whereas the others 22a and 22b showed comparative activity to illicinin A [bib_ref] Establishment of an epidermal growth factor-dependent, multipotent neural precursor cell line, Nakagaito [/bib_ref] or were more potent, thereby indicating that an allyl group in 22 is essential for exerting neurotrophic activity. In addition, the presence and position of the prenyl group in 22 were shown to play an important role in neurotrophic activity.
Typical neolignans, magnolol (6) and honokiol (7), were introduced in the early section of this review as having interesting neurotrophic properties. Macranthol [bib_ref] Nonpeptide neurotrophic agents useful in the treatment neurodegenerative diseases such as Alzheimer's..., Akagi [/bib_ref] [bib_ref] New sesqui-neolignan from the pericarps of Illicium macranthum, Kouno [/bib_ref] and isodunnianol (24) [bib_ref] Two new sesquineolignans from the bark of Illicium dunnianum, Kouno [/bib_ref] , another neolignans bearing one phenyl group called sesquineolignan, showed neuroprotective activity at 5-10 μM [bib_ref] Structure and neurotrophic activity of novel sesqui-neolignans from the pericarps of Illicum..., Moriyama [/bib_ref] and neurite outgrowth-promoting Proposed neurotrophic mechanism of honokiol (7) in cortical neurons. IP 3 and DAG production, cytoplasmic free Ca 2+ increase, and ERK1/2 phosphorylation are identified as effects of honokiol. The involvement of PLC, CaMK II, and MEK is shown using their specific inhibitor, U73122, KN93, and PD98059, respectively activity at 0.1-10 μM in primary cultured rat cortical neurons [bib_ref] Two new sesquiterpenoids and two new prenylated phenylpropanoids from Illicium fargesii, and..., Moriyama [/bib_ref] along with anti-AChE activity with an IC50 value of 13.0 μM [bib_ref] Sesquilignans and sesquiterpenoid from the stem barks of Illicium simonsii and their..., Dong [/bib_ref]. In addition, macranthol (25) exerted an antidepressant-like activity in mice by increasing the expression of hippocampal BDNF [bib_ref] Antidepressant-like effect of macranthol isolated from Illicium dunnianum tutch in mice, Li [/bib_ref] , and the mechanism of its mediated antidepressant-like action was verified to be associated with BDNF-TrkB and downstream activation of the PI3K/Akt-Bcl-2/caspase-3 signal pathway [bib_ref] Macranthol promotes hippocampal neuronal proliferation in mice via BDNF-TrkB-PI3K/Akt signaling pathway, Luo [/bib_ref]. Merrillianoid (26), a unique caged-shaped neolignan possessing a benzo-2,7-dioxa[3,2,1]octane moiety that was isolated as a racemic form from the leaves of I. merrillianum, influenced the NGF-induced neurite outgrowth of PC12 cells at concentrations from 1 to 10 μM, possibly by interacting with the TrkA receptor and downstream activation of ERK1/2 and MEK in the Ras/ERK signal cascade [bib_ref] Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two..., Tian [/bib_ref].
The fruits of I. anisatum (Japanese star anise, "shikimi") are known to be very toxic. Ingestion of these fruits causes convulsive symptoms, frequently leading to death. In 1952, Lane succeeded in the isolation of the pure toxic principle named anisatin [bib_ref] Mastigophorenes: novel dimeric isocuparane-type sesquiterpenoids from the liverwort Mastigophora diclados, Fukuyama [/bib_ref] , and its complete structure was later established by Yamada and Hirata [bib_ref] Isolation and structure of neoanisatin, Takada [/bib_ref]. Kawano et al., who continued to investigate the toxic substance in I. anisatum, succeeded in systematic studies on the chemical components in Illicium plants. Later, Schmidt and our group joined the chemical and biological studies of Illicium plants. A number of unique seco-prezizaane-type sesquiterpenes or so-called Illicium sesquiterpenes have been reported exclusively by the above three groups, and the occurrence of Illicium sesquiterpenes has been found to be limited to the genus Illicium [bib_ref] Chemistry and neurotrophic activity of seco-prezizaane-and anislatone-type sesquiterpenes from Illicium species, Fukuyama [/bib_ref]. Following the extensive chemical studies on Illicium sesquiterpenes, we turned our attention to neurotrophic properties but not neurotoxic activity for these sesquiterpenes. The isolation of sesquiterpenes from Illicium species, guided by the enhancement of ChAT activity, neurite outgrowth promotion and neuroprotective activity in primary cultured rat cortical neurons resulted in the discovery of a number of neurotrophic sesquiterpenes as shown in [fig_ref] Figure 12: Anisatin [/fig_ref].
Isodunnianin [bib_ref] Novel neurotrophic isocuparane-type sesquiterpene dimers, mastigophorenes A, B, C and D, isolated..., Fukuyama [/bib_ref] was the first neurotrophic Illicium sesquiterpene isolated from the wood of Illicium tashiroi collected in Ishigaki Island, Japan, and it was shown to moderately enhance neurite-sprouting during the development of neurons and increase ChAT activity at 10 μM in primary fetal rat cortical neurons [bib_ref] Isodunnianin: a new sesquiterpene enhancing neurite outgrowth in primary culture of fetal..., Fukuyama [/bib_ref]. The structure of 28 was elucidated on the basis of the published NMR data of dunnianin [bib_ref] New pseudoanisatin-like sesquiterpene lactones from the bark of Illicium dunnianum, Kouno [/bib_ref] , but later was corrected to 28 according to the revised structure of dunnianin [bib_ref] New sesquirepene lactones from Illicium floridanum, Schmidt [/bib_ref]. Two majucin-type sesquiterpenes, (2S)-hydroxy-3,4-dehydroneomajucin [bib_ref] Structures of ent-herbertane sesquiterpenoids displaying antifungal properties from the liverwort Herberta adunca, Matsuo [/bib_ref] and jiadifenin (31), isolated from Illicium jiadifengpi, significantly promote the neurite outgrowth of primary cultured rat cortical neurons at concentrations ranging from 0.1 to 10 μM [bib_ref] New seco-prezizaane-type sesquiterpenes, jiadifenin with neurotrophic activity and 1,2-dehydroneomajucin from Illicium jiadifengpi, Yokoyama [/bib_ref]. Jiadifenin (31) could be transformed from (2S)hydroxy-3,4-dehydronemajucin (29) if the C-10 hydroxyl group is oxidized to a ketone. Thus, compound 30, which was derived from 29 by DMP, was exposed to epimerization conditions at C-1 with DBU, leading to the thermodynamically more stable product (1R)-30. Next, DMP or Jones oxidation of (1R)-30 [bib_ref] Total synthesis of (±)-jiadifenin and studies directed to understanding its SAR: probing..., Carcache [/bib_ref] gave rise to jiadifenin [bib_ref] First synthesis of mastigophorenes A and B, by biomimetic oxidative coupling of..., Bringmann [/bib_ref] as an equilibrated mixture at the C-10 position in good yield, followed by the addition of MeOH to the reaction mixture (Scheme 4). This chemical transformation confirmed the absolute configuration of jiadifenin. Another interesting majucin-subtype sesquiterpene, jiadifenolide [bib_ref] Total syntheses of neuroprotective mastigophorenes A and B, Fukuyama [/bib_ref] , and jiadifenoxolanes A (33) and B (34), were isolated from the same plant [bib_ref] Novel pentacyclic seco-prezizaane-type sesquiterpenoids with neurotrophic properties from Illicium jiadifengpi, Kubo [/bib_ref]. Jiadifenolide [bib_ref] Total syntheses of neuroprotective mastigophorenes A and B, Fukuyama [/bib_ref] and jiadifenoxolane A (33) were found to significantly enhance the neurite outgrowth in primary cultured rat cortical neurons, in particular, jiadifenolide exhibited more potent activity at concentrations as low as 0.01 μM as shown in [fig_ref] Scheme 1, Figure 3: Biosynthetic route to dimeric isocuparenes 1, 2, 3 and 4 based on... [/fig_ref]. These majucin-subtype sesquiterpenes were reported to promote neurite outgrowth of NGF-mediated PC12 cells but have no effect on PC12 cells in the absence of NGF [bib_ref] Total synthesis of (±)-jiadifenin and studies directed to understanding its SAR: probing..., Carcache [/bib_ref]. Moreover, jiadifenolide (32) promoted neurite extension and significantly increased the total neurite area and length in neuronal cells derived from human induced pluripotent stem (iPS) cells at concentrations ranging from 1 to 10 μM [bib_ref] Neurotrophic activity of jiadifenolide on neuronal precursor cells derived from human induced..., Shoji [/bib_ref].
MEB5 is a multipotent stem cell line that can differentiate into neurons, astrocytes, and oligodendrocytes and, thus, is regarded as a potential tool to investigate compounds effective for the differentiation of CNS multipotent neuronal stem cells [bib_ref] Establishment of an epidermal growth factor-dependent, multipotent neural precursor cell line, Nakagaito [/bib_ref] [bib_ref] Activin promotes astrocytic differentiation of a multipotent neural stem cell line and..., Satoh [/bib_ref] [bib_ref] Effects of leukemia inhibitory factor on the differentiation of astrocyte progenitor cells..., Nakagaito [/bib_ref]. Furthermore, we attempted to assess the induction of differentiation of MEB5 cells by jiadifenolide. As shown in [fig_ref] Figure 4: Neuroprotective activity of mastigophorenes A [/fig_ref] , jiadifenolide significantly induced neuronal differentiation of MEB5 cells at 10 μM rather than astrocytic differentiation, with leukemina inhibitory factor (LIF) specifically induced as shown in [fig_ref] Figure 4: Neuroprotective activity of mastigophorenes A [/fig_ref]. The number of neurons at all the tested concentrations was greater in cultures treated with jiadifenolide than in control cultures [fig_ref] Figure 4: Neuroprotective activity of mastigophorenes A [/fig_ref]. These results demonstrate that jiadifenolide promotes neuronal differentiation in the same manner as NGF.
Jiadifenolide [bib_ref] Total syntheses of neuroprotective mastigophorenes A and B, Fukuyama [/bib_ref] can be obtained straightforwardly from neomajucin (40) by DMP oxidation presumably through the proposed mechanism in Scheme 5 [bib_ref] Novel pentacyclic seco-prezizaane-type sesquiterpenoids with neurotrophic properties from Illicium jiadifengpi, Kubo [/bib_ref]. Jiadifenolide [bib_ref] Total syntheses of neuroprotective mastigophorenes A and B, Fukuyama [/bib_ref] and the other majucin-subtype sesquiterpenes 29-31 and 33 have attracted great attention from organic chemists due to their complex caged structural architecture and remarkable neurotrophic properties. Taking the structural similarity of the majucin-subtype sesquiterpenes into consideration, most synthetic strategies are divergent and comprehensive [bib_ref] Neurotrophic natural products: chemistry and biology, Xu [/bib_ref] [bib_ref] Illicium sesquiterpenes: divergent synthetic strategy and neurotrophic activity studies, Trzoss [/bib_ref] [bib_ref] An eight-step gramscale synthesis of (-)-jadifenoilde, Lu [/bib_ref] [bib_ref] Protecting-group-free total synthesis of (-)-jiadifenolide: development of [4 + 1] annulation toward..., Shen [/bib_ref] [bib_ref] Synthesis of neurotrophic secoprezizaane sesquiterpenes (1R, 10S)-2-oxo-3,4-dehydroneomajucin, (2S)-hydroxy-3,4-dehydroneomajucin, and (-)-jiadifenin, Cheng [/bib_ref] [bib_ref] Development of a terpene Feedstock-based oxidative synthetic approach to the Illicium sesquiterpenes, Hung [/bib_ref].
Studies on seco-prezizaane-type sesquiterpenes from the Illicium species have culminated in their classification into further subgroups on the basis of a lactone type as follows: anisatin-subtype, pseudoanisatin-subtype, minwanensin-subtype, majucin-subtype, pseudomajucin-subtype, cycloparvifloralone-subtype, anislactone-subtype and allo-cedranesubtype consisting of rare carbon skeletons. Merrilactone A (37), of the anislactone-subtype, from the pericarps of Illicium merrillianum was found to exhibit neurotrophic activity in primary cultured rat cortical neurons at a concentration as low as 0.1 μM [bib_ref] Merrilactone A, a novel neurotrophic sesquiterpene dilactone from Illicium merrillianum, Huang [/bib_ref]. The limited amount of 37 has prevented further biological studies. This encouraged us to attempt partial synthesis of merrilactone A (37) from anislactone B [bib_ref] Novel neurotrophic sesquiterpene-neolignans from Magnolia obovata, Fukuyama [/bib_ref] , which was available in a large quantity from the same plant [bib_ref] Structures of merrilactones B and C, novel anislactone-type sesquiterpenes from Illicium merrillianum,..., Huang [/bib_ref]. First, a solution of 42 in trifluoroacetic acid (TFA) was refluxed to bring about the lactone transformation to the C-4 hydroxyl group with dehydration of the C-1 hydroxyl group, giving rise to 43 in good yield. Then, epoxidation of 43 with m-chloroperoxybenzoic acid (m-CPBA) proceeded in a highly stereoselective fashion to give 44, which was treated with p-toluenesulfonic acid (p-TsOH) to produce merrilactone A (37) in 78% yield (Scheme 6). Following our reports, a number of excellent total syntheses of merrilactone A were published, and one should take a look at references [bib_ref] Total syntheses of sesquiterpenes from Illicium species, Urabe [/bib_ref] [bib_ref] Total synthesis of (±)-anislactone A and (±)-merrilactone A, Greaney [/bib_ref] for the details of each synthesis.
Tashironin [bib_ref] Total syntheses of (-)-herbertenediol, (-)-mastigophorene A, and (-)-mastigophorene B. Combined utility of..., Degnan [/bib_ref] and 11-O-debenzoyltashironin (36) were isolated from I. tashiroi [bib_ref] Tashironin, a plausible biosynthetic precursor of anisatin-type sesquiterpenes, Fukuyama [/bib_ref] and I. merrillianum [bib_ref] Structure and neurotrophic activity of seco-prezizaane-type sesquiterepenes from Illicium merrillianum, Huang [/bib_ref]. Tashironin and its congeners [bib_ref] Two new compounds and anti-HIV active constituents from Illicium verum, Song [/bib_ref] have a tricarbocyclic skeleton corresponding to the key intermediate, allo-cedrane in the biosynthesis of seco-prezizaane-type sesquiterpenes. Among allo-cedrane-type sesquiterpenes 11-O-debenzoyltashironin (36) exhibits solely neurotrophic features in primary cultured rat cortical neurons at 0.1-10 μM and the presence of a free acetal group at the C-11 position is essential for having neurotrophic activity [bib_ref] Structure and neurotrophic activity of seco-prezizaane-type sesquiterepenes from Illicium merrillianum, Huang [/bib_ref]. In 2017, illisimonin A, which could be classified as a rare rearranged allocedrane, was isolated from the fruits of I. simonsii and was reported to show neuroprotective effects against oxygen-glucose deprivation-induced cell injury in SH-SY5Y cells with an EC50 value of 27.72 μM [bib_ref] Illisimonin A, a caged sesquiterpenoid with tricyclo[5.2.1.0 1,6 ]decane skeleton from the..., Ma [/bib_ref]. Recently, the absolute configuration of 38 was revised by its enantioselective synthesis [bib_ref] Total synthesis and structure revision of (-)-illisimonin A, a neuroprotective sesquiterpenoid from..., Burns [/bib_ref]. The first example of a seco-prezizaane-type norsesquiterpene, (2R)-hydroxy-norneomajucin (39) was isolated from I. jiadifengpi and was added to a list of Illicium sesquiterpenes with neurotrophic activity [bib_ref] The first examples of seco-prezizaane-type norsesquiterpenoids with neurotrophic activity from Illicium jiadifengpi, Kubo [/bib_ref]. The biosynthesis of 39 could be initiated by oxidation of the hydroxyl group at the C-10 position of neomajucin 40 to give a highly strained α-keto-δ-lactone, which would cause decarboxylation to lose one carbon, thereby leading to the less strained five-membered lactone 39. As shown in Scheme 7, when 2-O-acetyl-(2S)-hydroxyneomajucin [bib_ref] Neurotrophic sesquiterpene-neoligans from Magnolia obovata: structure and neurotrophic activity, Fukuyama [/bib_ref] was oxidized with Jones reagent, decarboxylation occurred spontaneously resulting in direct formation of the γ-lactone 46, from which 39 was readily accessible after several reactions [bib_ref] The first examples of seco-prezizaane-type norsesquiterpenoids with neurotrophic activity from Illicium jiadifengpi, Kubo [/bib_ref]. MEB5 cells were first cultured in the presence EGF at the density of 1.8 × 10 4 cells/cm 2 , and then medium was changed to EGF-free medium. After 4 days in the absence of EGF, the cells were double labeled with antibodies to class III β-tubulin (Tuj-1, red) and glial fibrillary acidic protein (GFAP, green). B The percentage of the neuronal (red) and astrocytic (green) cells; the cells were maintained for 1 day in the proliferation medium, and then transferred to the differentiation medium containing vehicle (control) or jiadifenolide and cultured another 7 days. After immunostaining for Tuj-1 and GFAP, neuron, astrocyte, and total cell numbers were counted, and the ratio to total cells was calculated. Data were expressed as means ± SE (n = 5) Neurotrophic sesquiterpenes 28-33 and 36-39 [fig_ref] Figure 12: Anisatin [/fig_ref] have significant structural homology with anisatin [bib_ref] Mastigophorenes: novel dimeric isocuparane-type sesquiterpenoids from the liverwort Mastigophora diclados, Fukuyama [/bib_ref] and picrotoxinin (49) which cause convulsions but do not have neurotrophic properties. Picrotoxinin has been validated to elicit convulsion by binding to the GABA A receptor and chloride anion blockade of the Cys-loop family of glutamate-gated chloride channels. Anisatin [bib_ref] Mastigophorenes: novel dimeric isocuparane-type sesquiterpenoids from the liverwort Mastigophora diclados, Fukuyama [/bib_ref] and veranisatin [bib_ref] Hippocampal and cortical neuronal growth mediated by the small molecule natural product..., Khaing [/bib_ref] were also identified as picrotoxin-like, noncompetitive GABA-antagonists [bib_ref] Anisatin, a potent GABA antagonist, isolated from Illicium anisatum, Kudo [/bib_ref] [bib_ref] Neurotropic components from star anise (Illcium verum Hook, fil.), Nakamura [/bib_ref]. Many of these convulsant terpenes contain γ-or δ-lactone motifs. Even the simple β-alkyl lactone β-EMGB exhibits convulsive activity by binding to the same site as picrotoxin. Based on structural homology with picrotoxinin (49) and anisatin [bib_ref] Mastigophorenes: novel dimeric isocuparane-type sesquiterpenoids from the liverwort Mastigophora diclados, Fukuyama [/bib_ref] [fig_ref] Figure 5: Neolignans 6-8 and sesquiterpene-neolignans 9-15 from Magnolia obovata 1 3 [/fig_ref] , it has been postulated that neurotrophic Illicium sesquiterpenes would enhance neurite outgrowth by modulation of the Cys-loop family of GABA A receptors, and a mechanistic link may exist between convulsant terpenes and neurotrophic Illicium sesquiterpenes [bib_ref] Neurite outgrowth enhancement by jiadifenolide: possible target, Shevi [/bib_ref].
In but more data are needed to validate this hypothesis in which convulsive and neurotrophic sesquiterpenes share a common target [bib_ref] Synthesis of (-)-11-O-debenzoyltashronin: neurotrophic sesquiterpenes cause hyperexcitation, Ohtawa [/bib_ref].
In 2018, Witkin et al. reported that jiadifenolide (32) and 11-O-debenzoyltashironin (36) did not cause convulsions in mice nor did they enhance or diminish convulsions induced by pentylenetetrazole (PTZ) although picrotoxinin [bib_ref] Neuronal growth promoting sesquiterpene-neolignans; syntheses and biological studies, Cheng [/bib_ref] and tetramethylenedisulfotetramine (TETS) both induced convulsions. Furthermore, jiadifenolide and 11-O-debenzoyltashironin were verified to be less potent and less efficacious antagonists of GABA receptors than either picrotoxinin or TETS [bib_ref] Pharmacological characterization of the neurotrophic sesquiterpene jiadifenolide reveals a non-convulsant signature and..., Witkin [/bib_ref].
The underlying molecular mechanisms by which jiadifenolide (32) and its analogous Illicium sesquiterpenes exert their neurotrophic effects remain unknown despite the abovementioned hypothesis that they share a common target with the convulsant compounds including anisatin and picrotoxinin. We previously reported that jiadifenolide significantly promotes neurite outgrowth and cell growth as well as prevents death of neuronal precursor cells derived from human pluripotent stem cells (hiPSCs) [bib_ref] Neurotrophic activity of jiadifenolide on neuronal precursor cells derived from human induced..., Shoji [/bib_ref]. By in silicon molecular network analysis of our comprehensive RNA sequencing results on 32-treated human neuronal cells using KeyMolnet software, 32 was found to activate cellular communication network factor (CCN) signaling pathways by upregulating the mRNA expression of CCN2. In addition, the CCN2 protein was confirmed to exhibit neurotrophic effects and promote phosphorylation of the p44/42 MAPK protein in human neuronal cells. This result suggests that the molecular mechanism by which 32 exerts its neurotrophic effect is linked with CCN signaling [bib_ref] Jiadifenolide induces the expression of cellular communication network factor (CCN) genes, and..., Shoji [/bib_ref]. It should be noted that this is the first discovery to connect neurotrophic effects with CCN signaling.
## Miscellaneous natural products with neurotrophic effects
Vibsane-type diterpenoids rarely occur as natural products and have been limited to the isolation from Viburnum species thus far. The carbon skeletons of these diterpenes are further classified into three subtypes: those with an elevenmembered ring, those with a seven-membered ring, and the rearranged types (neovibsanins) [bib_ref] Chemistry and biological activities of vibsane-type diterpenoids, Fukuyama [/bib_ref] [bib_ref] Chemical diversity of vibsane-type diterpenoids and neurotrophic activity and synthesis of neovibsanin, Miwa [/bib_ref]. Among the three subtypes of vibsane-type diterpenoids, neovibsanins A (50) and B [bib_ref] Efficient synthesis and structure-activity relationship of honokiol, a neurotrophic biphenyl-type neolignan, Esumi [/bib_ref] and their congeners 52-55 with modifications on the prenyl group, as shown in , significantly enhanced the neurite outgrowth of NGF-mediated PC12 cells at concentrations ranging from 5 to 40 μM [bib_ref] NGF-potentiating vibsane-type diterpenoids from Viburnum sieboldii, Kubo [/bib_ref] [bib_ref] Structure of seven new vibsane-type diterpenoids from Viburnum awabuki, Kubo [/bib_ref]. A good synthetic achievement of neovibsanin B was accomplished by Imagawa et al. [bib_ref] Total synthesis of (±)-neovibsanin B, Imagawa [/bib_ref] , who then extended their synthetic efforts to structure-activity relationship studies, resulting in elucidation of the minimum structure required for displaying neurite outgrowth activity in NGF-mediated PC12 cells (compound 56) [bib_ref] Syntheses of structurally-simplified and fluorescently-labeled neovibsanin derivatives and analysis of their neurite..., Imagawa [/bib_ref].
The Brazilian medicinal plant Ptychopetalum olacoides is known as a nerve tonic and is used for the treatment of chronic degenerative conditions of the central nervous system. From the MeOH extract of this plant, a number of clerodane diterpenes 57-62 and 64-65 were isolated, among which ptychonal hemiacetal, ptychonal (58), 6α,7αdihydroxyannonene [bib_ref] Magnolol and honokiol prevent learning and memory impairment and cholinergic deficit in..., Matsui [/bib_ref] , and 7α,20-dihydroxyannonene (62) exhibited NGF-potentiating activity in PC12 cells in the presence of NGF (20 ng/mL) at concentrations ranging from 0.1 to 40 μM [bib_ref] Clerodane diterpenoids with NGF-potentiating activity from Ptychopetalum olacoides, Tang [/bib_ref] [bib_ref] Novel NGF-potentiating diterpenoids from a Brazilian medicinal plant, Ptychopetalum olacoides, Tang [/bib_ref]. On the other hand, compounds 59, 60, and 63-65 had no effect on PC12 cells in the presence or absence of NGF. Compound 61 is the most potent NGF potentiator of these active compounds. The adjacent hydroxyl groups in 61 presumably contribute to its increased activity because the acetonide 63 loses activity at the same concentrations. In addition, the furan ring plays an important role in the appearance of NGF-potentiating activity since 64 and 65, with modifications to the furan ring, showed no activity at all. NGF-potentiating activity. However, 67-70 seemed to be more potent NGF potentiators than 66, which had toxicity at concentrations higher than 30 μM [bib_ref] Spirocyclic nortriterpenoids with NGF-potentiating activity from the fruits of Leonurus heterophyllus, Liu [/bib_ref].
The polycyclic prenylated acylphloroglucinol (PPAP) family comprises a highly oxygenated and densely substituted bicyclo[3.3.1]nonane skeleton bearing prenyl or geranyl side chains that are rich in the family Clusiaceae (Guttiferae). Hypericum perforatum, commonly known as St. John's wort, has attracted attention due to its antidepressant activity [bib_ref] Polycyclic polyprenylated acylphloroglucinols, Ciochina [/bib_ref]. In the course of chemical studies on PPAPs of Garcinia subelliptica (Guttiferae) [bib_ref] Subellinone, a polyisoprenylated phloroglucinol derivative from Garcinia subelliptica, Fukuyama [/bib_ref] [bib_ref] Garsubellins, polyisoprenylated phloroglucinol derivatives from Garcinia subelliptica, Fukuyama [/bib_ref] , we found that garsubellin A (71) could increase the ChAT activity, a key enzyme for physiologic acetylcholine synthesis in the nervous system, by up to 154% at 10 μM in comparison with the control in primary cultured P10 rat septal neurons [bib_ref] Garsubellin A, a novel polyprenylated phloroglucin derivative, increasing choline acetyltransferase (ChAT) activity..., Fukuyama [/bib_ref]. Owing to its important biological activity and architectural structure, garsubellin A (71) stimulated substantial synthetic effort, and the excellent synthetic achievements have been reported [bib_ref] Polycyclic polyprenylated acylphloroglucinols, Ciochina [/bib_ref] [bib_ref] Synthesis of polyprenylated acylphloroglucinols using bridgehead lithiation: the total synthesis of racemic..., Ahmad [/bib_ref] [bib_ref] Stereoselective total synthesis of garsubellin A, Uwamori [/bib_ref] [bib_ref] Programmable meroterpene synthesis, Shen [/bib_ref]. More efficient synthetic methods of 71, however, still have to be developed to supply sufficient quantities to explore its full biological characterization [fig_ref] Figure 7: Morphology of cultured rat cortical neurons demonstrated with anti-MAP2 immunochemical staining [/fig_ref].
A unique group of the neolignans, such as americanol A (72), isoamericanol A (73), americanin A (72a), isoamericanin A (73a), americanoic acid A methyl ester [bib_ref] Isolation, synthesis, and neurite outgrowth-promoting activity of illicinin A from the flowers..., Takaoka [/bib_ref] and isoamericanoic acid A methyl ester [bib_ref] New sesqui-neolignan from the pericarps of Illicium macranthum, Kouno [/bib_ref] , which are characterized by having a 1,4-benzodioxane ring and have diverse and significant biological activities, occur exclusively in the seeds of Phytolacca americana L. (Phytolaccaceae) [bib_ref] The structure of new lignans from the seeds of Phytolacca americana, Woo [/bib_ref] [bib_ref] Structure of isoamericanin A, a prostaglandin I 2 inducer, isolated from the..., Hasegawa [/bib_ref]. In particular, americanol A and isoamericanol A were found to enhance not only ChAT activity but also neurite outgrowth at 10 μM in primary cultured fetal rat hemispheres [bib_ref] Structures of americanol A and isoamericanol A having a neurotrophic property from..., Fukuyama [/bib_ref]. Neolignans 72 and 73 would be formed by oxidative dimerization of the corresponding monomeric unit, coniferyl alcohol. In fact, caffeic acid was subjected to horseradish peroxidase (HRP)-catalyzed oxidative conditions to give rise to dicarboxylic acids 74 and 75, which were converted to 72 and 73, respectively, followed by sequential reductions [bib_ref] Convenient syntheses of neurotrophic americanol A and isoamericanol A by HRP catalyzed..., Matsumoto [/bib_ref]. We reexamined the neurotrophic activities of 72-77 in primary cultured rat cortical neurons. In addition to 72 and 73, americanoic acid A methyl ester (76) was found to exhibit potent neurite outgrowth activity at 0.1 μM; whereas, the activities of compounds 74, 75 and 77 were comparable with control cultures. Although compounds 74 and 75 had no effects on neurite outgrowth, they induced significant neuritogenesis such as increasing the number of neurite branches in the concentration range from 0.1 to 10 μM in a similar manner to basic fibroblast growth factor (bFGF) as shown in [fig_ref] Figure 9: Distribution of fluorescent derivatives in primary cultured rat cortical neurons [/fig_ref] [bib_ref] Structures of 1,4-benzodioxane derivatives from the seeds of Phytolacca americana and their..., Takahashi [/bib_ref].
Novel polyoxygenated benzofuran derivatives, namely, ribisin A (78), ribisin B (79), ribisin C (80), and ribisin D (81), were isolated from the fruiting bodies of Phellinus ribis, which is used in the East Asian countries as a traditional medicine for enhancing immunity and gastrointestinal cancer. Beznofurans 78-81 showed marked neurite outgrowth-promoting activity in NGF-mediated Neovibsanin-subtype diterpenes with neurotrophic effects PC12 cells at concentrations ranging from 1 to 30 μM; whereas, none of these four compounds had morphological effects on PC12 cells in the absence of NGF. Their absolute configurations on the chiral positions were elucidated by applying the CD exciton chirality method to the readily derived p-bromobenzoate [bib_ref] Nerve growth factor-potentiating benzofuran derivatives from the medicinal fungus Phellinus ribis, Liu [/bib_ref]. Chemoenzymatic total syntheses of 78-81 were achieved by two groups [bib_ref] Chemoenzymatic total syntheses of ribisins A, B, and D, polyoxygenated benzofuran derivatives..., Lan [/bib_ref] [bib_ref] Chemoenzymatic synthesis of (-)-ribisins A and B from dibenzo[b,d]furan, Boyd [/bib_ref] , and therefore, the absolute stereochemistry of ribisin C (80) was revised as its ent-form.
A prenylated and geranylated biphenyl derivative, clusiparalicoline A (82), was isolated from the roots of Clusia paralicola and showed modest cytotoxicity in the KB cell line [bib_ref] New biphenyl compounds with DNA strand-scission activity from Clusia paralicola, Seo [/bib_ref]. We achieved the first synthesis of 82 by applying sequential palladium-catalyzed Stille and Suzuki reactions, and synthetic 82 was assessed by our neuronal cell assay. As a result, 82 was found to exhibit potent neurite outgrowth-promoting activity at concentrations from 0.1 to 1.0 μM in primary cultured rat cortical neurons, but the application of a higher dose than 10 μM induced the death in all neurons [bib_ref] The first total synthesis and neurotrophic activity of clusiparalicoline A, a prenylated..., Takaoka [/bib_ref]. Although 82 shows cytotoxicity at high concentrations, the biaryl moiety of 82 may have some positive effects on the development and survival of neurons in the same manner as honokiol and magnolol, which have a specific affinity for neuronal cells [bib_ref] Neurotrophic sesquiterpene-neoligans from Magnolia obovata: structure and neurotrophic activity, Fukuyama [/bib_ref].
Piperine (1-piperoylpiperidine), a pungent nitrogenous substance, is a main alkaloid in various piper species. These piper plants are commonly used as household spices as well as important traditional medicine in many Asian countries. With regard to piperine on cognitive function, recent pharmacological studies have shown that piperine possesses cognitive-enhancing activity [bib_ref] Piperine, the potential functional food for mood and cognitive disorders, Wattanathorn [/bib_ref] and improves both memory impairment and neurodegeneration in rats with cognitive defects induced by the ethylcholine aziridinium ion (AF64A) [bib_ref] Piperine, the main alkaloid of Thai black pepper, protects against neurodegeneration and..., Chonpathompikunlert [/bib_ref]. These reports prompted us to evaluate the neurotrophic properties of the fruits of the Javanese long pepper, Piper retrofractum, which are called Cabe Jawa and are used as one of the ingredients in the Indonesian natural medicine jamu. The methanolic extract of P. retrofractum [fig_ref] Figure 7: Morphology of cultured rat cortical neurons demonstrated with anti-MAP2 immunochemical staining [/fig_ref] Other terpenes that enhance neurite outgrowth of NGF-mediated PC12 cells fruits exhibited neurite outgrowth-promoting activity in NGF-mediated PC12 cells. Bioassay-guided fractionation resulted in the isolation of the active component, piperodione [bib_ref] Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two..., Tian [/bib_ref] , which is the first example of piperine oxidized at the C-2 and C-5 positions. Compound 83 showed potent NGF-potentiating activity in PC12 cells at concentrations ranging from 0.1 to 10 μM, but piperine had no effect on PC12 cells at the same dose as 83 [bib_ref] Evaluation of constituents of Piper retrofractum Fruits on neurotrophic activity, Kubo [/bib_ref]. Our study has demonstrated that piperine is not the active constituent responsible for the observed neurotrophic activity of P. retrofractum fruits. Recently, the total synthesis of piperodione (83) was reported from two groups, and 83 was verified to have NGFpotentiating activity but not cytotoxicity at all [bib_ref] First total synthesis of piperodione and analogs, Sommerwerk [/bib_ref] [bib_ref] Total synthesis of natural product piperodione and its analogues, Tiwari [/bib_ref]. Now, with large quantities of 83 in hand, further pharmacological studies of 83 are in progress. Dunnett's t test vs. control, **p < 0.01 [bib_ref] Structures of 1,4-benzodioxane derivatives from the seeds of Phytolacca americana and their..., Takahashi [/bib_ref] Finally, it should be noted that a comprehensive review on (-)-talaumidin, a neurotrophic 2,5-biaryl-3,4-dimethylhydrofuran lignan, has been published quite recently. Given the increasing interest in neurotrophin-mimic compounds, we suggest that readers refer to a recent review covering another topic of neurotrophic natural products.
# Conclusions
In this review, we have introduced neurotrophin-mimicking natural products discovered by our group and have focused on the chemical and biological features of neurotrophic compounds but have not emphasized their synthetic achievements, because there are already some excellent reviews on this subject [bib_ref] Neurotrophic natural products: chemistry and biology, Xu [/bib_ref] [bib_ref] Total synthesis of (±)-anislactone A and (±)-merrilactone A, Greaney [/bib_ref] [bib_ref] Application of total synthesis to problems in neurodegenereation: fascinating chemistry along the..., Wilson [/bib_ref] that summarize the overview of their organic syntheses. Endogenous neurotrophins (NGF, BDNF etc.) are a class of polypeptidyl agents that promote neurogenesis, neuron survival, process outgrowth, and synaptic connectivity in the development of the neuronal system and neuronal plasticity in adult neurons. As mentioned in the introduction, the application of neurotrophins to treat neurodegenerative diseases suffers serious drawbacks in practice due to their unfavorable properties. Consequently, a small molecule non-peptidyl agent that mimics the functions of neurotrophic factors would be an attractive alternative for the treatment and/or protection of neurodegenerative diseases. This idea prompted us to search for plant-derived small molecules that have neurotrophic effects on three neuronal cells: PC12 cells, primary cultured rat cortical neurons, and MEB5 neuronal stem cells. Beginning in 1988, structurally unique isocuparane dimers, mastigophorenes A (1) and (2) were discovered as neurotrophic compounds by our group. Since then, a number of compounds with neurotrophic activity have been discovered from various plants. However, many active compounds have not gained access to detailed biological investigations, in particular, to in vivo animal models and their underlying neurotrophic mechanisms remain obscure because insufficient quantities of material are an often-encountered problem associated with natural products. To overcome this issue, many groups have been engaged in the synthesis of neurotrophic natural products. Jiadifenolide (32) is a good example. Chemical synthesis has supplied a sufficient quantity of 32 to undertake detailed biological studies, which leads to the possibility of clinical utility for jiadifenolide.
Through this review, we hope that organic chemists and medicinal chemists will share their interests and efforts with neurotrophin-mimicking small molecules, which are expected to not only play a critical role in chemical control over each step in the neural circuit life model (neurogenesis, differentiation, neurite outgrowth, death), as proposed in , but also make a positive contribution to protecting cognitive impairment in a superaged society.
[fig] Figure 1: Protocol of searching for neurotrophic compounds by the assay system using three cells: PC12, primary cultured rat cortical neurons, and MEB5 [/fig]
[fig] Scheme 1, Figure 3: Biosynthetic route to dimeric isocuparenes 1, 2, 3 and 4 based on one-electron oxidative coupling from (-)-herbertenediol (5) Enhancement of neurite outgrowth by mastigophorenes A (1) and B (2) in primary cultured rat cortical neurons in serum-free DMEM medium supplemented with B-27. a 0.5% EtOH, b 1μM mastigophorene A, c 1μM mastigophorene B [/fig]
[fig] Figure 4: Neuroprotective activity of mastigophorenes A (1) and B(2) in primary cultured rat cortical neurons in serum-free DMEM medium supplemented with N-2. After the neuronal cells (2 × 10 5 cells cm −2 ) were cultured for 3 days in the presence of 0.5% EtOH (control) and compounds 1 and 2, neuronal viability was assessed by the WST-8 reduction assay. The data are expressed as means SE (n = 4); *p < 0.05, **p < 0.015, ***p < 0.005 versus control. the 4′-hydroxy group and the 5-allyl group are essential for honokiol-mediated neurite outgrowth-promoting activity(Figs. 7, 8). Based on the results of this SAR study, Gree and Chandrasekhar et al. synthesized24 derivatives [/fig]
[fig] Figure 5: Neolignans 6-8 and sesquiterpene-neolignans 9-15 from Magnolia obovata 1 3 [/fig]
[fig] Figure 7: Morphology of cultured rat cortical neurons demonstrated with anti-MAP2 immunochemical staining. a Neurons in the presence of 0.5% ethanol as vehicle control; b neurons in the presence of 0.1μM 7; c neurons in the presence of 1μM 7a; d neurons in the presence of 0.1μM 7d 1 3 [/fig]
[fig] Figure 9: Distribution of fluorescent derivatives in primary cultured rat cortical neurons. Rat cortical neurons were incubated in the presence of 5-μM 7g, 7h and 7MCME at 37 °C for 60 min followed by fluo-rescence imaging under microscope. Ex = 330 nm, Em = 431 nm for 7g and 7MCME. Ex = 470 nm, Em = 560 nm for 7 h [/fig]
[fig] 2017 ,: Shenvi et al. succeeded in the synthesis of (-)-11-O-debenzoyltashironin (36) and compared the effects of neurotrophic compounds 32 and 36, and convulsive compounds 27 and 49 on primary cultures of rat cortical neurons in terms of the hyperexcitation expected from the antagonism of inhibitory channels. Although all four compounds similarly caused hyperexcitation of cortical neurons and inhibited GABA-evoked currents, the GABA antagonistic effects of 32 and 36 were tenfold weaker than those of 27 and 49. Neurite outgrowth enhancement by 32 and 36 might be accounted for by a mechanism of chronic depolarization Scheme 5 Direct oxidative conversion of 40 to 32 Scheme 6 Partial synthesis of 37 from anislactone B (42) Scheme 7 Chemical conversion from 45 to 39 [/fig]
[table] 1: Protocol of searching for neurotrophic compounds by the assay system using three cells: PC12, primary cultured rat cortical neurons, and MEB5 [/table]
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Plausible Role of Estrogens in Pathogenesis, Progression and Therapy of Lung Cancer
Citation: Musial, C.; Zaucha, R.; Kuban-Jankowska, A.; Konieczna, L.; Belka, M.; Marino Gammazza, A.; Baczek, T.; Cappello, F.; Wozniak, M.; Gorska-Ponikowska, M. Plausible Role of Estrogens in Pathogenesis, Progression and Therapy of Lung Cancer. Int. J. Environ. Res. Public Health 2021, 18, 648. https://doi.
# Introduction
In the developed countries, lung cancer is the most frequent malignancy and is responsible for about 1 million deaths annually. The overall survival rate involving this tumor is about 10%. The decisive trigger is long-term smoking. Available data indicate that only 20% of lung cancer cases develop in non-smokers. Other environmental factors include pollution, exhaust fumes, ionizing radiation, mycotoxins, second hand smoke, occupational exposure to chemicals such as chromium, nickel, asbestos, polycyclic aromatic hydrocarbons, arsenic, vinyl chloride and radioactive gas-radon . Susceptibility to the disease is also genetically determined. The WHO classification distinguishes two main types of lung cancer: small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). The latter is divided into subtypes including squamous cell carcinoma and adenocarcinoma.
(NSCLC). The latter is divided into subtypes including squamous cell carcinoma and adenocarcinoma.
## Lung cancer-short review
Lung cancer is the leading cause of mortality in women and men worldwide, and is considered to be a major global epidemic.
In Europe, lung cancer ranks second in terms of incidence. According to the data, as many as 23% of lung cancer cases worldwide occur in Europe. In 2018, an estimated 1.8 million people developed lung cancer.shows a graph showing the number of lung cancer cases (statistics for 2018). In Austria, lung cancer is the second most common cancer among men-2940 cases, and the third most common cancer among women-2202 cases. In Belgium, 9400 people were diagnosed with lung cancer in 2018. Similarly to the Austrian population, lung cancer is the second most common cancer among Belgian men-15% of all cases, and the third most common cancer among Belgian women-8%. Moreover, it is the most common cause of cancer deaths among both women (15%) and men (30%). In France, lung cancer is the second most common cancer in both women and men. In Germany, more than 66,000 cases of lung cancer were diagnosed in 2018-more than 27,000 women and over 39,600 men. According to the data, in Poland, lung cancer has been diagnosed twice as often among men than among women. In the United Kingdom, lung cancer is diagnosed in 1 in 17 women and 1 in 13 men. Statistical analysis shows that in Sweden, lung cancer causes the highest mortality in both women and men. The data presented inindicate that in 2018, Hungary had the highest rate of lung cancer. The vast majority of lung cancer cases are caused by smoking tobacco, with smoking being a documented factor in the development of lung cancer . Free radical oxygen is the main component of tobacco smoke. The substance may cause oxidation of guanine DNA nucleobases to form 8-oxoguanine (OGG1). Published data suggest that there is an increased risk of lung cancer in smokers with low OGG1 activity. Tobacco smoke contains over 60 carcinogens, including aromatic hydrocarbons such as nitrosamines and benzopyrene, carbon monoxide (chad), tar, phenol, cresol, formaldehyde and hydrogen cyanide. Polycyclic aromatic hydrocarbons activated by cytochrome P450 enzymes can bind to DNA. Enzymes that catalyze the glutathione-S reaction protect against a DNA reaction that causes the formation of DNA adducts. Chronic and too frequent adduct formation might cause gene mutations that could lead to the development of lung cancer. Nicotine inhibits apoptosis and promotes tumor cell growth in lung epithelial cells. Available studies indicate that non-smokers are more likely to survive than smokers, regardless of other prognostic factors. Quitting smoking has a direct impact on reducing The vast majority of lung cancer cases are caused by smoking tobacco, with smoking being a documented factor in the development of lung cancer . Free radical oxygen is the main component of tobacco smoke. The substance may cause oxidation of guanine DNA nucleobases to form 8-oxoguanine (OGG1). Published data suggest that there is an increased risk of lung cancer in smokers with low OGG1 activity. Tobacco smoke contains over 60 carcinogens, including aromatic hydrocarbons such as nitrosamines and benzopyrene, carbon monoxide (chad), tar, phenol, cresol, formaldehyde and hydrogen cyanide. Polycyclic aromatic hydrocarbons activated by cytochrome P450 enzymes can bind to DNA. Enzymes that catalyze the glutathione-S reaction protect against a DNA reaction that causes the formation of DNA adducts. Chronic and too frequent adduct formation might cause gene mutations that could lead to the development of lung cancer. Nicotine inhibits apoptosis and promotes tumor cell growth in lung epithelial cells. Available studies indicate that non-smokers are more likely to survive than smokers, regardless of other prognostic factors. Quitting smoking has a direct impact on reducing lung cancer risk. Exposure to tobacco smoke in the environment increases the risk of lung cancer by as much as 10% to 15%.
Other factors that contribute to the development of lung cancer include exposure to air pollution, chronic infections, asbestos, radon gas, viruses (JC virus, simian virus 40, BK virus, cytomegalovirus and human papillomavirus) and sex hormones .
Susceptibility to the disease is also genetically determined. Gene mutations, which result in changes of protein expression, such as Bax, p53 or Bcl-2, have a significant impact on the prognosis of lung cancer patients. The disease onset is very insidious without any early symptoms. In more advanced stages, the main symptoms at diagnosis include: chronic cough, chest pain, shortness of breath, recurrent pneumonia and hemoptysis. Lung cancer spreads easily to the bones causing pathological fractures and bone pain, to the liver causing significant weight loss, jaundice, nausea and abdominal pain, to the adrenal glands-which is symptomless or leads to endocrine alterations-or to the brain causing seizures, paresis, balance disorders, sensory disturbances, convulsions and headache.
There are two main histological classes of neoplasms derived from respiratory epithelial cells: small cell lung cancer (SCLC) in 15% of cases and non-small cell lung cancer (NSCLC) further divided into adenocarcinoma, squamous cell carcinoma and large cell carcinoma in the other 85% of all cases [3,. The most aggressive is SCLC, which Other factors that contribute to the development of lung cancer include exposure to air pollution, chronic infections, asbestos, radon gas, viruses (JC virus, simian virus 40, BK virus, cytomegalovirus and human papillomavirus) and sex hormones .
Susceptibility to the disease is also genetically determined. Gene mutations, which result in changes of protein expression, such as Bax, p53 or Bcl-2, have a significant impact on the prognosis of lung cancer patients. The disease onset is very insidious without any early symptoms. In more advanced stages, the main symptoms at diagnosis include: chronic cough, chest pain, shortness of breath, recurrent pneumonia and hemoptysis. Lung cancer spreads easily to the bones causing pathological fractures and bone pain, to the liver causing significant weight loss, jaundice, nausea and abdominal pain, to the adrenal glands-which is symptomless or leads to endocrine alterations-or to the brain causing seizures, paresis, balance disorders, sensory disturbances, convulsions and headache.
There are two main histological classes of neoplasms derived from respiratory epithelial cells: small cell lung cancer (SCLC) in 15% of cases and non-small cell lung cancer (NSCLC) further divided into adenocarcinoma, squamous cell carcinoma and large cell carcinoma in the other 85% of all cases [3,. The most aggressive is SCLC, which rapidly spreads to the regional lymph nodes and parenchymal organs vessels. Another characteristic includes extreme predilection to the brain and other parenchymal organs. One of the first cell lines to analyze molecular biology of lung cancer was established in the 1960s, while the SCLC cell line was established in the 1970s. Lung cancer treatment, depending on the type and stage of cancer, includes surgery, chemotherapy, targeted therapy and radiation therapy. Cisplatin is the backbone of the most efficient chemotherapy regimens, usually combined with either gemcitabine, vinorelbine, etoposide or taxanes.
Nowadays, in locally advanced NSCLC, which is not amenable towards radical surgery, the best treatment results are obtained by using upfront concomitant chemoradiotherapy followed by a maintenance immune checkpoint inhibitor. The results of phase III prospective randomized clinical trial PACIFIC showed that durvalumab-an anti-PDL-1 monoclonal IgG1 kappa antibody-significantly prolonged overall survival, as compared with placebo (stratified hazard ratio for death, 0.68; 99.73% CI, 0.47 to 0.997; p = 0.0025). Interestingly, a meta-analysis evaluating the effectiveness of anti-PD1 inhibitors and standard chemotherapy in female patients did not show a clear benefit from immune checkpoint inhibitors. In contrast, male patients had a 24% reduction in the risk of progression. These results raise the question about the gender-related mechanisms and the value of sex as an independent prognostic factor for anti-PD1 or anti-PD-L1 blockade. Female patients may have more potent immune systems or develop immune-resistant lung tumors.
Patients diagnosed with adenocarcinoma harboring druggable mutations are currently offered molecularly targeted medications directed against EGFR, ALK, ROS, Her2 new, MET, TRK or any other even less frequent alterations. In SCLC, the treatment progress is less evident than in NSCLC, but immunotherapy impacting the CTLA4 or PD signaling can be added to standard chemotherapy and combined with radiation.
Better treatment results in lung cancer female patients have been noticed in several clinical trials. Pinto et al. retrospectively reviewed available data to explore differences in gender outcomes in NSCLC. The meta-analysis showed a 27% reduction in the risk of death in female patients. In six trials evaluating EGFR TKI (tyrosine kinase inhibitors) in adenocarcinoma of the lung, there was a 10% reduction in the risk of progression in women compared to men (HR = 0.44 vs. 0.34 for men and women, respectively). However, it is essential to mention that important prognostic factors such as ethnicity or smoking status have not been included. Another meta-analysis by Pujol et al. did not show any differences in benefit from cetuximab (an anti-EGFR antibody) between genders in the subgroup analysis.
Regarding another biological compound-anti-angiogenic bevacizumab-the results of clinical trials are inconclusive. A meta-analysis by showed no correlation between gender and the treatment effect. Female patients with lung adenocarcinoma harboring ALK that was rearranged and treated with ALK inhibitors such as crizotinib or ceritinib obtain similar benefits as men.
## Sex differences in lung cancer
In contrast to men, available epidemiological data indicate an upward trend in the incidence of lung cancer in women, despite a 50 percent reduction of women smokers. The decisive role is attributed to female sex hormones, mainly estrogen, which is a steroid. The main hormone referred to as the reproductive hormone is 17-β-Estradiol-E2, which is synthesized in the ovaries under the influence of the luteinizing and follicular hormones.
There are two types of estrogen receptors (ER): ER alpha (ERα, also known as ESR1) and ER beta (ERβ, ESR2). Many studies have shown a correlation between hormone replacement therapy and the risk of cancer mortality in women. The ERβ receptor has been demonstrated as essential in healthy lung tissues, where it is necessary to maintain the extracellular matrix. The ERβ estrogen receptor is characterized by genomic and non-genomic activity. The non-genomic effect concerns vasodilatation. It becomes apparent 5 to 20 min after exposure to estrogen, and does not require changes in gene expression. In contrast, the genomic effect of estrogens involves protection against atherosclerosis and inhibition of the response to injury. In addition, high ERβ expression is associated with a poor prognosis of advanced NSCLC. mRNA analyses were performed, comparing lung tumors with low and high levels of ERβ receptors. Tumors exhibiting high ERβ expression have been characterized as signalers via fibroblast growth factors, which are the autocrine signaling loop and contribute to the progression of lung cancer and pluripotency of human embryonic stem cells. Moreover, cancer stem cells (CSCs) are responsible for both primary tumor growth and metastasis. Wnt pathway, Notch pathway and Hh pathway routes are responsible for differentiation and pluripotency of CSCs. In vitro studies indicate that in the lung, ER estrogen receptors may interact with the epidermal growth factor receptor (EGFR) during carcinogenesis. A clinical study of 180 women showed an increased risk of developing adenocarcinoma in patients receiving hormone replacement therapy (HRT). It was also noticed that women taking HRT for extended time periods are more susceptible to the harmful effects of tobacco smoke. Genetic factors are another important determinant of developing lung cancer, which is independent of the smoking status. Notably, available data indicate that it is the reason why women bear a generally higher risk than men. One of the main well-studied factors is the role of CYP1A1 (cytochrome P450) gene expression. The CYP1A1 gene codes for the phase I enzyme, which is involved in the metabolism of polycyclic aromatic hydrocarbons (PAH), contained in tobacco smoke as well as other types of smoke produced by burning different products. This mechanism prevents the precarcinogen from turning carcinogenic. Circulating female steroid hormones are believed to influence the modulation of PAH enzyme expression due to interaction with receptors in the patients' lungs. In addition, it has been shown that the expression of steroid receptors is much more common in women than in men. Available clinical studies indicate that overexpression of the CYP1A1 gene has a clear effect on the increased risk of lung cancer in women. It is also known that due to impaired DNA repair mechanisms, platinum-based chemotherapy has a better therapeutic effect. The antitumor activity of platinum drugs is mainly based on the mechanism of deformation of the DNA structure, thanks to the formation of stable DNA adducts.
## Estrogens short review
Steroid hormones are endogenous estrogens that include estrone (E1), estriol (E3) and 17β-estradiol (E2). These structural and biogenic hormones are derived from cholesterol C17. LDL-cholesterol is the major reactant necessary for the synthesis of steroid hormones, which is called steroidogenesis. Estrogens also have a positive effect on the cardiovascular system, among other factors, by lowering total cholesterol, shaping the blood lipid profile and also affecting the musculoskeletal system by stimulating the repair process of damaged muscle fibers. Notably, during menopause, there is a decrease in estrogen levels, which results in a decrease in muscle mass, as well as osteoporosis. The lowest biological activity is shown by estriol, which is considered to be the weakest of estrogens, being a product of estrone and estradiol metabolism. Estrone, in turn, exhibits a markedly higher biological Cholesterol is metabolized in a number of enzymatic pathways. The process of their creation depends on the aromatization of androgens. In addition, they have the ability to bind to the protein receptor (ER) as well as to diffuse through the cell membrane. Direct penetration through the cell membrane into the cytosol occurs due to the properties of the lipophilic structure. In addition, estrogens are included in the group of pleiotropic hormones.
The two main estrogens also named "parent" estrogens-estrone, and estradiol-are low-molecular steroids of lipophilic nature acting as agonists of estrogen receptors ERα and ERβ. However, estrogen-like activity should be also attributed (with varying extent) to a range of estrogen metabolites, usually referred to as EM. The parent estrogens are irreversibly oxidized in the cytochrome P450 dependent pathway by hydroxylation at the C-2, C-4 and C-16 positions of the steroid ring forming hydroxylated metabolites. The main and mots studied metabolites include 2-hydroxyestrogen (2-OH-E), 4-hydroxyestrogen (4-OH-E) and 16-hydroxyestrogen (16α-OH-E) have significant estrogenic activity. Those metabolites are further transformed by conjugation with a methyl group, glucuronic acid, and sulfuric acid (forming methoxy-metabolites, glucuronates and sulfates, respectively). Thus, many authors point out the necessity of studying a wide panel of estrogens, including minor metabolites in order to fully understand their influence on human physiology as well as the etiology and progression of various pathological states. This relatively new approach needs easily accessible and reliable bioanalytical methods to determine their concentrations in human biofluids and tissues. From the analytical point of view, this is not a straightforward task for several main reasonsFirstly, the analytical technique needs to have enough selectivity to differentiate between chemically similar compounds, so an efficient separation technique is required. To achieve that, chromatography is applied, with high performance liquid chromatography coupled to mass spectrometry as a method of choice. Practically, it is not possible to separate lipophilic parent estrogens, their hydroxylated metabolites, and much more polar conjugates with glucuronic and sulfuric acids. In order to avoid the development validation of separate LC-MS methods for polar and nonpolar analytes, enzymatic hydrolysis is typically involved as a sample preparation step. β-glucuronidase/sulfatase from Helix pomatia has been proven to sufficiently hydrolyze estrogen metabolites. The involvement of enzymatic cleavage enables us to gain detailed information about a wide range of estrogen metabolites. Secondly, due to the low levels of many of the above mentioned metabolites, the high sensitivity of the analysis is a critical issue. Sensitive quan-tification depends on the detection method and sample preparation. Mass spectrometry, despite being expensive, can detect estrogen compounds down to the pmol/L level. On the other hand, extensive clean-up of a sample with simultaneous preconcentration of analytes is beneficial to improve sensitivity and avoid interfering compounds. Improvement at the sample preparation step in estrogen analysis is thus still required. The application of novel selective materials, including sorbents processed by using 3D-printing, can be a promising approach, especially in a high throughput format. More selective extraction utilizing specific sorbent-analyte interactions can potentially further improve quantification of a wide range of estrogens. In particular, boronate affinity solid-phase microextraction, as was previously claimed to be useful for diol-containing compounds, seems to be an attractive approach. The main role of estrogens in a woman's body is to shape secondary and tertiary sexual characteristics, which affects the development of external genitalia, as well as the fallopian tubes, uteri, vaginas and nipples. Estrogens also perform a key function in the male body-they condition the development of the male reproductive system.
Estrogens also have a positive effect on the cardiovascular system, among other factors, by lowering total cholesterol, shaping the blood lipid profile and also affecting the musculoskeletal system by stimulating the repair process of damaged muscle fibers. Notably, during menopause, there is a decrease in estrogen levels, which results in a decrease in muscle mass, as well as osteoporosis. The lowest biological ac-tivity is shown by estriol, which is considered to be the weakest of estrogens, being a product of estrone and estradiol metabolism. Estrone, in turn, exhibits a markedly higher biological activity. Finally, the type of estrogen with the highest activity is a 17β-estradiol, with a potency of about 5 to 10 times greater than the former type. Both types of estrogen receptors-ERα and ERβ-have a relationship with the heat shock proteins (HSP) complex. In addition, estrogen receptors have the ability to form heterodimers and homodimers.
## Estrogens in etiopathogenesis and therapy of lung cancer
It is well known that estrogens can cause carcinogenicity. The impact of estrogens is noted in female cancers, for example breast cancer, or in the case of modulation of genetic mutations. However, based on available clinical studies, it is also hypothesized that the mechanism of action of the estrogen pathway in lung cancer is similar to the one established for breast cancer.
Available studies indicate that estrogen affects lung carcinogenesis via non-genomic and genomic signaling. In genomic signaling, homodimers and heterodimers are formed acting as ligands, which bind to the ER nucleus. In contrast, non-genomic signaling works by means of the mitogen-activated protein kinase (MAPK1) pathways through the ER.
The endogenous metabolite of 17β-estradiol (E2) resulting from the hydroxylation and methylation of the second-position is 2-methoxyestradiol (2ME, (17beta)-2-methoxyestra-1,3,5 (10)-triene-3,17-diol). This metabolite inhibits angiogenesis by reducing endothelial cell proliferation. In addition, 2ME is an antiproliferative and anti-angiogenic agent.
The metabolite 2ME inhibits carcinogenic cell growth due to tubulin binding. In vitro studies show that 2-methoxyestradiol inhibits a wide range of non-cancer and cancer cell lines. It has also been shown in vitro that 2ME inhibits several stages of the andiogenic cascade, thereby inhibiting proliferation and inducing tumor cell apoptosis. Cell line growth inhibition was achieved in the lung cancer line of human origin A459 and H460 p53 wild-type. Minor changes after treatment with 2ME occurred in H322 p53 and H358 type p53 cell lines. Western Blot analysis was performed, which resulted in a significant increase in p53 protein after treatment with 2-ME. The main change observed during the study involving treatment with 2ME was an eight-fold increase in endogenous p53 protein.
The level of mutated p53 protein remained unchanged. The p53 protein is the major tumor suppressor responsible for regulating the cell's life cycle and apoptosis.
The Charité University Clinic in Berlin conducted a study to confirm the inhibition of the growth of various cell lines with 2ME, including lung cancer. Orally administered 2-ME was combined with gene therapy and an adenovirus expressing the p53 gene was administered intravenously. The results demonstrated that lung cancer cells that were resistant to cisplatin were particularly sensitive to 2ME.
Several experiments have been devoted to the metabolite 4-hydroxyestrogen (4-OH-E), which is a CYP1B1 product, and has mutagenic and carcinogenic effects. Studies indicate that tobacco smoke stimulates the metabolism of 17β-estradiol to the toxic metabolite 4-OH-E. In addition, 4-OH-E levels are elevated in patients with lung cancer as compared to healthy controls. It has been hypothesized that the 4-OH-E metabolite affects oncogene mutation in the lungs and also activates ER signaling, which increases the risk of lung cancer. Last year at the Research Institute of Fox Chase Cancer Center in Philadelphia, it was discovered that the human lung can metabolize estrogen to 4-hydroxyestrogen.
Studies show differential expression of nuclear ER-β in NSCLC. Nuclear expression of ER-α and ER-β was determined by immunohistochemistry. The study identified ER-β nuclear expression in NSCLC tumor tissue and control tissue correctly, in both women and men. In men, nuclear ER-β expression was found to be more frequent in adenocarcinomas of the lung.
In the case of NSCLC, research indicates stimulation of tumor growth through the expression of ER forms that interact with the epidermal growth factor receptor (EGFR). Importantly, EGFR supports the growth of NSCLC and breast cancer. The available data indicate the responsibility of HER2 and EGFR for a number of states of endocrine immunity. Moreover, in response to estrogen, the ERs proliferate as a result of their interaction with ER-containing vascular endothelial cells.
Estrogens regulate the expression of miRNAs, which are found in small non-coding RNAs containing about 21-25 nucleosites. The miRNA finds application in distinguishing between different subtypes of lung cancer. Studies have reported that miR-124a is characteristic of NSCLC lung adenocarcinoma and miR-205 for squamous cell carcinoma, while miR-375 and miR-21-5p are highly expressed in SCLC. In addition, the histological patterns of growth of lung adenocarcinomas were analyzed, showing a significant influence of miRNA expression. In tumors, the presence of solid components in tumors was demonstrated when miR-212, miR-27a and miR-132 were expressed. However, in order to demonstrate the possible benefits of miRNA targeted therapy in lung cancer patients, more comprehensive studies should be conducted.
Research indicates that estradiol can be synthesized locally in NSCLC, analogous to breast cancer tissue. Moreover, on the basis of the obtained results, it was proved that the concentration of estradiol in the NSCLC tissues was significantly 3.7 times higher in men than in women after menopause. Researchers say that the essence of this phenomenon are the circulating androgens produced by aromatase, which in the case of NSCLC and estradiol production could be the leading substrates. The study determined the estradiol concentration in 59 NSCLC cases, followed by in vitro A549 NSCLC cell cultures. Forty-three of the subjects showed an increase in the concentration of estradiol in the neoplastic tissues compared to the non-neoplastic lung tissues of the patients. However, in the case of in vitro studies, the increase in the proliferation of cell cultures of both A549 + ER-α and A549 + ER-β was determined. Moreover, both cell cultures were found to express aromatase. Importantly, studies show an increase in A549 cell proliferation during testosterone use. Therefore, it is suggested on the basis of the obtained studies that if estrogens, and more specifically oestradiol occurring inside cancerous tumors by aromatase, including NSCLC, and favor their development, anti-estrogen therapy would be an effective therapy in the fight against cancer.
Both NSCLC and breast cancer are entities that frequently occur in everyday pathological diagnosis. However, it should be remembered that both disease entities in the form of lung metastatic breast cancer and primary lung cancer are treated completely differently. Typical immunohistochemical markers in the differential diagnosis of breast cancer are: HER2-tyrosine kinase receptor encoded by HER2-growth-promoting protein, ER, MAMG-mammaglobion, GATA3-GATA 3 binding protein (zinc finger transcription factor) and PgR-the steroid hormone progesterone receptor. However, in the case of NSCLC, the most frequently used immunohistochemical markers are: TTF-1-thyroid transcription factor 1, Napsin A, CK7-cytokeratin 7, p63, p40 and CK5-cytokeratin 5. In addition, molecular tests for mutations are performed to diagnose NSCLC activators in the EGFR gene. Due to the limited research on the immunohistological expression of NSCLC markers, a study was conducted using clinical variables and staining results for CK5/6, p40, TTF-1 and napsin A. An analysis of 1291 samples of NSCLC patients with successively diagnosed adenocarcinoma (ADC) was performed-636 patient samples, squamous cell carcinoma (SqCC)-536 patient samples, large cell carcinoma (LLC)-65 patient samples, polymorphic carcinoma (PC)-34 patient samples, and large cell neuroendocrine carcinoma (LCNEC)-20 patient samples. Most of the patients had disease stages I to III. In addition, 380 patients were women with more ADC than SqCC, while the remaining 911 patients were men. ER-positive tumors were found to be much more common in women than in men. On the basis of the conducted studies, the expression of all five markers was found in many patients, thus it can be concluded that the interpretation of tumor markers is important in the differential diagnosis.
Estrogen is known to induce ERβ-mediated cell growth in NSCLC. Moreover, high levels of circulating interleukins 6 (IL6) are associated with poor prognosis for NSCLC; however, the determination of the specific role of IL6 in NSCLC is not fully understood and requires a lot of research. One of the studies assessed both the biological effects as well as the expression of interleukins in NSCLC cells after treatment with 17β-estradiol (E2). The expression of IL6/ERβ in 289 NSCLC samples was determined via immunohistochemistry. The study included A549 and H1793 non-small cell lung cancer cells. Cells were treated with E2. Their expression levels were determined sequentially by means of ELISA, western blotting and immunofluorescence staining. The study also used an animal xenograft model to determine and observe differences in IL6 and ERβ expression in NSCLC tumor growth. Research showed an increased increase in both ERβ and IL6, which was closely related, the researchers indicated, to either increased metastasis or decreased differentiation. Indeed, the study showed ERβ mediated regulation of IL6 expression through the PI3K/AKT and MAPK/ERK pathways due to the use of E2. Importantly, an increase in malignancy of NSCLC cells was also found due to the regulation of E2 on IL6/ERβ.
Experimental research indicate that the ER potentially promotes NSCLC progression via modulation of the membrane receptor signaling network, composed of the GSK3β/βcatenin, Notch1 and EGFR pathways. Furthermore, one of the treatments for lung cancer may be anti-estrogen therapy. Additionally, 17-β-estradiol is produced by aromatase activity, which in turn influences the control of estrogen levels in the lung cancer microenvironment. Clinical studies suggest that aromatase inhibitors are a good therapeutic option for lung adenocarcinoma. Aromatase inhibitors are classified into classes I and II: (I) irreversible steroid inhibitors; (II) non-steroidal inhibitors. Sulfotransferases activated by e.g., dexamethasone are also used in the treatment of hormonedependent tumors. Preclinical studies indicate inhibition of A549 cell tumor growth, while lowering estrogen levels. Fulvestrant, an estrogen receptor degradator, is also used in NSCLC research. According to the data, fulvestrant causes greater sensitization of the NSCLC tumor to chemotherapy and reduces the mesinochemical features. Due to the fact that one of the leading elements of the patient's immune profile are steroid hormones, next to chemotherapy, radiotherapy or surgery, immunotherapy is an effective lung cancer treatment strategy. The development of personalized medicine is conditioned by numerous preclinical and clinical studies taking into account sex differences or the expression of hormonal markers, in which the response to therapy in patients with NSCLC, survival as well as pathological and clinical features are tested.
## Current clinical trials registered for non-small lung cancer and estrogens with completed status with results
Currently, as of 20 December 2020, 3 clinical trials are registered on clinicaltrials.gov for lung cancer: (Alisertib); in lung cancer, the chemo-sensitive, chemo-resistant population was analyzed, in breast cancer, ER2 and ER2 were analyzed. HR + = positive estrogen or progesterone receptor, both SCLC and NSCLC patients received 50 mg of MLN8237 orally twice daily for 7 days, consecutively 14 days off.
# Conclusions
Extensive data, in vitro and in vivo studies indicate a significant role of the female sex hormone β-estradiol in the etiopathogenesis, clinical treatment and prognosis of NSCLC. This manuscript focuses on a review of the available data describing the hormonal difference between the sexes in the development of lung cancer.
Estrogen activity in the growth of NSCLC tumors has been confirmed by a number of studies, and lowering the level of estrogen hormones could have a positive effect on antitumor activity in this area.
There have been several reports suggesting an upward trend in the incidence of lung cancer in women. Compared to men, a much more common tumor suppressor p53 mutation was observed in women with NSCLC.
There is enough in vivo and in vitro evidence that female sex hormones are an important factor in the development of neoplastic tumors, which are mitogenic and carcinogenic. Research indicates that women are more predisposed and exposed to adenocarcinoma, while men are more likely to suffer from squamous cell carcinoma.
There are two types of estrogen receptors in lung cancer cells: ERα and ERβ. The ERα receptor has been detected in a number of lung cancer cell lines, and interestingly, the ERβ receptor is a prognostic marker in NSCLC.
The manuscript presents the molecular basis of lung cancer in women. The authors point to a number of genetic abnormalities that may be closely related to the increased incidence of lung cancer among women. Advances in medicine and molecular diagnostics create an opportunity for more effective anti-cancer therapies and detection of lung cancer at an earlier stage. Role of estrogens in pathogenesis and diagnosis of lung cancer therefore still needs to be elucidated. The review was based on extensive literature emphasizing the important role of estrogen and estrogen receptors in the progression and development of NSCLC. |
Persistent Changes in Circulating and Intestinal γδ T Cell Subsets, Invariant Natural Killer T Cells and Mucosal-Associated Invariant T Cells in Children and Adults with Coeliac Disease
Coeliac disease is a chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals. The only current therapy is a lifelong gluten free diet. While much work has focused on the gliadin-specific adaptive immune response in coeliac disease, little is understood about the involvement of the innate immune system. Here we used multi-colour flow cytometry to determine the number and frequency of γδ T cells (Vδ1, Vδ2 and Vδ3 subsets), natural killer cells, CD56 + T cells, invariant NKT cells, and mucosal associated invariant T cells, in blood and duodenum from adults and children with coeliac disease and healthy matched controls. All circulating innate lymphocyte populations were significantly decreased in adult, but not paediatric coeliac donors, when compared with healthy controls. Within the normal small intestine, we noted that Vδ3 cells were the most abundant γδ T cell type in the adult epithelium and lamina propria, and in the paediatric lamina propria. In contrast, patients with coeliac disease showed skewing toward a predominant Vδ1 profile, observed for both adult and paediatric coeliac disease cohorts, particularly within the gut epithelium. This was concurrent with decreases in all other gut lymphocyte subsets, suggesting a specific involvement of Vδ1 cells in coeliac disease pathogenesis. Further analysis showed that γδ T cells isolated from the coeliac gut display an activated, effector memory phenotype, and retain the ability to rapidly respond to in vitro stimulation. A profound loss of CD56 expression in all lymphocyte populations was noted in the coeliac gut. These findings demonstrate a sustained aberrant innate lymphocyte profile in coeliac disease patients of all ages, persisting even after elimination of gluten from the diet. This may lead to impaired immunity, and could potentially account for the increased incidence of autoimmune co-morbidity.
# Introduction
Innate, or unconventional, lymphocytes such as γδ T cells, CD56 + T cells, natural killer (NK) cells, invariant NK T (iNKT) cells and mucosal associated invariant T (MAIT) cells, comprise part of a complex immunosurveillance system, where infected, damaged, or otherwise abnormal cells are rapidly recognised and eliminated. Depending on the context of their activation, innate lymphocytes can also display immunoregulatory properties, e.g. invariant natural killer T (iNKT) cells can produce IFN-γ or IL-4 depending on the nature of antigen encountered and the cytokine environment [bib_ref] Going both ways: Immune regulation via CD1d-dependent NKT cells, Godfrey [/bib_ref]. The role of innate lymphocytes in the pathogenesis of coeliac disease (CD) remain unknown, but it has been reported that NK cells and iNKT cells are reduced in blood and gut of CD patients, and display a diminished capacity for cytokine production. Mucosal associated invariant T (MAIT) cells are also implicated in mucosal immunity, recognising and responding to a diverse set of bacterial and fungal antigens, including microbial vitamin metabolites [bib_ref] Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1, Treiner [/bib_ref] [bib_ref] Human MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi IL-17-secreting T cells, Dusseaux [/bib_ref] [bib_ref] MR1 presents microbial vitamin B metabolites to MAIT cells, Kjer-Nielsen [/bib_ref]. The role of MAIT cells in CD has not been previously investigated however. Infiltration of T cells into the small intestinal epithelium is one of the earliest events in CD development [bib_ref] Quantitation of intraepithelial lymphocytes in human jejunum, Ferguson [/bib_ref]. Both αβ and γδ T cells are present in this infiltrate, but while αβ T cell levels return to normal upon exclusion of gluten from the diet, γδ T cells remain elevated [bib_ref] Quantitation of intraepithelial lymphocytes in human jejunum, Ferguson [/bib_ref] [bib_ref] Numbers of T cell receptor (TCR) alpha beta+ but not of TcR..., Kutlu [/bib_ref] [bib_ref] Small intestinal CD8+TCRgammadelta+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients..., Bhagat [/bib_ref]. The significance of this and the specific role of γδ T cells in the gut remain unknown. There are 3 main γδ T cell subsets in humans -Vδ1, Vδ2 and Vδ3. Within the peripheral blood, the majority of γδ T cells possess an invariant Vγ9Vδ2 T cell receptor, whereas the Vδ1/Jδ1encoded chain predominates in healthy gut tissue [bib_ref] Do human Peyer's patches contribute to the intestinal intraepithelial gamma/delta T-cell population?, Farstad [/bib_ref]. The Vδ1 subset is reportedly expanded in the intestinal epithelium in CD [bib_ref] Gamma delta T cell receptor-positive cells of the human gastrointestinal mucosa: occurrence..., Trejdosiewicz [/bib_ref] [bib_ref] Phenotypical and functional characterization of small intestinal TcR gamma delta + T..., Rust [/bib_ref] [bib_ref] T cell receptor heterogeneity in gamma delta T cell clones from intestinal..., De Libero [/bib_ref] [bib_ref] TCR gamma/delta + and CD8+TCR alpha/beta + intraepithelial lymphocytes (IEL) express proliferation..., Halstensen [/bib_ref] [bib_ref] Intraepithelial T cells of the TcR gamma/delta+ CD8-and V delta 1/J delta..., Halstensen [/bib_ref] and expresses NKG2A and TGF-β, suggesting an immunoregulatory role [bib_ref] Small intestinal CD8+TCRgammadelta+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients..., Bhagat [/bib_ref] , but data regarding other γδ subsets in the intestine is lacking, or contradictory [bib_ref] Predominance of T cell receptor V delta 3 in small bowel biopsies..., Falk [/bib_ref] [bib_ref] Analysis of gamma delta V region usage in normal and diseased human..., Bucht [/bib_ref] [bib_ref] Repertoire analysis of human peripheral blood lymphocytes using a human V delta..., Peyrat [/bib_ref]. Since murine γδ T cell subsets differ distinctly from human, and the majority of work on γδ T cells in humans involves the Vδ2 subset, clarification and distinction of the roles discrete γδ subsets play is important, particularly if these cells are to be successfully exploited for immunotherapy [bib_ref] Human gammadelta-T cells in adoptive immunotherapy of malignant and infectious diseases, Lopez [/bib_ref] [bib_ref] T-APCs: a novel tool for immunotherapy?, Moser [/bib_ref]. Phenotypic and genetic analyses indicate that different γδ T cell subsets may have different, perhaps even opposing roles [bib_ref] Distinct gene expression in human Vdelta1 and Vdelta2 gammadelta T cells following..., Kress [/bib_ref] , and developmental pathways [bib_ref] Vdelta1 and Vdelta2 gammadelta T cells express distinct surface markers and might..., Rosa [/bib_ref].
In this study we used multi-parameter flow cytometry to characterise the frequency and phenotype of a number of novel innate lymphocyte populations in the blood and gut of adult and paediatric patients with CD. By comparing profiles of healthy control donors and CD patients, we were able to identify persistent alterations in innate lymphocyte populations, as a first step toward elucidating the potential roles for these cells in CD pathogenesis.
# Materials and methods
# Ethics statement
## Study populations
Blood samples were obtained from 76 adult patients attending the coeliac clinic at St James's Hospital, Dublin (SJH), and 78 control subjects recruited from healthy, age-and sex-matched research staff. Controls reported no family history of CD and tested negative for tissue transglutaminase antibodies (tTG). Within the coeliac donor cohort, 56 individuals were defined as treated coeliac donors (TCD), i.e. adhering to a gluten free diet, with accompanying tTG negative serology for at least one year prior to sample collection. The remaining 20 coeliac patients were classified as untreated coeliac donors (UCD) -a population of tTG positive patients on a glutencontaining diet. Duodenal biopsies were obtained from 8 TCD, 6 UCD and 7 control subjects. The control cohort comprised patients undergoing routine endoscopy at SJH, who reported no family history of CD, showed minimal histological inflammation, and tested tTG negative. Blood samples and duodenal biopsies were also obtained from 22 paediatric UCD and 38 age-and sex-matched control patients undergoing endoscopy at Our Lady's Children's Hospital Crumlin (OLCH).
In all cases, CD diagnosis was confirmed according to clinical histological and serological analyses. Patients with a known history or subsequent diagnosis of inflammatory bowel disorders were excluded from recruitment/analysis.
All samples were collected with the informed consent of individuals over 18 years of age, or with assent of those under 18 years with consent from guardians, as appropriate.
## Sample collection
Blood was harvested in EDTA and serum Vacutainer tubes (BD Biosciences).
Duodenal biopsies (3-6 sections) were collected into sterile pots containing calcium-and magnesium-free Hank's Balanced Salt Solution (HBSS) (Sigma-Aldrich) supplemented with 5% v/v foetal calf serum (FCS) (BioWest). Tissue biopsies were processed within 15 minutes of collection.
## Preparation of intestinal tissue
Small intestinal epithelial and lamina propria cells were released from biopsies as previously described [bib_ref] Diverse populations of T cells with NK cell receptors accumulate in the..., O'keeffe [/bib_ref]. Briefly, duodenal biopsies were agitated for 1 hour in a shaker at 37°C in HBSS supplemented with 5% v/v FCS, 1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) and 1 mM dithiothreitol (DTT) (Fisher). The resulting epithelial singlecell suspension was filtered through a 40 µM filter (BD Biosciences), washed in complete RPMI 1640 solution (Gibco, with Glutamax, supplemented with 10% v/v FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B Fungizone and 0.02 M HEPES). Tissue remaining in the filter was collected into complete cRPMI solution containing 130 U/ml collagenase (Type IV-S, Sigma-Aldrich), and rotated for 3 hours at 37°C. Cells were then filtered, washed and enumerated by ethidium bromide and acridine orange staining. T cells isolated from the intestinal epithelium consistently stained >90% positive for the integrin CD103.
## Antibodies and flow cytometry
Absolute numbers of T cells, B cells and NK cells in unprocessed blood samples were determined by TruCount MultiTEST mAb (BD Biosciences) staining and analysis on a BD FACSCanto flow cytometer. For lymphocyte phenotyping, whole blood was stained directly according to BD FACS Lysing Solution recommendations. Dissociated intestinal cells were resuspended in approximately 50 µl phosphate buffer containing 1% v/v BSA, and 0.02% v/v sodium azide, prior to staining. Fluorochrome-labelled monoclonal antibodies (mAb) used for phenotyping included: CD3-Pacific Blue, γδTCR-PE, Vδ2-PE, CD56-PE/Cy7, Vα7.2-APC, CD161-FITC, CD45RA-FITC, Vα24-Jα18-PE (clone 6B11), CD27-APC (all from BioLegend), Vδ3 (unconjugated antibody, clone 11.5B; Beckman Coulter), Vδ1-FITC (ThermoScientific, clone δTCS1). Vδ3 mAb was conjugated to APC dye using a Lightening-Link conjugation kit (Innova Biosciences, UK). Cells were stained, fixed in 1% paraformaldehyde (Santa Cruz Biotechnology) and analysed on a CyAn flow cytometer (Beckman Coulter). NK cells were defined as CD3 -/CD56 + lymphocytes, CD56 + T cells as CD3 + /CD56 + , MAIT cells as CD3 + /CD161 + /Vα7.2 + , and iNKT cells as CD3 + /6B11 + .
# Intracellular cytokine analysis
Small intestinal epithelial cells were isolated, washed and resuspended in cRPMI medium, and immediately plated at a density of 1x10 6 cells per ml. Cells were stimulated with either medium only or 10 ng/ml phorbol myristate acetate (PMA) and 1 µg/ml ionomycin (both from Sigma-Aldrich), and incubated for 4 hours at 37°C, 5% CO 2 in the presence of 10 µg/ml Brefeldin A (Sigma-Aldrich). Cells were then harvested, stained for cell surface markers, fixed in 1% paraformaldehyde (Santa Cruz Biotechnology), permeabilised using 0.2% w/v Saponin (Sigma-Aldrich), incubated with the following intracellular cytokine antibodies: IFN-γ-Pacific Blue and IL-10-APC (BioLegend), IL-17A-PerCP/Cy5.5, IL-21-PE, and IL-22-PECy7 (eBioscience). Cells were analysed on a CyAn flow cytometer (Beckman Coulter).
## Statistical analyses
Prism GraphPad software was used for data analysis, and differences in control and coeliac donor data were evaluated using a Mann Whitney U test. Statistical advice was provided for this study by Centre for Support and Training in Analysis and Research (CSTAR), University College, Dublin.
# Results
## Innate lymphocytes are decreased in circulation of adult, but not paediatric coeliac patients
Flow cytometric analysis of whole blood revealed significant decreases in almost all circulating innate lymphocyte subsets for the adult, but not paediatric, CD cohort when compared to normal controls [fig_ref] Figure 1: Circulating innate lymphocytes are depleted in adults, but not children, with CD [/fig_ref]. Adult coeliacs showed marked decreases in all three main γδ T cell subsets (Vδ1, Vδ2 and Vδ3) both in proportion of total T cells and absolute cell number. iNKT and MAIT cells were also reduced in adult CD patient circulation, albeit not as dramatically as γδ T cells. Interestingly, these decreases were observed even when patients were adhering to a GFD, showed negative serology and reported an improvement in symptoms (i.e. treated CD).
## Innate lymphocyte subset frequencies are perturbed in both adult and paediatric cd patient small intestine
We noted that Vδ3 + cells accounted for the majority of healthy intestinal γδ T cells, in both the epithelium and lamina propria of control donors of all ages, with the exception of the paediatric epithelium, where proportions of Vδ1 + and Vδ3 + cells were similar [fig_ref] Figure 2: Innate lymphocyte subsets are altered in the intestinal epithelium and lamina propria... [/fig_ref]. The average Vδ3 + cell frequency observed for paediatric control donors was 7.5% in epithelium and 6.4% in lamina propria, compared to Vδ1 + cell frequencies of 7.8% and 3.5% respectively. In comparison, in adult control donors, average Vδ3 + cell frequencies were 3.9% in epithelium and 3.4% in lamina propria compared to Vδ1 + cell averages of 1.9% and 1.4%, respectively. Unlike Vδ1 + cells, which were most abundant in the gut epithelium, Vδ3 + cells were similarly well represented in both the epithelium and lamina propria. In both adult and paediatric cohorts, we observed that the frequency of Vδ1 + cells was elevated in CD gut epithelium, in line with previous literature [bib_ref] Gamma delta T cell receptor-positive cells of the human gastrointestinal mucosa: occurrence..., Trejdosiewicz [/bib_ref] [bib_ref] Phenotypical and functional characterization of small intestinal TcR gamma delta + T..., Rust [/bib_ref] [bib_ref] T cell receptor heterogeneity in gamma delta T cell clones from intestinal..., De Libero [/bib_ref] [bib_ref] TCR gamma/delta + and CD8+TCR alpha/beta + intraepithelial lymphocytes (IEL) express proliferation..., Halstensen [/bib_ref] [bib_ref] Intraepithelial T cells of the TcR gamma/delta+ CD8-and V delta 1/J delta..., Halstensen [/bib_ref]. In contrast to the expansion of Vδ1 + cells in CD, Vδ3 + cells did not expand, but were instead significantly reduced in the paediatric lamina propria. Intestinal Vδ2 + cell frequencies were low in the control donor gut and remained unchanged in CD, except for a moderate increase observed in the paediatric lamina propria. iNKT cells were strikingly reduced in the paediatric CD epithelium when compared to controls, though this trend was not observed in the lamina propria, nor in adult CD patients. MAIT cell frequencies were significantly reduced throughout the paediatric gut, particularly in the lamina propria (a known MAIT cell niche), and a similar trend was noted in adult donors.
## Small intestinal γδ t cells from paediatric cd patients display a functional effector memory phenotype and are capable of rapid activation upon in vitro stimulation
Having observed the abundance of Vδ1 + cells in the coeliac gut, we undertook further characterisation of γδ T cells in blood and biopsies taken from paediatric patients [fig_ref] Figure 3: γδ T cells from the coeliac small intestine display a functional "effector... [/fig_ref]. Memory status was evaluated by analysis of CD45RA and CD27 markers expressed by γδTCR + cells in paediatric blood and gut [fig_ref] Figure 3: γδ T cells from the coeliac small intestine display a functional "effector... [/fig_ref]. Although much variation was seen between individuals, overall frequencies of circulating γδ T cells bearing naïve, central memory (T CM ), effector memory (T EM ) and terminally differentiated (T EMRA ) phenotypes were similar in CD patients and controls. However, in the coeliac gut, strong skewing toward a predominant T EM phenotype (CD45RA -/ CD27 -) was observed in the γδ T cell compartment, with a concomitant reduction in the proportion of naïve and T CM cells. This effect was most striking in the lamina propria, where in some donors almost 100% of γδ T cells displayed the T EM phenotype. Similar findings were observed for non-γδ T cells (data not shown). We also analysed the cytokine producing ability of γδTCR + cells from the small intestinal epithelium (γδiEL) stimulated in vitro [fig_ref] Figure 3: γδ T cells from the coeliac small intestine display a functional "effector... [/fig_ref]. Unstimulated γδiEL from CD patients demonstrated a distinct lack of background cytokine production when compared to control γδiEL (as seen for IFN-γ expression, [fig_ref] Figure 3: γδ T cells from the coeliac small intestine display a functional "effector... [/fig_ref] , and also observed for IL-10, IL-17A, IL-21 and IL-22; data not shown). Upon in vitro stimulation, γδiEL were capable of rapid and potent upregulation of IFN-γ, at levels similar to those seen for non-γδ T cells (latter data not shown). Taken together, the data provide evidence for the presence of activated and functional γδ T cells in the coeliac gut.
## Cd56 downregulation is not restricted to nk cells in the cd gut
We initially observed that NK cells (defined as CD3 -/CD56 + lymphocytes) were reduced in the small intestinal epithelium and lamina propria of CD patients , leftmost column, , in agreement with published data [bib_ref] Dynamics of non-conventional intraepithelial lymphocytes-NK, NKT, and gammadelta T-in celiac disease: relationship..., Calleja [/bib_ref]. However, further analyses revealed that this apparent reduction reflected a more general loss of CD56 expression, by T cells (including Vδ1 + cells) as well as non-T cells , and MAIT cells (data not shown). As well as an observed decrease in the percentage CD56 + cells , analysis of mean fluorescence intensity (MFI) further demonstrated an accompanying decrease in CD56 expression intensity, seen for T cell populations but not NK cells, in the coeliac gut . Similar reductions were noted in adult coeliac patient blood and gut (data not shown), when compared to controls.
# Discussion
In this study we show that the human duodenum hosts several innate lymphocyte populations, including γδ T cell subsets, iNKT cells and MAIT cells, and the relative abundance of these cells is dramatically and persistently altered in CD, for both paediatric and adult cohorts. Using multi-colour flow cytometry, we undertook a simultaneous comparison of innate lymphocyte subsets within the blood, gut epithelium and lamina propria compartments. Our findings provide further detail to support previous studies which showed similar abnormalities in innate lymphocyte populations in CD, specifically, observed decreases in γδ T cells, NK cells and iNKT cells in blood and gut -with the exception of γδ T cells, which are persistently increased in CD small intestinal epithelium [bib_ref] Numbers of T cell receptor (TCR) alpha beta+ but not of TcR..., Kutlu [/bib_ref] [bib_ref] Small intestinal CD8+TCRgammadelta+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients..., Bhagat [/bib_ref] [bib_ref] Dynamics of non-conventional intraepithelial lymphocytes-NK, NKT, and gammadelta T-in celiac disease: relationship..., Calleja [/bib_ref] [bib_ref] Circulating T lymphocyte subsets in coeliac disease (CoD) patients and healthy family..., Kerttula [/bib_ref]. We corroborate the observation that γδ T cell levels (specifically, Vδ1 cells) remain elevated in the coeliac gut even when a patient adopts a GFD and reverts to serological and histological normalcy. Extending these observations, we further show that all major subsets of γδ T cells (Vδ1, Vδ2 and Vδ3), and the newly described MAIT cell subset, are also significantly affected in CD.
We observed a reduction in MAIT cells in both adult and paediatric blood and gut, similar to observations made in the context of HIV [bib_ref] Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection, Cosgrove [/bib_ref] [bib_ref] Activation, exhaustion, and persistent decline of the antimicrobial MR1-restricted MAIT-cell population in..., Leeansyah [/bib_ref]. In HIV, increased permeability of the gut and associated microbial translocation are thought to drive MAIT cell influx and activation. It is hypothesised that the level of microbial exposure encountered in the HIV-infected gut induces cell death in MAIT cells however, which would account for the observed reduction in MAIT cells in both blood and gut, compared to uninfected controls. A similar scenario likely occurs in the coeliac gut, where gut barrier function is compromised and bacterial overgrowth is common. We also noted a decrease in iNKT cells in coeliac disease. In the healthy gut of children and adults, iNKT cells accounted for <1% of T cells, similar to the frequency noted in blood. It should be noted that in this study iNKT cells were defined solely by phenotypic markers (CD3 + Vα24/Jα18 + ). For a definitive iNKT cell characterisation however, α-GalCer/CD1d tetramers are the gold standard. However our observations agree with those of Grose and colleagues, who found that Va24 + T cells, Va24 + /Vb11 + T cells and α-GalCer/CD1d tetramer + iNKT cells were numerically deficient and functionally impaired in coeliac disease.
It is well documented that Vδ1 cells comprise a major cell population in the normal intestine [bib_ref] Intraepithelial T cells of the TcR gamma/delta+ CD8-and V delta 1/J delta..., Halstensen [/bib_ref]. However, we report here for the first time that Vδ3 cells also make up a significant proportion of γδ T cells within the small intestine, in healthy donors of all ages. Vδ3 cells accounted for the majority of γδ T cells in healthy adult intestine, in both the epithelium and lamina propria regions, and in the lamina propria of paediatric donors (Vδ1 cells were slightly more abundant in the paediatric epithelium). Thus, while Vδ1 and Vδ3 cells are relatively rare in healthy blood, they represent a major innate lymphocyte population in the healthy human gut. The function and antigen specificity of Vδ3 cells are poorly defined, but our group recently reported that Vδ3 cells respond to glycolipid antigens by killing CD1d + target cells and releasing cytokines that promote T H 1, T H 2 and T H 17 responses [bib_ref] Cutting edge: CD1d restriction and Th1/Th2/Th17 cytokine secretion by human Vδ3 T..., Mangan [/bib_ref]. Vδ1 cells have also been shown to recognise sulfatide presented by CD1d tetramers, with some clones demonstrating CD1d recognition in the absence of the sulfatide antigen [bib_ref] The majority of CD1d-sulfatide-specific T cells in human blood use a semiinvariant..., Bai [/bib_ref]. Recognition of CD1d by tissue-resident γδ T cell subsets is not surprising given the broad expression of this molecule in the intestine -on dendritic cells, macrophages, B cells and intestinal epithelial cells [bib_ref] Role of NKT cells in the digestive system. III. Role of NKT..., Zeissig [/bib_ref]. Vδ2 cells, on the other hand, are the most abundant type of γδ T cell in blood, but least abundant in the gut and do not appear to respond to CD1d (our unpublished observations). This finding highlights the need for specific identification of γδ T cell subsets in order to more accurately elucidate discrete functions of these different cell types.
Coeliac patients of all ages demonstrated a skewing from a predominant Vδ3 to a predominant Vδ1 phenotype throughout the gut. This was due to an increase in Vδ1 cells and concurrent reduction in Vδ3 cell frequency. It is interesting to note that this increase of Vδ1 + T cells in the CD gut, concurrent with a decrease in circulation, directly opposes findings from other inflammatory bowel disorders, where an increase in Vδ1 + T cells is noted in blood [bib_ref] Increased frequency of abnormal gamma delta T cells in blood of patients..., Söderström [/bib_ref] [bib_ref] Increase of circulating gamma/delta T lymphocytes in the peripheral blood of patients..., Giacomelli [/bib_ref] , but not within the inflamed gut [bib_ref] Gamma delta T cell receptor-positive cells of the human gastrointestinal mucosa: occurrence..., Trejdosiewicz [/bib_ref] [bib_ref] Intestinal gamma/delta receptor-bearing T lymphocytes in celiac disease and inflammatory bowel diseases..., Savilahti [/bib_ref]. Our analysis of CD103, a putative gut homing and activation marker [bib_ref] Tissue-infiltrating lymphocytes analysis reveals large modifications of the duodenal "immunological niche" in..., Cianci [/bib_ref] , revealed similar expression patterns on Vδ1 and non-γδ T cells in blood and gut compartments (data not shown). This similarity held for both coeliac and control donors, except for epithelial Vδ1 cells, which demonstrated elevated CD103 expression in coeliac donors compared to controls. This increase may be explained by TGF-β production by Vδ1 cells [bib_ref] Small intestinal CD8+TCRgammadelta+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients..., Bhagat [/bib_ref] , which is known to enhance CD103 expression [bib_ref] Tissue-infiltrating lymphocytes analysis reveals large modifications of the duodenal "immunological niche" in..., Cianci [/bib_ref]. Further analysis of γδ T cells within the coeliac small intestine revealed a predominant 'effector memory' (CD27 -/ CD45RA -) phenotype. Although initially this phenotype was used to describe Vδ2 cells in blood [bib_ref] Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes..., Dieli [/bib_ref] , the authors' definition of such cells, 'abounding at sites of inflammation' and displaying 'immediate effector functions' fit with our findings of an expanded population capable of rapid IFN-γ expression. A corresponding altered phenotype was not observed in blood. We observed a similar effector memory phenotype between γδ and non-γδ T cells (data not shown), in agreement with [bib_ref] Phenotypical characterization of peripheral blood T cells in patients with coeliac disease:..., Kerttula [/bib_ref] , who also reported an increase in effector memory (defined by CD45RO) Vδ1 cells in coeliac patients, . CD56 expression is profoundly downregulated in the coeliac gut, but not blood, both in percentage of cell expression, and expression intensity. CD56 expression was analysed on non-T cells, non-γδ T cells, and Vδ1 T cells in terms of frequency (A) and mean fluorescence intensity (B). Flow cytometry plots are also shown for representative control and coeliac donors (C). For simplicity, data is shown for a paediatric population only, but similar trends were noted in the adult cohort. *P <0.05, **P<0.01, ***P<0.001, error bars show standard error of the mean. doi: 10.1371/journal.pone.0076008.g004 with a similar trend seen for αβ T cells [bib_ref] Phenotypical characterization of peripheral blood T cells in patients with coeliac disease:..., Kerttula [/bib_ref]. Despite these observed similarities with inflammatory αβ T cells, evidence points toward an immunoregulatory role for Vδ1 cells [bib_ref] Small intestinal CD8+TCRgammadelta+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients..., Bhagat [/bib_ref] [bib_ref] A role for human skin-resident T cells in wound healing, Toulon [/bib_ref]. We propose a scenario where Vδ1 cells expand in the coeliac gut in response to signals resulting from epithelial cell damage, such as elevated MICA/B expression [bib_ref] Recognition of stressinduced MHC molecules by intestinal epithelial gammadelta T cells, Groh [/bib_ref] [bib_ref] Distinct pattern of human Vdelta1 gammadelta T cells recognizing MICA, Li [/bib_ref]. Activated Vδ1 cells then exert regulatory effects via TGF-β and NKG2A expression, contribute to T cell suppression [bib_ref] Small intestinal CD8+TCRgammadelta+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients..., Bhagat [/bib_ref] [bib_ref] Tumorinfiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms..., Peng [/bib_ref] [bib_ref] Human peripheral gammadelta T cells possess regulatory potential, Kühl [/bib_ref] , antitumour immunosurveillance [bib_ref] The presence of small intestinal intraepithelial gamma/delta T-lymphocytes is inversely correlated with..., Verbeek [/bib_ref] , and contribute to gut repair [bib_ref] A role for human skin-resident T cells in wound healing, Toulon [/bib_ref] [bib_ref] Epidermal T cells and wound healing, Havran [/bib_ref]. Vδ1 cells lack expression of markers typical of regulatory T cells (our unpublished observations [bib_ref] Tumorinfiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms..., Peng [/bib_ref] [bib_ref] Human peripheral gammadelta T cells possess regulatory potential, Kühl [/bib_ref] , suggesting these cells have a different mode of action to traditional regulatory T cells. Interestingly, [bib_ref] Tumorinfiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms..., Peng [/bib_ref] showed that Vδ1 cell suppressive action was abrogated upon TLR-8 ligation. Since TLR-8 is known to recognise viral as well as bacterial components [bib_ref] TLR8: the forgotten relative revindicated, Cervantes [/bib_ref] , this mechanism could potentially explain the tentative link between rotavirus and CD [bib_ref] Rotavirus infection frequency and risk of celiac disease autoimmunity in early childhood:..., Stene [/bib_ref] [bib_ref] In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor..., Zanoni [/bib_ref].
As well as changes in specific innate subsets, we also noted a profound decline in CD56 expression in the gut of coeliac donors, compared to healthy donors, which extended across all CD56-expressing lymphocytes, including CD3 + , CD3 -, all three γδ T cell subsets and MAIT cells (albeit as a preliminary finding for the latter, as only 5 coeliac donors were tested). Loss of CD56 expression was observed both in terms of positive cell frequency and expression intensity. Expression of CD56 by CD3 -lymphocytes is most frequently used to define NK cells, whereas expression of CD56 by CD3 + lymphocytes identifies a population of T cells capable of potent non-antigen-specific cytotoxicity and cytokine release [bib_ref] Activation-induced expression of CD56 by T cells is associated with a reprogramming..., Kelly-Rogers [/bib_ref]. Expression of CD56 by CD3 + and CD3 -lymphocytes has previously been described in the gut and shown to be associated with a cytotoxic, nonproliferative, T H 1 phenotype [bib_ref] CD56 marks an effector T cell subset in the human intestine, Cohavy [/bib_ref]. CD56 + cells are depleted in inflamed, but not uninvolved, IBD colonic tissue, and in the liver during chronic HCV infection [bib_ref] Decrease in hepatic CD56(+) T cells and V alpha 24(+) natural killer..., Deignan [/bib_ref]. This decrease has been attributed to concurrent expansion of inflammatory IFN-γproducing CD4 + αβ T cells. A similar scenario could potentially occur in CD, where expansion of gluten-reactive αβ T cells in the small intestine has been demonstrated [bib_ref] Investigation of the putative immunodominant T cell epitopes in coeliac disease, Ellis [/bib_ref]. The immunological significance of this CD56 downregulation remains to be established, but nevertheless highlights the need for improved phenotypic characterization of NK cells in tissue. Other NK cell markers such as CD16 or natural cytotoxicity receptors (e.g. NKp44 or NKp46) may be useful in this regard [bib_ref] Human natural killer cells, Caligiuri [/bib_ref].
CD is linked to an increased incidence of autoimmune disorders [bib_ref] Incidence of autoimmune diseases in celiac disease: protective effect of the gluten-free..., Cosnes [/bib_ref] [bib_ref] Duration of exposure to gluten and risk for autoimmune disorders in patients..., Ventura [/bib_ref]. One explanation proposed for this is a dysregulation in communication between innate and adaptive arms of the immune system [bib_ref] Systemic autoimmune disorders in celiac disease, Fasano [/bib_ref]. In this study we describe significant and persistent changes in innate lymphocyte profiles in CD patients compared to controls. While the function of these cells and their individual contributions to CD pathogenesis remain to be fully elucidated, the diminishment of Vδ2, Vδ3, iNKT, MAIT, NK and other CD56-expressing cells in the coeliac gut is likely to compromise immune defences within the damaged epithelial barrier. Due to persistent elevation within the histologically improved gut after gluten elimination, we propose that Vδ1 cells play roles in regulation and repair within the small intestine, and potentially pose a useful therapeutic target. Our findings further underline the differences in human γδ T cell subsets and highlight the need to discriminate between these subsets in future research.
[fig] Figure 1: Circulating innate lymphocytes are depleted in adults, but not children, with CD. Scatterplots showing percentages (left column) and absolute numbers (right column) of innate lymphocyte subsets in fresh whole blood, comparing coeliac donors to matched controls for (A) paediatric (n=38 for controls, n=21 for UCD), and (B) adult (n=78 controls, n=56 TCD, n=18 UCD) populations. All subsets are expressed as a percentage of the total T cell population. TCD = treated coeliac donors (on gluten free diet), UCD = untreated coeliac donors (diet contains gluten). Dotplots show flow cytometry data for representative control, treated and untreated coeliac donors (C), where Vα7.2 + /CD161 + populations describe MAIT cells. *P <0.05, **P<0.01, ***P<0.001, error bars show standard error of the mean. doi: 10.1371/journal.pone.0076008.g001 [/fig]
[fig] Figure 2: Innate lymphocyte subsets are altered in the intestinal epithelium and lamina propria of both paediatric and adult coeliac donors, compared with controls. Scatterplots showing percentages of small intestinal innate lymphocyte subsets in (A) epithelium, and (B) lamina propria for paediatric (n=34 controls, n=19 UCD) and adult coeliac (n=7 controls, n=8 TCD, n=6 UCD) donors and matched control subjects. All subsets are expressed as a percentage of the total T cell population. TCD = treated coeliac donors (on gluten free diet), UCD = untreated coeliac donors (diet contains gluten). Dotplots show flow cytometry data from gut epithelial cells (C), and lamina propria cells (D) for representative control and coeliac donors, where Vα7.2 + /CD161 + populations describe MAIT cells. *P <0.05, **P<0.01, ***P<0.001, error bars show standard error of the mean. doi: 10.1371/journal.pone.0076008.g002 [/fig]
[fig] Figure 3: γδ T cells from the coeliac small intestine display a functional "effector memory" phenotype. (A) Differential expression of CD45RA and CD27 by γδ T cells was used to analyse naïve (CD45RA+/CD27+), central memory (TCM; CD45RA-/ CD27+), effector memory (TEM; CD45RA-/CD27-) and terminally differentiated (TEMRA; CD45RA+/CD27-) subsets in the blood, small intestinal epithelium and lamina propria from paediatric patients. (B) Expression of IFN-γ by unstimulated and stimulated epithelial γδ T cells, control n=5, coeliac n=4. *P <0.05, **P<0.01, ***P<0.001, error bars show standard error of the mean. doi: 10.1371/journal.pone.0076008. [/fig]
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TENET 2.0: Identification of key transcriptional regulators and enhancers in lung adenocarcinoma
Lung cancer is the leading cause of cancer-related death and lung adenocarcinoma is its most common subtype. Although genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations driving lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We found over 32,000 enhancers that appear differentially activated between normal lung and lung adenocarcinoma. Among the identified transcriptional regulators inactivated in lung adenocarcinoma vs. normal lung, NKX2-1 was linked to a large number of silenced enhancers. Among the activated transcriptional regulators identified, CENPA, FOXM1, and MYBL2 were linked to numerous cancer-specific enhancers. High expression of CENPA, FOXM1, and MYBL2 is particularly observed in a subgroup of lung adenocarcinomas and is associated with poor patient survival. Notably, CENPA, FOXM1, and MYBL2 are also key regulators of cancer-specific enhancers in breast adenocarcinoma of the basal subtype, but they are associated with distinct sets of activated enhancers. We identified individual lung adenocarcinoma enhancers linked to CENPA, FOXM1, or MYBL2 that were associated with poor patient survival. Knockdown experiments of FOXM1 and MYBL2 suggest that these factors regulate genes involved in controlling cell cycle progression and cell division. For example, we found that expression of TK1, a potential target gene of a MYBL2-linked enhancer, is associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma epigenomes, highlighting novel potential targets for clinical intervention.Author summaryAlthough genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Here we investigated epigenetic alterations that might contribute to lung adenocarcinoma progression. We used a bioinformatics approach called TENET 2.0 to identify key regulators and enhancers altered in lung adenocarcinoma compared to normal lung. We identified NKX2-1 as a transcriptional regulator inactivated in lung adenocarcinoma, linked to a large number of enhancers silenced in cancer. Among the activated transcriptional regulators, CENPA, MYBL2, and FOXM1 were linked to numerous cancer-specific enhancers and high expression of these regulators was observed in a subgroup of lung adenocarcinomas showing poor patient survival. Investigating downstream effects of these key regulators, we also determined individual enhancers associated with survival and their potential target genes, including TK1. Our findings suggest that abnormal expression of key regulators drives epigenetic deregulation in lung adenocarcinoma, promoting future development of novel biomarkers and therapeutic targets.PLOS GENETICSKey transcriptional regulators and enhancers in lung adenocarcinoma PLOS Genetics | https://doi.org/10.
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# Introduction
Lung cancer is the second most commonly diagnosed form of cancer and the leading cause of cancer-related death in both men and women [bib_ref] Cancer statistics, Siegel [/bib_ref]. Lung adenocarcinoma (LUAD) arises in the alveolar epithelium of the lung and comprises almost 50% of all lung cancer cases in the United States. Major risk factors for LUAD include tobacco smoking, inherited genetic factors, diet, alcohol consumption, exposure to sources of ionizing radiation and environmental contaminants [bib_ref] Risk factors for lung cancer worldwide, Malhotra [/bib_ref] [bib_ref] Cigarette smoking and lung cancer-Relative risk estimates for the major histological types..., Pesch [/bib_ref]. These risk factors induce molecular and cellular changes in alveolar epithelial cells, leading these purported cells of origin to form LUAD. A number of somatic genetic alterations such as KRAS, EGFR, NF1, and BRAF mutations, gene fusions involving ALK, EML4, and ROS1, and copy number variations of the KRAS and EGFR genes have been identified and utilized in the development of targeted therapies for LUAD [bib_ref] Molecular pathology of lung cancer: Key to personalized medicine, Cheng [/bib_ref] [bib_ref] Non-small cell lung cancer: Current treatment and future advances, Zappa [/bib_ref]. However, approximately a quarter of LUAD cases do not possess any of these genetic alterations [bib_ref] Comprehensive molecular profiling of lung adenocarcinoma: The cancer genome atlas research network, Collisson [/bib_ref] , suggesting that other molecular changes likely contribute to lung cancer development.
Epigenomic features do not affect the sequence of DNA, but can affect the transcriptional output of genes in a cell-type specific manner by altering the activity of regulatory elements such as promoters (which are located proximal to the transcription start site of genes) and enhancers (which can be found at a great genomic distance (distal) from their target genes). Promoters and enhancers play critical roles in ensuring cell type specificity by controlling gene expression through the binding of transcription factors and recruitment of the transcriptional machinery [bib_ref] The role of general initiation factors in transcription by RNA polymerase II, Roeder [/bib_ref] [bib_ref] The importance of detailed epigenomic profiling of different cell types within organs, Stueve [/bib_ref]. Disruption of epigenetic marks and altered activity of regulatory elements may lead to the development of cancer [bib_ref] Epigenetics of lung cancer: a translational perspective, Molina-Pinelo [/bib_ref]. The epigenetic state at regulatory elements can be determined by measuring levels of histone 3 lysine 4 trimethylation (H3K4me3, a promoter mark) or histone 3 lysine 27 acetylation (H3K27ac, an enhancer mark), using chromatin immunoprecipitation (ChIP)-seq. Epigenetic states can also be assessed by using open chromatin assays such as DNase-seq, NOMe-seq (Nucleosome Occupancy and Methylome), or ATAC-seq (assay for transposase-accessible chromatin) [bib_ref] Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals..., Rhie [/bib_ref]. DNA methylation levels can be used to infer accessibility of open chromatin regions at regulatory elements since active promoters and enhancers tend to be unmethylated [bib_ref] Inferring regulatory element landscapes and transcription factor networks from cancer methylomes, Yao [/bib_ref] [bib_ref] Identification of activated enhancers and linked transcription factors in breast, prostate, and..., Rhie [/bib_ref]. Moreover, the binding of activated transcription factors affects DNA methylation states of targeted regulatory elements [bib_ref] Inferring regulatory element landscapes and transcription factor networks from cancer methylomes, Yao [/bib_ref] [bib_ref] Identification of activated enhancers and linked transcription factors in breast, prostate, and..., Rhie [/bib_ref] [bib_ref] Transcriptional competence and the active marking of tissue-specific enhancers by defined transcription..., Xu [/bib_ref] [bib_ref] DNA methylation of distal regulatory sites characterizes dysregulation of cancer genes, Aran [/bib_ref] [bib_ref] ELmer v.2: An r/bioconductor package to reconstruct gene regulatory networks from DNA..., Silva [/bib_ref].
We previously developed the Tracing Enhancer Networks using Epigenetic Traits (TENET) method which can identify differentially activated enhancers and their associated transcriptional regulators (TRs) using tumor vs. normal tissue samples. TENET incorporates information from ChIP-seq and open chromatin assays to determine the location of enhancers in normal and tumor tissues, and uses the DNA methylation levels of probes in the identified regions as an indicator of enhancer activity. Then, TENET uses gene expression data from the same samples to identify transcriptional regulators whose expression levels are highly correlated with the DNA methylation level of each enhancer. Using TENET, biomarkers and potential oncogenic drivers of breast cancer (e.g. GATA3, ESR1, FOXA1), prostate cancer (e.g. FOXA1, HOXC6, HOXB13), and kidney cancer (e.g. GLIS1, MAF, RUNX1) have been identified [bib_ref] Identification of activated enhancers and linked transcription factors in breast, prostate, and..., Rhie [/bib_ref]. These results illustrate the utility of the TENET method to identify key transcriptional regulators associated with tumorigenesis. However, computational time and power associated with identification of the key transcriptional regulators of the original TENET method was not optimal. Here, we have significantly improved the method, updating the databases, including new algorithms to identify epigenetic traits associated with mortality, and greatly decreasing the computational time needed. We then applied the improved version of the method (TENET 2.0) to numerous epigenome and transcriptome datasets from lung and lung cancer and identified key transcriptional regulators and enhancers associated with lung adenocarcinoma, providing novel potential targets for clinical intervention.
# Results
## Identification of differentially activated enhancers in normal lung versus lung adenocarcinoma
Each cell type has a distinct transcriptome, which is established by the levels and activities of transcriptional regulators that bind to regulatory elements and control the expression of numerous target genes. Among regulatory elements, the activity of enhancers is most closely linked to cell identity, as they are often bound by cell-type specific transcriptional regulators [bib_ref] The abundance and function of regulatory sequences beyond promoters, Bulger [/bib_ref]. We developed TENET 2.0 to identify key transcriptional regulators whose expression levels are associated with changes in DNA methylation levels at enhancers in normal vs. tumor tissue samples [fig_ref] Fig 1: A workflow of TENET 2 [/fig_ref]. TENET 2.0 now utilizes human reference genome hg38 and includes updated databases of human genes (GENCODE v22) [bib_ref] Improving GENCODE reference gene annotation using a high-stringency proteogenomics workflow, Wright [/bib_ref]. To comprehensively characterize and identify transcriptional regulators altered in tumors, we used the transcription factor database specified by Lambert et al.. We developed TENET 2.0 to have increased processing speed, compared to the original version, and have also included new algorithms to assess the relationship with patient survival, among others.
To study transcriptional enhancer networks in LUAD using TENET 2.0, we first identified lung-relevant enhancer regions. Alveolar epithelial cells (AECs) are the presumed cells of origin of LUAD [bib_ref] Non-small-cell lung cancers: A heterogeneous set of diseases, Chen [/bib_ref]. There are two types of alveolar epithelial cells: cuboidal type 2 cells (AT2), which are involved in surfactant production and serve as facultative progenitors post-injury, and large, delicate type 1 cells (AT1), which cover the majority of the alveolar surface and mediate gas exchange. While AT2 cells are the suspected cells of origin of lung adenocarcinoma, the possible role of AT1 cells has not been well investigated due to the difficulty in manipulating these fragile cells. Thus, we incorporated both populations of these cells into our study. We first purified human AT2 cells and then used an in vitro differentiation protocol (which mimics aspects of normal lung re-epithelialization) to derive AT1-like cells [bib_ref] Positional integration of lung adenocarcinoma susceptibility loci with primary human alveolar epithelial..., Yang [/bib_ref] [bib_ref] Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation, Marconett [/bib_ref]. We then generated H3K27ac ChIP-seq data from the AT2 cells (day 0), transitional cells (day 4), and differentiated AT1-like cells (day 6). We also used H3K27ac ChIP-seq data from normal lung tissue samples and LUAD cells downloaded from the Roadmap Epigenomics Project [bib_ref] Integrative analysis of 111 reference human epigenomes, Roadmap Epigenomics Consortium [/bib_ref] , the Encyclopedia of DNA Elements Project (ENCODE) [bib_ref] An integrated encyclopedia of DNA elements in the human genome, Dunham [/bib_ref] [bib_ref] The Encyclopedia of DNA elements (ENCODE): Data portal update, Davis [/bib_ref] , and the Data-Base of Transcriptional Start Sites (DBTSS) [bib_ref] DBTSS/DBKERO for integrated analysis of transcriptional regulation, Suzuki [/bib_ref]. Because tumorigenesis might activate enhancers that are not normally active in the lung, we also included H3K27ac ChIP-seq from 98 different cell types collected from REMC [bib_ref] Integrative analysis of 111 reference human epigenomes, Roadmap Epigenomics Consortium [/bib_ref] and ENCODE [bib_ref] An integrated encyclopedia of DNA elements in the human genome, Dunham [/bib_ref] [bib_ref] The Encyclopedia of DNA elements (ENCODE): Data portal update, Davis [/bib_ref]. We next delineated the open chromatin regions where the transcription factors bind within each enhancer, using ATAC-seq peaks generated in-house from AECs, ATAC-seq peaks from LUAD tissues and cell lines downloaded from other studies [bib_ref] Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug..., Guler [/bib_ref] [bib_ref] The open chromatin landscape of non-small cell lung carcinoma, Wang [/bib_ref] , DNaseI hypersensitive sites from LUAD cell lines, and a collected list of DNaseI hypersensitive sites from 125 different tissues and cell lines from ENCODE [bib_ref] An integrated encyclopedia of DNA elements in the human genome, Dunham [/bib_ref] [bib_ref] The Encyclopedia of DNA elements (ENCODE): Data portal update, Davis [/bib_ref]. A list of datasets we used for this study can be found in S1 identified enhancer and open chromatin regions can be found in S2 [fig_ref] Table: List of FOXM1 and MYBL2 motifs [/fig_ref] Finally, DNA methylation probes from the Illumina Infinium Human Methylation 450K (HM450) array that are contained within the open chromatin region of each enhancer were selected. As enhancers are bound by cell-type specific transcription factors and more cell-type and individual specific than promoters [bib_ref] Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals..., Rhie [/bib_ref] , we focused on enhancers for our analyses using only probes located >1.5 kb from transcription start sites. In all, we identified 76,765 "enhancer probes" that can be studied using lung tissue samples (S3A ; on average, one probe was found per open chromatin region in each enhancer.
Having collected the above information, we next assessed the differential activities of all of the enhancers in normal lung vs. LUAD tumor samples. For this, we collected DNA methylation data for the enhancer probes (n = 76,765) from 453 LUAD tissue samples and 21 histologically normal lung tissue samples adjacent to tumors from The Cancer Genome Atlas (TCGA) [bib_ref] Comprehensive molecular profiling of lung adenocarcinoma: The cancer genome atlas research network, Collisson [/bib_ref] (S4 . By comparing the DNA methylation level (as a reflection of enhancer activity) of each probe in the normal vs. tumor samples, we classified the enhancer probes into 4 groups: methylated ("constitutively inactive"), i.e. highly methylated in both normal and tumor samples; unmethylated ("constitutively active"), i.e. lowly methylated in both normal and tumor samples; hypermethylated ("normal-specific"; inactivated in LUAD), i.e. showing low methylation in normal samples but higher methylation in tumor samples; and hypomethylated ("cancer-specific"; activated in LUAD), i.e. showing high methylation in normal samples, but lower methylation in tumor samples. For example, the unmethylated probe cg05156800, located in an enhancer region on chr1p36.11 near the 3'UTR of EXTL1, marks an enhancer that is active in both normal lung and LUAD tumors [fig_ref] Fig 2: Identification of differentially-methylated enhancer probes [/fig_ref]. In contrast, the hypermethylated probe cg24149590 in an intergenic region on chr14q24.3 is located in an enhancer, active in normal lung but not in LUAD [fig_ref] Fig 2: Identification of differentially-methylated enhancer probes [/fig_ref]. Hypomethylated probe cg04683210, located in an intron of MACROD1 on chr11q13.1 marks an enhancer that is active LUAD but not in normal AECs [fig_ref] Fig 2: Identification of differentially-methylated enhancer probes [/fig_ref]. Using this classification scheme, we identified 4,344 unmethylated, 6,830 methylated, 9,056 hypermethylated, and 23,583 hypomethylated enhancer probes. An excess of identified hypomethylated probes suggests that enhancer activation is a common molecular alteration in LUAD [fig_ref] Fig 2: Identification of differentially-methylated enhancer probes [/fig_ref] and S3B .
## Identification of key transcriptional regulators dysregulated in lung adenocarcinoma
Having identified over 32,000 differentially activated enhancer probes between normal lung and LUAD, we next used matched gene expression data to test the association between the expression of each known human transcriptional regulator (n = 1,639) and the level of DNA methylation (as a measure of accessibility and thus activity) of each enhancer probe, using TENET 2.0. We identified 1) inactivated transcriptional regulators that showed a correlation of lower expression with increased DNA methylation of enhancer probes in a subset of LUAD samples (candidate tumor suppressors), and 2) activated transcriptional regulators that showed a correlation of higher expression with decreased DNA methylation of enhancer probes in a subset of LUAD samples (candidate oncogenes) (S1 Most of the known 1,639 human transcriptional regulators we interrogated were linked to relatively few cell-type specific enhancer probes [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref]. However, a subset of transcriptional regulators was found to be linked to many cell-type specific enhancer probes [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref].
We found that 31 inactivated transcriptional regulators were found to be linked to 10 or more hypermethylated enhancer probes (S5A . For example, NKX2-1 and HNF1B were linked to 123 and 50 hypermethylated enhancer probes, respectively [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref] , S5A . NKX2-1, the top transcriptional regulator inactivated in LUAD, linked to the largest number of enhancers silenced in LUAD, is known to play an important role in lung development and maintenance of AEC cell identity [bib_ref] Lung development: orchestrating the generation and regeneration of a complex organ, Herriges [/bib_ref]. NKX2-1 also acts as an activator of HOP (Hsp70/Hsp90 Organizing Protein), a potential tumor suppressor gene in lung cancer, inhibiting epithelial to mesenchymal transition [bib_ref] Homeobox gene HOP has a potential tumor suppressive activity in human lung..., Chen [/bib_ref]. HNF1B is previously reported to act as a tumor suppressor in several tumors, including renal cancer, ovarian cancer, and prostate cancer [bib_ref] Germline hepatocyte nuclear factor 1α and 1β mutations in renal cell carcinomas, Rebouissou [/bib_ref] [bib_ref] Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines, Terasawa [/bib_ref] [bib_ref] A review on hepatocyte nuclear factor-1beta and tumor, Yu [/bib_ref]. Our finding that lower expression of HNF1B is linked to many inactivated enhancers in LUAD suggests that it may also act as a tumor suppressor in lung cancer.
On the other hand, we found 101 activated transcriptional regulators linked to 50 or more hypomethylated probes (S5C . The top activated transcriptional regulators were CENPA, FOXM1, TCF24, and MYBL2, which were linked to 875, 845, 843, and 840 cancerspecific enhancer probes, respectively [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref] , S5C . These transcriptional regulators likely have the largest influence on the transcriptomes of lung adenocarcinoma tumors by changing the activities of many enhancers. Therefore, we further investigated the identified activated transcriptional regulators associated with many cancer-specific activated enhancers. To determine whether these transcriptional regulators control the activity of distinct enhancers or cooperate with each other to regulate the same set of enhancers, we generated an interaction map displaying the association of the 3,682 cancer-specific enhancer probes linked to at least one of the 101 transcriptional regulators [fig_ref] Fig 4: Interaction of key transcriptional regulators activated in LUAD [/fig_ref]. Interestingly, CENPA, FOXM1, and . The overlap between these transcriptional regulators is much higher than with other key transcriptional regulators identified (e.g. TCF24, SOX2). Examination of the expression levels of each of the 101 top-ranked transcriptional regulators showed that the expression levels of CENPA, FOXM1, and MYBL2 were highly correlated with each other (r 2 >0.7) across all profiled LUAD samples [fig_ref] Fig 4: Interaction of key transcriptional regulators activated in LUAD [/fig_ref] red brackets, S5F . We validated these results using an additional transcriptomic dataset obtained from other lung tumor tissue samples from ORIEN (Oncology Research Information Exchange Network) (www.oriencancer.org) (S2 . These results suggest that these 3 transcriptional regulators may work together to activate a common set of cancer-specific enhancers.
## Identification of transcriptional regulators whose expression is associated with poor patient survival
To further investigate the role of key transcriptional regulators activated in LUAD, we more closely examined gene expression levels in normal vs. tumor tissues. Of the top 12 transcriptional regulators, CENPA, FOXM1, and MYBL2 were among the most highly expressed and displayed the largest differences in expression between tumor and normal tissues; each was >8 times more highly expressed in LUAD as compared to normal lung [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref]. Next, we examined the association of transcriptional regulator expression with patient survival, and we found that high expression levels of CENPA, FOXM1, and MYBL2 were the most significantly associated with poor patient survival in the TCGA LUAD cohort [fig_ref] Fig 4: Interaction of key transcriptional regulators activated in LUAD [/fig_ref]. We validated these results for CENPA, MYBL2, and FOXM1 using an additional survival dataset obtained from other LUAD samples [bib_ref] Online survival analysis software to assess the prognostic value of biomarkers using..., Gyorffy [/bib_ref] (S5 . Expression of CENPA, FOXM1, and MYBL2 did not appear to be very strongly associated with age, sex, or cancer stage. However, we found that history of tobacco exposure was correlated with the gene expression of each of the three transcriptional regulators (S6A [fig_ref] Fig 6: LUAD and BRCA subgroups with activated CENPA, FOXM1 and MYBL2-linked enhancers [/fig_ref]. Additionally, we found that high total mutation burden was similarly associated with increased expression of these genes in the LUAD tumor samples (S6B
## Cenpa, foxm1, and mybl2 are activated in a subgroup of lung adenocarcinoma and breast adenocarcinoma
Tumor samples with higher expression of CENPA, FOXM1, and MYBL2 appear to harbor relatively more cancer-specific enhancers, suggesting that tumors highly expressing CENPA, FOXM1, and MYBL2 may have distinct enhancer profiles (S7A To investigate this, we generated a DNA methylation heatmap of the enhancer probes linked to these three transcriptional regulators [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref]. We observed a subgroup consisting of LUAD samples that are broadly hypomethylated across these enhancers, and that possess relatively high expression of these three transcriptional regulators together [fig_ref] Fig 6: LUAD and BRCA subgroups with activated CENPA, FOXM1 and MYBL2-linked enhancers [/fig_ref]. These samples did not appear to be associated with age, sex, cancer stage, purity, or cancer stage, but they were slightly associated with smoking history in the TCGA dataset, especially current smoking, as well as total mutational burden (S8A- [fig_ref] Fig 8: Identification of genes regulated by FOXM1 and MYLB2 [/fig_ref]. We saw no apparent association between genetic alterations to KRAS, EGFR, NF1, or BRAF and activation of specifically activation of KRAS signaling increases expression of FOXM1 [bib_ref] Foxm1 Mediates Cross Talk between Kras/Mitogen-Activated Protein Kinase and Canonical Wnt Pathways..., Wang [/bib_ref] , and that MYBL2 can be regulated by EGFR [bib_ref] Co-regulation of B-Myb expression by E2F1 and EGF receptor, Hanada [/bib_ref]. We therefore examined the total number of cancer-specific enhancer links in samples with and without KRAS or EGFR genetic alterations and in the highest quartile and remaining quartiles of expression of FOXM1 and MYBL2, respectively. Samples with the highest quartile of FOXM1 and MYBL2 expression possessed a significantly greater number of cancer-specific enhancer links, however, KRAS or EGFR genetic alteration status was not associated with a significant difference in the number of these links regardless of the FOXM1 and MYBL2 expression level (S7B We also observed that a subgroup of LUAD In previous analyses, we found that FOXM1 and MYBL2 were activated in breast adenocarcinoma (BRCA) [bib_ref] Identification of activated enhancers and linked transcription factors in breast, prostate, and..., Rhie [/bib_ref]. Having now identified these as key regulators in LUAD, we sought to determine if different enhancers are linked to FOXM1 and MYBL2 in the two cancer types. We reanalyzed the BRCA data using TENET 2.0 (S3C and S3D , and found that CENPA, FOXM1, and MYBL2 were among the top transcriptional regulators in BRCA when ranked by number of linked probes (S9A However, only a subset of TENET-identified CENPA, FOXM1, and MYBL2-linked enhancer probes were shared between both datasets (S9B This suggests that although some cancer-specific enhancers are common to LUAD and BRCA (S9C [fig_ref] Fig 7: Examples of CENPA/FOXM1/MYBL2-linked enhancer probes associated with survival rate [/fig_ref] , the enhancers regulated by CENPA, FOXM1, and MYBL2 are largely different between tumor types. To further characterize the BRCA enhancers linked to CENPA, MYBL2, and FOXM1, we generated heatmaps of DNA methylation for enhancer probes linked to any of the three transcriptional regulators in BRCA. Interestingly, we found that BRCA tumor samples that belong to the basal subtype have higher expression of these three transcriptional regulators as well as a larger number of hypomethylated CENPA, FOXM1, and MYBL2-linked enhancers than other BRCA subtypes (i.e. luminal A, luminal B, Her2, normal-like) [fig_ref] Fig 6: LUAD and BRCA subgroups with activated CENPA, FOXM1 and MYBL2-linked enhancers [/fig_ref].
## Identification of cenpa/foxm1/mybl2-linked enhancers associated with poor patient survival and their potential target genes
We next wondered whether high expression of CENPA, FOXM1, and MYBL2 and the presence of more activated enhancers was clinically relevant. Therefore, we examined the subgroup of LUAD samples, which had high expression of the three transcriptional regulators as well as many enhancer links (over 290 cancer-specific CENPA, FOXM1, or MYBL2 enhancer links), called "highly linked" samples for correlations to overall patient survival (S8A These "highly linked" samples showed significantly poorer survival outcomes than lowly linked samples (S8D To further investigate whether any particular cancer-specific enhancers were linked to patient outcome, we performed survival analyses using cancer-specific enhancer probes linked to CENPA, FOXM1, or MYBL2. We found 101 enhancer probes for which lower levels of DNA methylation were associated with poor patient survival (Log-rank p<0.05) (S3B . Examples of enhancer probes linked to patient survial included cg03535253, located on chr14q32.12 in the 3'UTR of the BTBD7 gene, cg06956006, located on chr17q21.2 in an intron of the ACLY gene, and cg04016113 in an intron of the SFXN5 gene on chr2p13.2. Each is located in the vicinity of an active enhancer region in LUAD cells not present in normal AEC, and patients with low levels of methylation of each of these probes (indicating the activation of the enhancer regions) showed significantly poorer survival outcomes [fig_ref] Fig 7: Examples of CENPA/FOXM1/MYBL2-linked enhancer probes associated with survival rate [/fig_ref]. We next aimed to identify genes and signaling pathways potentially regulated by CENPA, FOXM1, and MYBL2. To this end, we first identified genes within 1 Mb of each of the enhancer probes since most enhancer-promoter interactions occur within a topologically associating domain (TAD) that is less than 1Mb in size [bib_ref] A high-resolution 3D epigenomic map reveals insights into the creation of the..., Rhie [/bib_ref]. From these, we selected the genes that were significantly upregulated in tumor relative to normal as potential targets of these enhancers. For example, we found that the SPR gene was a potential target of the enhancer probe cg0416113 [fig_ref] Fig 7: Examples of CENPA/FOXM1/MYBL2-linked enhancer probes associated with survival rate [/fig_ref]. SPR (sepiapterin reductase) is located~177kb upstream of the enhancer probe. A recent study showed that SPR depletion inhibited liver cancer cell proliferation and promoted cancer cell apoptosis in vivo [bib_ref] Sepiapterin reductase promotes hepatocellular carcinoma progression via FoxO3a/Bim signaling in a nonenzymatic..., Wu [/bib_ref] , suggesting its role as an oncogene. Gene ontology (GO) analyses revealed that target genes potentially regulated by CENPA, FOXM1, and MYBL2 are involved in cell cycle, cellular response to DNA damage stimulus, chromosome organization, and DNA repair (S8A and S8B .
To identify genes and signaling pathways regulated by FOXM1 and MYBL2, known transcription factors, we performed knockdown experiments for FOXM1 and MYBL2 in A549 . Genes shown represent potential target genes within 1 Mb of CENPA/FOXM1/MYBL2-linked enhancers whose activation is significantly associated with poor patient survival. Expression of the gene TK1 is highlighted by the red arrow. (E) Diagram of A549 H3K27ac mark overlapping the MYBL2-linked probe cg09580922 and its potential target gene TK1 (see S10 . cells, a LUAD cell line. More than a thousand genes were differentially expressed upon knockdown of either FOXM1 or MYBL2 or both [fig_ref] Fig 8: Identification of genes regulated by FOXM1 and MYLB2 [/fig_ref] , S9A-S9C . GO analyses of the genes downregulated after knocking down FOXM1 or MYBL2 or both indicated that these genes are involved in cell cycle and cell division, supporting the gene predictions made from degerulated genes near the activated enhancers (S8C-S8E . We determined which of the significantly downregulated genes from the siRNA knockdowns were located within 1 Mb of the enhancer probes we had previously linked to these transcriptional regulators [fig_ref] Fig 8: Identification of genes regulated by FOXM1 and MYLB2 [/fig_ref]. These genes likely represent the direct target genes of the enhancers.
Of particular interest is the gene TK1, which showed a~40% reduction in expression after MYBL2 knock down (adjusted p = 2.506x10 -7 ) (S9B and S9C . TK1, encoding a protein that plays an important role in thymidine metabolism, is located~188 kb from the MYBL2linked enhancer probe cg09580922 on chr17q25.3 [fig_ref] Fig 8: Identification of genes regulated by FOXM1 and MYLB2 [/fig_ref]. Low methylation of cg09580922 is strongly associated with poor patient survival [fig_ref] Fig 8: Identification of genes regulated by FOXM1 and MYLB2 [/fig_ref] , as is high expression of TK1 [fig_ref] Fig 8: Identification of genes regulated by FOXM1 and MYLB2 [/fig_ref]. The promoter of TK1 and cg09580922 are both located in the same TAD according to Hi-C maps from both A549 as well as GM12878, another cell line for which a high resolution Hi-C dataset is available (S10 . This suggests that a cancer-specific enhancer potentially regulated by MYBL2 may increase the expression of TK1. A complete list of enhancers and their potential target genes confirmed by knockdown experiments and located in the same TAD can be found in S9D Table.
# Discussion
We have developed TENET 2.0, a method to characterize enhancer networks controlled by transcriptional regulators that are potential tumor suppressors or oncogenic drivers. Using H3K27ac ChIP-seq and open chromatin datasets, we identified enhancers active in lung cells. Then, using DNA methylation levels at the identified enhancers in hundreds of normal vs. LUAD tissue samples [bib_ref] Comprehensive molecular profiling of lung adenocarcinoma: The cancer genome atlas research network, Collisson [/bib_ref] , we identified over 32,000 differentially activated enhancers. By integrating DNA methylation and gene expression data, we identified key transcriptional regulators (e.g. NKX2-1, CENPA, FOXM1, and MYBL2) that are linked to many cell-type specific enhancers. We further found that high expression of CENPA, FOXM1, and MYBL2 is associated with poor survival in patients with LUAD and with broad enhancer activation in a distinct group of LUAD tumors. We found a subgroup of BRCA tumor samples which also showed activation of BRCA enhancers linked to these three transcriptional regulators, and basal-subtype tumors were particularly enriched in that subgroup. We then identified LUAD-specific enhancers that are linked to the three transcriptional regulators and whose increased activities are correlated with poor survival. For example, the enhancer marked by probe cg09580922 appears to regulate the TK1 gene, whose high expression is associated with poor patient survival.
TENET 2.0, which now has updated databases, including new algorithms to identify epigenetic traits associated with mortality with greatly decreased computational time, allowed us to identify dysregulated transcriptional regulators and enhancers in LUAD. Key inactivated transcriptional regulators, which are potential tumor suppressors, include NKX2-1 [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref]. Low expression of NKX2-1 was observed in a subgroup of LUAD samples (S8A was linked to over a hundred inactivated enhancers. NKX2-1, also known as thyroid transcription factor 1 (TTF1), regulates transcription of genes specific for the thyroid and lung. NKX2-1 is reported to be involved in lung development, and it inhibits epithelial to mesenchymal transition, supporting its role as a tumor suppressor [bib_ref] Suppression of lung adenocarcinoma progression by Nkx2-1, Winslow [/bib_ref] [bib_ref] MicroRNA-33a mediates the regulation of high mobility group AT-hook 2 gene (HMGA2)..., Rice [/bib_ref]. Besides NKX2-1, we identified that HNF1B, a previously reported tumor suppressor found in other cancer types [bib_ref] Germline hepatocyte nuclear factor 1α and 1β mutations in renal cell carcinomas, Rebouissou [/bib_ref] [bib_ref] Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines, Terasawa [/bib_ref] [bib_ref] A review on hepatocyte nuclear factor-1beta and tumor, Yu [/bib_ref] , STAT6, and SP100 were inactivated and linked to many silenced enhancers in a subgroup of LUAD [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref].
Of the transcriptional regulators activated in LUAD, CENPA, FOXM1, and MYBL2 were linked to the activation of hundreds of cancer-specific enhancers. These transcriptional regulators are therefore potential cancer driver oncogenes. CENPA, which has a histone-binding domain, directs the assembly of active kinetochores together with centromere-specific-DNAbinding factors. A recent study in cervical and colorectal cancer cells reported that CEPNA can also bind to DNaseI hypersensitive sites [bib_ref] CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human..., Athwal [/bib_ref]. MYBL2 (a.k.a. B-MYB), a member of the MYB family, regulates cell cycle genes by binding to regulatory elements [bib_ref] The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene..., Sadasivam [/bib_ref]. FOXM1, a member of the Forkhead family of pioneer transcription factors [bib_ref] FOXM1 binds directly to non-consensus sequences in the human genome, Sanders [/bib_ref] , is involved in the proper development of several different organ systems, including the lungs [bib_ref] Foxm1 Mediates Cross Talk between Kras/Mitogen-Activated Protein Kinase and Canonical Wnt Pathways..., Wang [/bib_ref]. It has been demonstrated to bind to enhancers in breast cancer cells [bib_ref] Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in..., Sanders [/bib_ref]. Here we showed that CENPA, FOXM1, and MYBL2 are upregulated together, potentially leading to the activation of many cancer-specific enhancers in a subgroup of LUAD. The subgroup of LUAD with both high expression of CENPA, FOXM1, and MYBL2 and broad enhancer activation had worse patient survival outcomes. This subgroup also appears to have a higher proportion of smokers, which may be related to the observed epigenomic changes, and higher tumor mutational burden [bib_ref] Association of Tumor Mutational Burden With DNA Repair Mutations and Response to..., Chae [/bib_ref]. It has been previously suggested that FOXM1 may act as a regulator for genes involved in DNA damage response and repair [bib_ref] FOXM1: An emerging master regulator of DNA damage response and genotoxic agent..., Zona [/bib_ref]. Besides these 3 transcriptional regulators, we identified other key transcriptional regulators, such as TCF24, SOX2, and ZNF695, each linked to over 500 enhancers activated in LUAD [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref] , providing many further avenues of investigation.
When we compared our LUAD data with that of a similar analysis of BRCA, CENPA, FOXM1, and MYBL2 were also found to be activated, particularly in basal-subtype tumors, supporting the idea that these factors work together in certain cancer subtypes. Previously, we showed that estrogen receptor and FOXA1, which are known to be activated in estrogen receptor-positive breast cancer subtypes (e.g. luminal A, luminal B), are not expressed in the basal subtype, but FOX and MYB motifs are enriched at enhancers in basal-like breast cancer cells [bib_ref] Nucleosome positioning and histone modifications define relationships between regulatory elements and nearby..., Rhie [/bib_ref]. FOXM1 and MYBL2 motifs were enriched at CENPA, FOXM1, and MYBL2-linked enhancers we found in lung cancer cells (91.8% for a FOXM1 motif, 60.3% for an MYBL2 motif) (S10 . Interestingly, CENPA, FOXM1, and MYBL2 appear to target different enhancers in BRCA and LUAD, potentially working with different co-factors [bib_ref] Cancer cell line specific co-factors modulate the FOXM1 cistrome, Wang [/bib_ref]. In spite of this difference, GO analysis of potential target genes for these enhancers revealed that both sets regulate similar cellular processes, including cell cycle control and DNA repair (S8A and S8B . Further studies to elucidate the function of these transcriptional regulators in tumor subgroups are needed to better understand their role in epigenetic deregulation of cancer cells.
Previous studies had implicated FOXM1 and MYBL2 in lung cancer [bib_ref] FOXM1 promotes lung adenocarcinoma invasion and metastasis by upregulating SNAIL, Wei [/bib_ref] [bib_ref] Expression and functional characterization of FOXM1 in non-small cell lung cancer, Zhang [/bib_ref] [bib_ref] Overexpression of MYBL2 promotes proliferation and migration of non-small-cell lung cancer via..., Xiong [/bib_ref] , but our analysis documents their profound effects on gene deregulation, potentially affecting hundreds of enhancers. As acquisition of cancer-specific enhancers can drive tumorigenesis, identifying key activated enhancers in cancer is highly relevant. Here, we identified 101 LUAD-specific enhancers linked to CENPA, FOXM1, and MYBL2 that show correlations with worse survival [fig_ref] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD [/fig_ref]. For example, we found that the enhancer probe cg04161113, whose activation (low DNA methylation) is associated with poor survival, is potentially regulating the SPR gene, which was recently reported as an oncogene in liver cancer [bib_ref] Sepiapterin reductase promotes hepatocellular carcinoma progression via FoxO3a/Bim signaling in a nonenzymatic..., Wu [/bib_ref].
Using knockdown experiments, we further identified potential target genes of these enhancers, which included genes involved in cell division and cell cycle control. These potential target genes included not only known oncogenes such as MYC, FBXL16, PHF5A, and KIF14 [bib_ref] KIF14 is a candidate oncogene in the 1q minimal region of genomic..., Corson [/bib_ref] [bib_ref] MYC amplification as a prognostic marker of early-stage lung adenocarcinoma identified by..., Iwakawa [/bib_ref] [bib_ref] PHD finger protein 5A promoted lung adenocarcinoma progression via alternative splicing, Mao [/bib_ref] [bib_ref] The F-box protein FBXL16 up-regulates the stability of C-MYC oncoprotein by antagonizing..., Morel [/bib_ref] but also genes (e.g. BRI3BP, RAB11FIP5) which are not yet reported to be involved in lung carcinogenesis (S9 . Of the downregulated genes after siRNA treatment, TK1 was the most significantly associated with survival rates (log-rank p = 1.194x10 -4 ). High expression of TK1 and low methylation of the nearby MYBL2-linked enhancer probe cg09580922 were associated with poor patient survival [fig_ref] Fig 8: Identification of genes regulated by FOXM1 and MYLB2 [/fig_ref] , and both appear to be located in the same TAD (S10 [fig_ref] Fig 1: A workflow of TENET 2 [/fig_ref] has been investigated as a diagnostic and prognostic biomarker for several types of cancer, including LUAD [bib_ref] Loss of thymidine kinase 1 inhibits lung cancer growth and metastatic attributes..., Malvi [/bib_ref]. Loss of TK1 has been shown to inhibit the growth and metastatic capabilities of LUAD in vitro as well as in mice through a reduction in expression of GDF15 [bib_ref] Loss of thymidine kinase 1 inhibits lung cancer growth and metastatic attributes..., Malvi [/bib_ref].
We have used TENET 2.0 to integrate epigenomic and transcriptomic profiles from hundreds of samples and have identified key transcriptional regulators and enhancers altered in LUAD. The lists of these enhancers, transcriptional regulators, and their potential target genes will be a useful resource for researchers aiming to better understand the molecular mechanisms driving carcinogenesis in different LUAD subgroups. Moreover, our findings may lead to new biomarkers as well as therapies that might target distinct LUAD subgroups associated with poor survival; small molecule inhibitors for MYB family members [bib_ref] Targeting the transcription factor Myb by small-molecule inhibitors, Uttarkar [/bib_ref] as well as FOXM1 have been developed but have not yet been tested in lung cancer [bib_ref] Thiostrepton selectively targets breast cancer cells through inhibition of forkhead box M1..., Kwok [/bib_ref] [bib_ref] Suppression of the FOXM1 transcriptional programme via novel small molecule inhibition, Gormally [/bib_ref]. Importantly, TENET 2.0 can be used to investigate molecular mechanisms underlying any cancer type for which gene expression and epigenetic data are available (http://github.com/suhnrhie/TENET_2.0).
# Materials and methods
# Ethics statement
Remnant human transplant lungs were obtained in compliance with USC Institutional Review Board protocol, approved for the use of human source material in research (HS-07-00660). As donors were deceased and de-identified, no patient consent was obtained or necessary.
## Cell culture
Human lung adenocarcinoma A549 cells (Cat # CRL-185, ATCC, Gaithersburg, MD) were grown at 37˚C with 5% CO2 in RPMI 1640 (Cat #10-040-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Cat # FBS-500, X&Y Cell Culture, MI, USA) and 100 units/ml of penicillin/streptomycin (formulated by Norris Comprehensive Cancer Center Media Core, CA, USA). Human AT2 cells were isolated from remnant transplant lung from deceased de-identified non-smoking donors in compliance with USC Institutional Review Board protocol for the use of human source material in research (HS-07-00660). As donors were deceased and de-identified, no patient consent was obtained or necessary. Lungs were processed as previously described [bib_ref] Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation, Marconett [/bib_ref]. The three donors were 25, 62, and 67-year-old males who died of non-lung related causes. AT2 cells were isolated from the samples, plated in 50% DMEM/F12 (Cat #D64421, Sigma, MO, USA), 50% DMEM high glucose (Cat #21063, GIBCO, MA, USA), supplemented with 10% FBS, penicillin/streptomycin, 50 ug/ml gentamycin (Cat #G1272, Sigma, MO, USA) and 2.5ug/ml amphotericin (Cat #A2411, Sigma, MO, USA), to allow differentiation to AT1-like cells, and isolated at three different time points (D0, D4, D6) as previously noted [bib_ref] Positional integration of lung adenocarcinoma susceptibility loci with primary human alveolar epithelial..., Yang [/bib_ref] [bib_ref] Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation, Marconett [/bib_ref] (S1 . Discovery, UK). Cells were transfected, cultured for 24 hours, and transfected again with the same concentration of siRNA, then incubated for an additional 24 hours before RNA was extracted using the Aurum Total RNA Mini Kit (Cat # 7326820, Bio-Rad, CA, USA). cDNA was synthesized using an iScript cDNA Synthesis Kit (Cat # 1708891, Bio-Rad, CA, USA) and expression levels of FOXM1 and MYBL2 were checked with qRT-PCR using SYBR Green Supermix (Cat # 1708886, Bio-Rad, CA, USA) with the listed primers (S11 . RNA-seq was performed using 150 bp paired-end sequencing using an Illumina HiSeq 4000 (GENEWIZ, South Plainfield, NJ, USA) for the single gene knockdown experiments, and using 100 bp paired-end sequencing using an Illumina NovaSeq 6000 (MedGenome, Foster City, CA, USA) for the double knockdown. RNA-seq reads were aligned to the human reference genome hg38 using the Genomic Data Commons Bioinformatics mRNA analysis pipeline. Read counts were generated for GEN-CODE v22 genes [bib_ref] Improving GENCODE reference gene annotation using a high-stringency proteogenomics workflow, Wright [/bib_ref] using the htseq-count function [bib_ref] HTSeq-A Python framework to work with high-throughput sequencing data, Anders [/bib_ref]. Differentially expressed genes were called using DESeq2 [bib_ref] Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Love [/bib_ref] with the lfcShrink function [bib_ref] Heavy-Tailed prior distributions for sequence count data: Removing the noise and preserving..., Zhu [/bib_ref]. Gene ontology analyses were performed using PANTHER [bib_ref] PANTHER version 14: More genomes, a new PANTHER GO-slim and improvements in..., Mi [/bib_ref] (S8C-S8E (see S1 Methods for more details).
## Sirna knockdown and rna-seq
## Chip-seq
ChIP-seq was performed on the D0, D4, and D6 AECs isolated from the 25-year-old and 62-year-old male subjects using H3K27ac antibody (Cat # 39133, Active Motif, CA, USA), as previously described [bib_ref] Positional integration of lung adenocarcinoma susceptibility loci with primary human alveolar epithelial..., Yang [/bib_ref] [bib_ref] Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation, Marconett [/bib_ref]. The ChIP-seq library from the 25-year-old individual was sequenced using 50 bp single-end reads on an Illumina HiSeq 2000 (S1 . Two technical replicates of A549 H3K27ac ChIP-seq data and two replicates of H3K27ac ChIP-seq data from lung tissue from a 53-year-old female donor generated by the ENCODE Consortium [bib_ref] An integrated encyclopedia of DNA elements in the human genome, Dunham [/bib_ref] [bib_ref] The Encyclopedia of DNA elements (ENCODE): Data portal update, Davis [/bib_ref] were used. H3K27ac ChIP-seq data from two additional lung tissue samples from 30-year-old female and 3-year-old male donors generated by the ROADMAP Consortium [bib_ref] Human DNA methylomes at base resolution show widespread epigenomic differences, Lister [/bib_ref] [bib_ref] The NIH roadmap epigenomics mapping consortium, Bernstein [/bib_ref] were also included (S1 . Finally, H3K27ac ChIP-seq data collected from 12 lung cancer lines from the DBTSS were downloaded and processed as well [bib_ref] DBTSS/DBKERO for integrated analysis of transcriptional regulation, Suzuki [/bib_ref]. ChIP-seq reads were aligned to the human reference genome hg38 and reproducible peaks were called, following the ENCODE ChIP-seq pipeline [bib_ref] ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia, Landt [/bib_ref] (see S1 Methods).
## Atac-seq
Intact nuclei from D0 AT2 cells were isolated from the 67-year-old male subject utilizing the protocol from Buenrostro et al. [bib_ref] ATAC-seq: A method for assaying chromatin accessibility genome-wide, Buenrostro [/bib_ref]. Briefly, intact nuclei were isolated and incubated with Tn5 transposase (Cat # FC-121-1030, Illumina, CA, USA). The transposed DNA was extracted and was amplified with PCR using NEBNext High-Fidelity PCR Master Mix (Cat # M0541S, New England Biolabs, MA, USA) and the resulting library was purified using a bead clean with AMPure XP Magnetic Beads (Cat # A63880, Beckman Coulter, CA, USA) and quality control was performed using a BioAnalyzer High-Sensitivity DNA Analysis kit (Cat # 5067-4626, Agilent, CA, USA). Data was sequenced as 75 bp paired-end reads on an Illumina HiSeq 2000. ATAC-seq data were processed using the ENCODE ATAC-seq pipeline (https://www. encodeproject.org/atac-seq/) (see S1 Methods). In addition, ATAC-seq peaks from 34 LUAD tissue samples were downloaded and lifted over to the hg38 reference genome using the Lift-Over tool available in the UCSC genome browser (https://genome.ucsc.edu/cgi-bin/ hgLiftOver) for TENET 2.0 analyses [bib_ref] The open chromatin landscape of non-small cell lung carcinoma, Wang [/bib_ref]. ATAC-seq peaks from an additional 22 LUAD tissue samples were addedalong with peaks from the PC-9 LUAD cell line [bib_ref] Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug..., Guler [/bib_ref] (S1 .
## Dnase-seq
Peaks of DNaseI hypersensitive sites in PC-9 and A549 cells processed by the ENCODE consortium were acquired [bib_ref] An integrated encyclopedia of DNA elements in the human genome, Dunham [/bib_ref] [bib_ref] The Encyclopedia of DNA elements (ENCODE): Data portal update, Davis [/bib_ref]. Those from A549 cells aligned to the hg19 human reference genome were lifted over to the hg38 reference genome using the LiftOver tool available in the UCSC genome browser (https://genome.ucsc.edu/cgi-bin/hgLiftOver) (S1 .
## Tenet program update and settings
Here we improved the original TENET program [bib_ref] Identification of activated enhancers and linked transcription factors in breast, prostate, and..., Rhie [/bib_ref] and developed TENET 2.0. TENET 2.0 uses a new reference genome (hg38) and gene annotation dataset (GENCODE v22) which covers >60,000 transcripts [bib_ref] Improving GENCODE reference gene annotation using a high-stringency proteogenomics workflow, Wright [/bib_ref]. It also includes a new dataset of 1,639 validated human transcription factors, the processing speed is increased, and useful functions were added to identify enhancers, genes, and tumor subgroups associated with survival. For enhancer analysis, we utilized H3K27ac ChIP-seq, ATAC-seq and DNase I hypersensivite site datasets. RNA-seq data along with DNA methylation data were downloaded for BRCA and LUAD samples from the TCGA [bib_ref] Comprehensive molecular profiling of lung adenocarcinoma: The cancer genome atlas research network, Collisson [/bib_ref] [bib_ref] Comprehensive molecular portraits of human breast tumours, Koboldt [/bib_ref] using the TCGAbiolinks package [bib_ref] TCGAbiolinks: An R/Bioconductor package for integrative analysis of TCGA data, Colaprico [/bib_ref] (see S1 Methods for more details on enhancer analysis, TCGA datasets, and TENET 2.0 program). TENET 2.0 program is available at http://github.com/suhnrhie/TENET_2.0.
## Heatmaps
For [fig_ref] Fig 4: Interaction of key transcriptional regulators activated in LUAD [/fig_ref] unsupervised clustering was performed and for [fig_ref] Fig 2: Identification of differentially-methylated enhancer probes [/fig_ref] pairwise correlation coefficients were calculated between each of the top LUAD transcriptional regulators identified and an unsupervised clustering was performed. [fig_ref] Fig 6: LUAD and BRCA subgroups with activated CENPA, FOXM1 and MYBL2-linked enhancers [/fig_ref] were generated and unsupervised clustering was performed. DNA methylation levels (β) ranging from 0 (unmeth) to 1 (meth) were plotted. Continuous variables, including gene expression, patient age, and tumor purity, were scaled using the function (X-X min )/(X max -X min ) with values equal to zero set to the minimum, non-zero value. Tumor purity values, including leukocytes unmethylation for purity, and overall derived consensus purity, were obtained from the Tumor purity dataset available from TCGAbiolinks package [bib_ref] TCGAbiolinks: An R/Bioconductor package for integrative analysis of TCGA data, Colaprico [/bib_ref] (see S1 Methods).
## Expression/correlation analyses
Expression values of key oncogenic transcriptional regulators from the adjacent normal and LUAD tumor samples were plotted, and Student's t-tests were performed to compare differential expression between normal and tumor groups. An one-way ANOVA analysis was performed to assess overall differences in transcriptional regulator expression between the smoking groups (67 never smokers, 278 former smokers, and 106 current smokers) and a Tukey Honest Significant Differences test was performed to assess significant differences between individual groups. Linear regression models were fit to predict expression of CENPA, FOXM1 and MYBL2 with respect to variables recorded for sample clinical information in the TCGA, including sample type, sex, age, smoking history, total pack years smoked, and race for samples which contained complete information for these variables. Independent RNA-seq data from 728 lung tumor tissues generated as part of the ORIEN were used to validate our correlation analyses. The correlation analyses were performed using the normalized RSEM values calculated following the ORIEN Total Cancer Care protocol (http://www.oriencancer.org/ ) accessed in May of 2020 [bib_ref] Patient Enrichment for Precision-Based Cancer Clinical Trials: Using Prospective Cohort Surveillance as..., Dalton [/bib_ref] [fig_ref] Fig 2: Identification of differentially-methylated enhancer probes [/fig_ref]
## Survival analyses
Survival analyses were performed comparing prognosis of patients with the highest and lowest quartiles of CENPA, FOXM1 and MYBL2 expression, linked-enhancer probe DNA methylation levels. Patient survival from samples within the "highly linked" group to those without any links to CENPA, FOXM1, and MYBL2 were also compared. Survival plots from Kaplan-Meier Plotter were performed on their website (https://kmplot.com/analysis/) [bib_ref] Online survival analysis software to assess the prognostic value of biomarkers using..., Gyorffy [/bib_ref] (see S1 Methods).
## Genetic alteration and mutation count analysis
Genetic alteration data for LUAD samples in the TCGA PanCancer Atlas was downloaded from the cBioPortal [bib_ref] Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal, Gao [/bib_ref] by selecting a query for mutations and putative copy-number alterations from GISTIC (https://software.broadinstitute.org/cancer/cga/gistic) for KRAS, EGFR, NF1, and BRAF. 445 of the 453 LUAD tumor samples in the TENET dataset contained information for these four alterations. Samples which were listed as having a "putative driver" mutation, amplification, deletion, or a fusion of each of the genes in this dataset were recorded as being positive for an alteration to that gene. Total mutation count data containing information for 447 of the 453 LUAD tumor samples was also downloaded from the cBioPortal repository [bib_ref] Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal, Gao [/bib_ref].
## Identification of potential target genes for cenpa/foxm1/mybl2-linked probes in luad and brca
Student's t-tests were performed for all genes in the LUAD and BRCA datasets, comparing expression in the tumor vs. normal samples. Genes that were significantly differentially expressed (fdr-corrected p<0.05) and upregulated specifically in the tumor samples were selected for further gene ontology (GO) analyses (S8A and S8B (see S1 Methods).
# Motif analysis
Minmeme motif files for FOXM1 or MYBL2, based on ChIP-seq experiments (3 from GSM12878 cells, MCF-7 cells, and SK-N-SH cells for FOXM1 and 1 from HepG2 cells for MYBL2), were downloaded from Factorbook (http://factorboook.org) in August of 2019, Additional minmeme motif files for FOXM1 and MYBL2 were downloaded from the HOCO-MOCO v11 database [bib_ref] HOCO-MOCO: Towards a complete collection of transcription factor binding models for human..., Kulakovskiy [/bib_ref]. Motif files we used are listed in S10 [fig_ref] Table: List of FOXM1 and MYBL2 motifs [/fig_ref] Using these motif files and FIMO program [bib_ref] FIMO: Scanning for occurrences of a given motif, Grant [/bib_ref] , we scanned DNA sequences within 1,117 bp, equivalent to half the average enhancer size as calculated from the lung enhancer regions (S2A , of FOXM1, MYBL2, or CENPA-linked enhancers (n = 1,338).
# Hi-c analysis
Using "ENCODE3-iced" data from A549 cells [bib_ref] An integrated encyclopedia of DNA elements in the human genome, Dunham [/bib_ref] and "Rao_2014-raw" data from GM12878 cells [bib_ref] A 3D map of the human genome at kilobase resolution reveals principles..., Rao [/bib_ref] , Hi-C heatmaps (25kb resolution, hg38) in S10 Fig were created from the 3D genome browser (http://promoter.bx.psu.edu/hi-c/view.php). Both datasets were processed and normalized using the pipeline, described in Wang et al. [bib_ref] The 3D Genome Browser: A web-based browser for visualizing 3D genome organization..., Wang [/bib_ref]. TAD information from A549 and GM12878 cells was downloaded from ENCODE and Rao et al. [bib_ref] A 3D map of the human genome at kilobase resolution reveals principles..., Rao [/bib_ref] , respectively (S1 .
## Supporting information
[fig] Fig 1: A workflow of TENET 2.0. First, DNA methylation probes marking enhancer regions of interest are identified by overlapping them with both H3K27ac ChIPseq datasets and open chromatin regions. Next, enhancer probes are classified based on their DNA methylation level in the tumor vs. normal samples and linked to the expression of genes to identify key transcriptional regulators (TRs). Using genetic alteration, Hi-C topologically associating domain (TAD), and clinical information, identified key TRs and TR-enhancer-gene networks are characterized. Additional gene expression and clinical data are used to validate findings of key TRs. Lung-related (REMC) [/fig]
[fig] Fig 2: Identification of differentially-methylated enhancer probes (A) Integrative Genomics Viewer (IGV) screenshots show 10 kb of the genomic context centered on example probes, with UCSC gene annotations (GENCODE v22) in the vicinity, the name and location of the probe, and the H3K27ac signal from AEC (normal) as well as A549 cells (LUAD cell line). The unmethylated probe shows an active enhancer region in both the AEC and A549 cells. The hypermethylated probe shows an active enhancer region found in only the AEC, indicating an enhancer that is inactive in tumors, while the hypomethylated probe displays marks in only A549 cells, indicating an enhancer that is activated in tumors. (B) Categorization of the identified enhancer probes by activity. https://doi.org/10.1371/journal.pgen.1009023.g002 [/fig]
[fig] Fig 3: Identification of key dysregulated transcriptional regulators in LUAD (A)The left histogram shows the number of inactivated (hypermethylated) enhancer probes per inactivated transcriptional regulator (TR), and the right shows the number of activated (hypomethylated) enhancer probes per activated TR. Most TRs were linked to relatively few enhancer probes. However, 31 inactivated TRs in LUAD were linked to 10 or more hypermethylated enhancer probes, and 101 activated TRs in LUAD were linked to 50 or more hypomethylated enhancer probes. (B) Number of MYBL2 showed considerable overlap in their sets of linked probes; over 75% of each of their linked probes was also linked to a probe in the set of at least one of the other two transcriptional regulators(Fig 4A-red box, S5E [/fig]
[fig] Fig 4: Interaction of key transcriptional regulators activated in LUAD (A) Interaction map of the top 101 transcriptional regulators and the 3,682 total unique hypomethylated probes linked to those genes. CENPA, FOXM1, and MYBL2 show strong overlap in linked probes (red box). (B) Heatmap of pairwise expression correlation values between each of the top 101 transcriptional regulators. FOXM1, CENPA, and MYBL2 show a high degree of correlation with each other (r 2 >0.7), but TCF24 (one of the top 4 most highly linked TRs; Fig 3B) does not (r 2 <0.1). https://doi.org/10.1371/journal.pgen.1009023.g004 Fig 5. CENPA, FOXM1 and MYBL2 are highly expressed in tumors and associated with poor patient survival. (A) Boxplots of expression of CENPA, FOXM1 and MYBL2 in 453 TCGA LUAD and 21 adjacent normal samples. All three genes were significantly upregulated in LUAD. (B) Kaplan-Meier survival plots comparing differences in survival between samples with the highest and lowest quartiles of CENPA, FOXM1 and MYBL2 expression. Survival was compared using TCGA LUAD samples. https://doi.org/10.1371/journal.pgen.1009023.g005 samples, representing those in the top 10% by number of links to CENPA, FOXM1, and MYBL2 showed poorer survival outcomes than samples which did not possess a link (S8D Fig). [/fig]
[fig] Fig 6: LUAD and BRCA subgroups with activated CENPA, FOXM1 and MYBL2-linked enhancers. (A) DNA methylation heatmap showing CENPA, FOXM1, and MYBL2 expression-linked LUAD-specific enhancer probes for normal and LUAD tissue samples. Clusters represent the largest two divisions in LUAD tumor samples as determined by unsupervised clustering. LUAD tumor samples in cluster b display generally higher expression of the 3 transcriptional regulators and broad hypomethylation of CENPA/FOXM1/MYBL2-linked probes. (B) DNA methylation heatmap showing CENPA, FOXM1, and MYBL2-linked breast cancer-specific enhancer probes for normal and BRCA tissue samples. BRCA PAM50 (Prediction Analysis of Microarray 50) subtypes are indicated in the middle bar. Of note is the cluster of samples on the right, comprised predominantly of BRCA tumor samples of the basal subtype, with relatively high expression of the three transcriptional regulators and broad hypomethylation of CENPA/FOXM1/MYBL2-linked probes, similar to what is seen in the subgroup of LUAD samples. https://doi.org/10.1371/journal.pgen.1009023.g006 [/fig]
[fig] Fig 7: Examples of CENPA/FOXM1/MYBL2-linked enhancer probes associated with survival rate. (A) Shown are three examples of lung cancer-specific enhancers linked to CENPA, FOXM1, or MYBL2 in LUAD. IGV screenshots show 10 kb of the genomic context centered on example probes, with GENCODE v22-annotated UCSC genes in the vicinity, the name and location of the probe, and the H3K27ac signal from normal AEC as well as lung tumor A549 cells. These hypomethylated probes show H3K27ac marks in A549 cells, indicating enhancers active in LUAD but not normal lung tissue. (B) Kaplan-Meier survival plots comparing differences in survival between samples with the highest and lowest quartiles of methylation of the enhancer probe. https://doi.org/10.1371/journal.pgen.1009023.g007 [/fig]
[fig] Fig 8: Identification of genes regulated by FOXM1 and MYLB2. Volcano plots showing gene expression changes after knocking down (A) FOXM1 or (B) MYBL2 or (C) Double (both FOXM1 and MYBL2). The knocked down genes (FOXM1 or MYBL2) are highlighted by a green or purple box, respectively. (D) Heatmap displaying fold change expression of significantly downregulated genes in the vicinity of cancer-specific enhancers associated with poor patient survival after FOXM1 (light blue) or MYBL2 (orange) or double (green) knockdown; log2(fold change) were plotted from dark blue to dark red (see [/fig]
[fig] S1: Fig. TENET 2.0 pictoral workflow. (A) DNA methylation levels of enhancer probes are used to assess differential activity of transcriptional regulator-linked enhancers. Enhancer probes are identified using H3K27ac ChIP-seq peaks overlapping with regions of open chromatin. Probes intersecting both of these regions are subsetted to those >1.5kb from GEN-CODE v22-annotated transcription start sites to avoid promoter regions. (B) TENET classifies enhancer probes based on their differential activity as measured by methylation level in normal vs. tumor samples. Methylated and unmethylated probes represent enhancers that are uniformly inactive and active, respectively. Hypermethylated probes represent enhancers that are inactive in cancer samples. These probes possess a low level of mean methylation in normal samples, but higher levels of methylation in a subset of tumor samples. Conversely, hypomethylated probes represent enhancers that are active in cancer samples, showing a decreased level of methylation in tumor vs. normal lung samples. (C) Analyses are focused on transcriptional regulators that are overexpressed in LUAD, resulting in increased activity of their regulated enhancer regions as represented by decreased DNA methylation. The expression of each transcriptional regulator and DNA methylation of each enhancer probe are assessed to find "linked" pairs with increased expression of the transcriptional regulator and decreased methylation of the probe in a subset of the tumor samples, relative to normal samples. (D) Transcriptional regulators with the most linked enhancers are of interest for study because they are more likely to have large-scale effects on genome-wide expression patterns. TENET 2.0 also includes new functions to identify tumor subgroups based on differences in the activation of enhancers linked to the top transcriptional regulators using heatmaps (E) and association with patient survival (F) as well as potential target genes of enhancers using topologically associating domain (TAD) information (G).(TIF) S2 Fig. Correlation analyses of key transcriptional regulators activated in lung cancer using ORIEN datasets. (A) Using ORIEN gene expression datasets from lung tumor tissue samples (n = 728), TR gene expression correlation analyses were performed. Barplots show the top 5 most correlated transcriptional regulators for CENPA (top), FOXM1 (middle), and MYBL2 (bottom). (B) TR gene expression scatterplots are shown for FOXM1 vs. MYBL2 (top), FOXM1 vs. CENPA (middle), and CENPA vs. MYBL2 (bottom). (TIF) S3 Fig. Expression of top 12 transcriptional regulators activated in LUAD. Boxplots of expression of remaining 9 of top 12 oncogenic transcriptional regulators in 453 TCGA LUAD tumor and 21 adjacent normal samples (CENPA, FOXM1, or MYBL2 are shown in Fig 5A). All genes were upregulated in LUAD tumors, but none as strongly as CENPA, FOXM1, or MYBL2. (TIF) S4 Fig. Survival analysis of top 12 transcriptional regulators activated in LUAD. Kaplan-Meier survival plots comparing differences in survival between samples with the highest and lowest quartiles of expression of the remaining 9 of top 12 cancer-specific transcriptional regulators by number of linked enhancers (CENPA, FOXM1, and MYBL2 are shown in Fig 5B). Survival was compared using TCGA LUAD samples. (TIF) S5 Fig. Replication of association of expression of highly-linked oncogenic transcriptional regulators with patient survival in LUAD using Kaplan-Meier Plotter. Kaplan-Meier survival plots comparing differences in survival between samples with the highest and lowest quartiles of expression of the top 12 oncogenic transcriptional regulators in LUAD cases using Kaplan-Meier Plotter (https://kmplot.com/analysis/) [36]. Again, expression of CENPA, FOXM1, and MYBL2 was the most strongly associated with patient survival amongst these transcriptional regulators. (TIF) S6 Fig. Smoking history is associated with CENPA, FOXM1, and MYBL2 expression in TCGA samples. (A) Boxplots of CENPA, FOXM1, and MYBL2 expression in 453 TCGA LUAD tumor samples stratified by smoking history. Tumor samples from current smokers had significantly higher expression of all three transcriptional regulator genes than those from former smokers and individuals who had never smoked (significant Tukey HSD p-values displayed; ��� p<0.005). (B) Boxplots show CENPA, FOXM1, and MYBL2 expression in all 453 TCGA LUAD tumor samples stratified by median total mutational count. Samples with higher mutational burden had higher expression of these transcriptional regulators. (TIF) S7 Fig. Association of total active enhancer links with three expression of three activated transcriptional regulators and common LUAD mutations. (A) Boxplots show the total number of links to activated enhancers on a per sample basis in LUAD samples with the highest quartile and lowest quartile of CENPA, FOXM1, and MYBL2 and expression. (B) Boxplots display differences in the total number of links to activated enhancers on a per sample basis in LUAD samples stratified by the presence and absence of a KRAS (right) or EGFR alteration (left), and with and without the highest quartile of expression of FOXM1 (right) or MYBL2 (left). There is a significant difference in the number of links between samples in the highest quartile of expression vs. the other three quartiles for both transcriptional regulators regardless of their alteration status, but no significant difference in the number of links between samples with and without either alteration but with the same expression level (Significant Tukey HSD p-values displayed; � = p<0.05, �� = p<0.01, ��� = p<0.005). (TIF) S8 Fig. Association of links to CENPA, FOXM1, MYBL2-linked enhancers with clinical data and subgroup analysis. (A) Heatmap of DNA methylation β-values for CENPA/FOXM1/ MYBL2-linked lung cancer-specific enhancers (n = 1,338) for normal and LUAD tissue samples. From top to bottom, samples are plotted with the age, sex, and cancer stage of the patients, smoking history status, expression of additional identified TRs TCF24, SOX2, and NKX2-1, leukocyte and overall tumor purity, presence of KRAS, EGFR, NF1 and BRAF alterations, log2-transformed mutational count, and sample link status. (B) Chi-square test results comparing smoking history in the more active cluster b, to the less active cluster a from Fig 6A. There is a much greater proportion of current smokers in cluster b than in cluster a. (C) ttest results comparing mean total mutation count of samples in cluster b to cluster a. Samples in cluster b have a significantly higher mean tumor burden than samples in cluster a. (D) Univariate Kaplan-Meier survival plot comparing difference in survival between the very highlylinked samples (marked in red in the link status bar), and samples that do not possess any links to CENPA/FOXM1/MYBL2-linked probes (marked in blue in the link status bar). (TIF) S9 Fig. Key transcriptional regulators identified in LUAD vs. BRCA and comparison of CENPA/FOXM1/MYBL2-linked probes in each dataset. (A) Barplot of the top 12 transcriptional regulators by number of links to activated enhancers identified using TENET 2.0 in LUAD (left) and BRCA (right). (B) Venn diagrams display overlap of probes linked to CENPA, FOXM1, and MYBL2 and (C) all hypomethylated probes in the LUAD vs. BRCA analyses. There is a considerably higher percentage of overlap between all hypomethylated probes than for probes linked only to CENPA, FOXM1, or MYBL2. (TIF) 0 Fig. Hi-C diagrams from A549 and GM12878 cells showing the cg09580922 and TK1 genomic region. Hi-C diagrams of A549 cells (top) and GM12878 cells (bottom) show the genomic context of the TK1/cg09580922 locus (middle) from chr17:77125000-79625000. In both cell lines, TAD boundaries (lower middle) show that both TK1 and cg09580922 are located in the same TAD. (TIF) Table. Datasets used in this study. (XLSX) S2 Table. List of enhancer and open chromatin regions identified in lung for this study. A) List of enhancer regions. B) List of open chromatin regions. (XLSX) S3 Table. List of HM450 enhancer probes. A) List of HM450 probes by enhancer type in LUAD. B) List of CENPA/FOXM1/MYBL2-linked enhancer probes in LUAD. C) List of HM450 probes by enhancer type in BRCA. D) List of hypomethylated enhancer probes in BRCA. (XLSX) S4 Table. List of TCGA IDs of LUAD and BRCA samples included in this study. (XLSX) S5 Table. Lists of top transcriptional regulators identified by TENET 2.0. A) List of top inactivated transcriptional regulator genes in LUAD. B) List of top inactivated transcriptional regulator genes in BRCA. C) List of top activated transcriptional regulator genes in LUAD. D) List of top activated transcriptional regulator genes in BRCA. E) Order of transcriptional regulator genes and enhancer probes as rows/columns for Fig 4A. F) Order of transcriptional regulator genes for Fig 4B. (XLSX) S6 Table. Regression analysis of CENPA/FOXM1/MYBL2 expression in LUAD. (XLSX) S7 Table. Lists of CENPA/FOXM1/MYBL2-linked enhancer probes in LUAD or BRCA. (XLSX) S8 Table. Gene Ontology categories enriched in potential target genes of CENPA, FOXM1, and MYBL2. A) GO analysis of genes linked to CENPA/FOXM1/MYBL2 linked enhancer probes in LUAD. B) GO analysis of genes linked to CENPA/FOXM1/MYBL2 linked enhancer probes in BRCA. C) GO analysis of genes significantly downregulated after siFOXM1 treatment in A549 LUAD cells. D) GO analysis of genes significantly downregulated after siMYBL2 treatment in A549 LUAD cells. E) GO analysis of genes significantly downregulated after double siFOXM1 and siMYBL2 treatment in A549 LUAD cells. (XLSX) S9 Table. List of differentially expressed genes identified using siRNA knockdown experiments. A) List of differentially expressed genes by siFOXM1 in A549 LUAD cells. B) List of differentially expressed genes by siMYBL2 in A549 LUAD cells. C) List of differentially expressed genes by double siFOXM1 and siMYBL2 in A549 LUAD cells. D) Potential genes regulated by CENPA/FOXM1/MYBL2-linked enhancers confirmed by siRNA experiment in A549 LUAD cell line. (XLSX) 0 [/fig]
[table] Table: List of FOXM1 and MYBL2 motifs. (XLSX) S11 Table. List of primer sequences used for RTqPCR. (XLSX) [/table]
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A Temperature-Insensitive Cladding-Etched Fiber Bragg Grating Using a Liquid Mixture with a Negative Thermo-Optic Coefficient
To compensate for the temperature dependency of a standard FBG, a cladding-etched FBG immersed with a liquid mixture having a negative thermo-optic coefficient is presented, and its characteristics are investigated. The Bragg wavelength of the cladding-etched FBG is shifted counter to the direction of the Bragg wavelength shift of a conventional FBG according to the mixing ratio of glycerin to water; thus, the temperature-dependent Bragg wavelength shift was almost compensated by using a liquid mixture of water (50%) and glycerin (50%) having the negative thermo-optic coefficient of −5 × 10 −4 °C −1 .
# Introduction
Fiber Bragg gratings (FBGs) are receiving much attention for fiber sensor applications due to their small size, absolute measurement capability, immunity to electromagnetic interference, wavelength multiplexing, and distributed sensing possibilities. Since they are readily made by controlling the period, length, amplitude, apodization, and chirp of a fiber grating, FBGs have been extensively studied as optical fiber sensors for measuring temperature [bib_ref] Characteristics of a fiber Bragg grating temperature sensor using the thermal strain..., Lee [/bib_ref] , strain [bib_ref] Design and application of a fiber Bragg grating strain sensor with enhanced..., Ren [/bib_ref] , pressure [bib_ref] Temperature-compensated high pressure FBG sensor with a bulk-modulus and self-demodulation method, Yang [/bib_ref] , acceleration [bib_ref] Flexural beam-based fiber Bragg grating accelerometers, Todd [/bib_ref] , OPEN ACCESS torsion [bib_ref] Torsion measurement using fiber Bragg grating sensors, Tian [/bib_ref] , flow [bib_ref] DP flow sensor using optical fiber Bragg grating, Lim [/bib_ref] , etc. FBG sensors offer high sensitivity, real-time processing, and long-term stability, as well as other important advantages.
However, due to the thermo-optic coefficient of silica and its thermal expansion coefficient, FBG sensors have a temperature-dependent Bragg wavelength shift of 13.7 pm/°C [bib_ref] Fiber Bragg gratings, Othonos [/bib_ref] ; therefore, the ability to eliminate the thermal effect remains a challenge, and considerable research has been carried out to compensate the temperature dependence by using two gratings [bib_ref] Bend measurement using Bragg gratings in multicore fibre, Gander [/bib_ref] and interrogation at two separate wavelengths [bib_ref] Simulations displacement and temperature measurement with cantilever-based fiber Bragg grating sensor, Dong [/bib_ref] , and bending effects [bib_ref] Temperature compensation of long-period fiber grating for refractive-index sensing with bending effect, Ng [/bib_ref]. These designs require additional components or the sensors must be positioned in a suitable geometrical structure to compensate for the temperature effect. Coating the cladding of an FBG with temperature-sensitive materials has also been found to have a significant effect on thermal sensitivity [bib_ref] Demonstration of etched cladding fiber Bragg grating-based sensors with hydrogel coating, Liu [/bib_ref]. In [bib_ref] Demonstration of etched cladding fiber Bragg grating-based sensors with hydrogel coating, Liu [/bib_ref] , the large thermal expansion of the coating-polymer induces an axial strain due to thermal stresses and changes the refractive index of the fiber core and fiber length, thereby improving the thermal sensitivity of the FBG. It is well known that the temperature dependence of the refractive index of an optical fiber core makes the Bragg wavelength shift to a longer wavelength with increasing temperature.
Recently, an etched FBG has been investigated for the measurement of refractive indexes using the thermo-optic coefficient of an external liquid [bib_ref] Determination of thermo-optic coefficient in liquids with fiber Bragg grating refractometer, Kamikawachi [/bib_ref] [bib_ref] Etched fiber Bragg gratings sensors for water-ethanol mixtures: A comparative study, Coradin [/bib_ref]. It is widely accepted that the etched cladding of an FBG undergoes a strong mode-coupling with surrounding materials, leading to a strong change of the effective refractive index of an FBG. Here, if a coating material with a negative thermo-optic coefficient is used on a cladding-etched FBG, the effective refractive index of the cladding-etched FBG can be lower, and as a result, the temperature dependence of the FBG can be significantly diminished.
In this paper, a new temperature-compensation method is presented and experimentally demonstrated in which a liquid mixture with a negative thermo-optic coefficient is used as an external coating material of a cladding-etched FBG. [fig_ref] Figure 1: Structure of the cladding-etched FBG [/fig_ref] shows the structure of the fabricated cladding-etched FBG etched almost to the fiber core to get the 0.3-μm-radius remained cladding (d), 61.2-μm-thickness removed cladding (t), and grating pitch of 535 nm (Λ).
## Experiments
An FBG is a type of distributed Bragg reflector having a periodic variation in the refractive index of an optical fiber core, which is fabricated by exposing a photosensitized fiber core to ultraviolet light. The reflected wavelength, the Bragg wavelength, is defined as: [bib_ref] Design and application of a fiber Bragg grating strain sensor with enhanced..., Ren [/bib_ref].
[formula] B e f f n λ = Λ (1) [/formula]
Generally, the grating period (Λ) and effective refractive index (n eff ) of a single-mode fiber core have a thermal response to the temperature applied to the fiber core. In the case of silica fibers, the thermal response is dominated by the refractive index change rather than the thermal expansion of the fiber core, accounting for more than 95% of the Bragg wavelength shift [bib_ref] Fiber grating sensors, Kersey [/bib_ref]. As a result of the change of refractive index, the Bragg wavelength shifts to the longer wavelength with a temperature sensitivity of 0.01 nm/°C. In addition, the Bragg wavelength of the cladding-etched FBG also depends on the refractive index of the external medium because the fiber mode profile and its effective refractive index are affected by evanescent wave coupling. [fig_ref] Figure 2: Effective refractive index and Bragg wavelength of cladding-etched single-mode FBG in relation... [/fig_ref] shows the effective refractive index of the cladding-etched single-mode FBG in relation to the refractive index of a liquid as an external medium (n ex ) for several remaining-cladding thicknesses (d). The eigenvalues were calculated by using the doubly clad theory [bib_ref] Propagation in doubly clad single-mode fibers, Monerie [/bib_ref] for the cladding-etched single-mode fiber that consisted of a fiber core, an etched cladding, and a liquid as an outer cladding. A standard single-mode optical fiber (SMF 28) with a cladding diameter of 125 μm, core diameter of 8.2 μm, and 0.36% relative refractive index difference was considered in the calculation. The refractive indices of the core and cladding were 1.449 and 1.444, respectively.
As shown in [fig_ref] Figure 2: Effective refractive index and Bragg wavelength of cladding-etched single-mode FBG in relation... [/fig_ref] , the effective refractive index of the fiber core decreases as the refractive index of the liquid as an external medium decreases. As the remaining-cladding thickness becomes smaller, the effective refractive index of the fiber core decreases more for a fixed refractive index of the liquid. When d = 0.3 μm and the refractive index of the liquid is 1.44, point A has the Bragg wavelength of 1,549.8 nm, which shifts to a shorter wavelength of 1,547.8 nm convertible to a Bragg wavelength shift of −2 nm by adjusting the refractive index of the liquid to 1.385 (point B). A properly designed liquid with a negative thermo-optic coefficient, the same magnitude with a positive thermo-optic coefficient of the fiber core, can counteract the temperature-dependent shifts of the Bragg wavelength of the silica fiber. Thus, this method directly compensates the thermal fluctuation of a Bragg wavelength of a FBG. [fig_ref] Figure 3: Experimental setup for cladding-etching process [/fig_ref] shows the experimental setup for the cladding-etching process in which the spectrum is monitored during and after the etching of an FBG connected to the 3 dB coupler. A broadband optical source (Agilent 83437A) and an optical spectrum analyzer (OSA, Ando, AQ6315A) were used to measure the reflected optical power through the 3 dB coupler. The FBG fiber was immersed and chemically etched in an aqueous solution of hydrofluoric acid (HF 40%) at 60 °C, and the Bragg wavelength shift relative to the initial Bragg wavelength was monitored in real time during the chemical etching process. With the approximate etching rate of 1.1 μm/min, the fiber cladding was etched almost to the fiber core for evanescent wave coupling with an external medium. As shown in [fig_ref] Figure 4: Measured Bragg wavelength before and after the cladding-etching process [/fig_ref] , during the cladding etching process, the effective refractive index of the fiber core decreases due to the evanescent wave coupling with the HF solution; thus, the Bragg wavelength of 1,550 nm shifts to the shorter wavelength of 1,547.4 nm, from which the remaining-cladding thickness can be derived, as seen in [fig_ref] Figure 2: Effective refractive index and Bragg wavelength of cladding-etched single-mode FBG in relation... [/fig_ref]. The remaining-cladding thickness was estimated to 0.3 μm. [fig_ref] Figure 5: Spectral responses as a function of temperature of [/fig_ref] shows the reflection spectra of the cladding-etched FBG with the estimated remaining-cladding thickness of 0.3 μm as a function of temperature when two kinds of liquid mixtures were used: water (7%) and glycerin (93%), and water (50%) and glycerin (50%). These are compared with the reflection spectra of a conventional non-cladding-etched FBG. Since the refractive indices of the liquid mixtures are dependent on the water and glycerin mixing ratio, the refractive indices of various weigh concentrations of water and glycerin were measured by using a commercial prism-coupling instrument.
The weigh concentration of water (7%) and glycerin (93%) was found to have a refractive index of 1.444, which is the same as that of fiber cladding, and that of water (50%) and glycerin (50%) was measured at 1.385. The cladding-etched FBG was immersed in a metal container on a hot plate filled with the liquid mixtures of water and glycerin, and the temperature of the liquid mixtures was varied from 30 °C to 70 °C in a controlled manner using a hot plate (MST basic 2987000, IKA).
A conventional standard FBG has a well-known temperature sensitivity of 0.01 nm/°C; hence, the non-cladding-etched FBG in [fig_ref] Figure 5: Spectral responses as a function of temperature of [/fig_ref] undergoes a Bragg wavelength of 1,549.5 nm shift to the longer wavelength of 1,549.9 nm when the temperature increases from 30 °C to 70 °C. As shown in [fig_ref] Figure 5: Spectral responses as a function of temperature of [/fig_ref] , the Bragg wavelength of 1,549.7 nm of the cladding-etched FBG with the liquid mixture of water (3%) and glycerin (93%) was shifted to a shorter wavelength, which shows a negative thermo-optic effect of the liquid mixture opposite to the positive thermo-optic effect of the standard FBG in [fig_ref] Figure 5: Spectral responses as a function of temperature of [/fig_ref]. With the greater thermo-optic effect of the liquid mixture than that of the silica FBG, the Bragg wavelength passed the central wavelength of 1,549.7 nm and shifted to a shorter wavelength. In [fig_ref] Figure 5: Spectral responses as a function of temperature of [/fig_ref] , in the liquid mixture of water (50%) and glycerin (50%) with the measured thermo-optic coefficient of −5 × 10 −4 °C −1 , the Bragg wavelengths of the proposed FBG were almost the same at 1,547.7 nm in the temperature range from 30 °C to 70 °C. Using this result, the Bragg wavelength shift dependent on the surrounding temperature can be avoided by the liquid mixture with a negative thermo-optic coefficient.
Experimentally we found out that the sharpness of the reflection spectrum of FBG degrades after wet etching. We inferred that non uniform thickness of fiber cladding after etching results in broadening of the reflection spectrum. Since etching speed of the silica fiber depends not only on the concentration of HF in etchant solution but also on the temperature of the liquid, non-homogeneous distribution in HF solution of the two physical parameters affecting etching speed may cause non uniform surface on etched fiber cladding surface. Our future research is to develop the etching technique to keep reflection spectrum after wet etching.
# Conclusions
We have studied the temperature insensitivity of the cladding-etched FBG fiber immersed in a liquid mixture with a negative thermo-optic coefficient. The obtained results demonstrate that when a cladding-etched FBG fiber is coated with properly selected liquid mixtures with negative thermo-optic coefficients, the thermal-dependency of the etched FBG fiber can be effectively compensated.
[fig] Figure 1: Structure of the cladding-etched FBG. [/fig]
[fig] Figure 2: Effective refractive index and Bragg wavelength of cladding-etched single-mode FBG in relation to the refractive index of the external medium for various remaining-cladding thicknesses. [/fig]
[fig] Figure 3: Experimental setup for cladding-etching process. [/fig]
[fig] Figure 4: Measured Bragg wavelength before and after the cladding-etching process. [/fig]
[fig] Figure 5: Spectral responses as a function of temperature of (a) non-cladding-etched FBG; (b) cladding-etched FBG in the liquid mixture of water (7%) and glycerin (93%); and (c) cladding-etched FBG in the liquid mixture of water (50%) and glycerin (50%). [/fig]
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Context conditioning in humans using commercially available immersive Virtual Reality
Despite a wealth of knowledge on how humans and nonhuman animals learn to associate meaningful events with cues in the environment, far less is known about how humans learn to associate these events with the environment itself. Progress on understanding spatiotemporal contextual processes in humans has been slow in large measure by the methodological constraint of generating and manipulating immersive spatial environments in well-controlled laboratory settings. Fortunately, immersive Virtual Reality (iVR) technology has improved appreciably and affords a relatively straightforward methodology to investigate the role of context on learning, memory, and emotion while maintaining experimental control. Here, we review context conditioning literature in humans and describe challenges to study contextual learning in humans. We then provide details for a novel context threat (fear) conditioning paradigm in humans using a commercially available VR headset and a cross-platform game engine. This paradigm resulted in the acquisition of subjective threat, threatconditioned defensive responses, and explicit threat memory. We make the paradigm publicly available and describe obstacles and solutions to optimize future studies of context conditioning using iVR. As computer technology advances to replicate the sensation of realistic environments, there are increasing opportunities to bridge the translational gap between rodent and human research on how context modulates cognition, which may ultimately lead to more optimal treatment strategies for anxiety-and stress-related disorders.Contextual information plays an important role in the development, maintenance, and treatment of anxiety and stress related disorders 1-3 . Much of what we know about the role of context in emotional learning and memory is informed by Pavlovian conditioning research, predominately in rodents. In context conditioning experiments, for instance, the subject learns to associate the presentation of a salient outcome, such as an electrical shock or food, with the environment it is in -typically the conditioning chamber -rather than with a discrete cue like a tone or a light. Behavioural and neurophysiological research on context conditioning in laboratory animals has increased our understanding of how the brain codes contextual representations and stores memories of threatening and rewarding environments. However, unlike Pavlovian conditioning that involves discrete cues, the translation of context conditioning research from rodents to humans has fallen short. This is in large measure due to a practical limitation; specifically, what is the human analogue to the rodent conditioning chamber? Posed another way: how does the experimenter effectively develop and incorporate complex environments composed of multiple spatiotemporal and sensory features while maintaining strong experimental control? As Pavlovian learning is thought to contribute to anxiety and stress disorders 4-6 , overcoming this limitation could contribute to more ecologically valid research on how contextual processes contribute to psychopathology. In the present article, we provide an overview of context conditioning research in the domain of threat (fear) learning, discuss practical and theoretical limitations to some of the existing context conditioning protocols in humans, and describe methods and results from a new threat context conditioning protocol using immersive virtual reality (iVR) that address those limitations. Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SCIeNtIfIC RePoRtS | 7: 8640 |
Pavlovian conditioning provides a valuable laboratory model to investigate the acquisition, expression, generalization, and inhibition of threat-related behaviour across species [bib_ref] Fear extinction and relapse: state of the art, Vervliet [/bib_ref] [bib_ref] Extending animal models of fear conditioning to humans, Delgado [/bib_ref]. Two Pavlovian conditioning paradigms have served as the workhorses to understand the mechanisms underlying learned threat and the role of contextual processes: cue conditioning and context conditioning. In standard aversive Pavlovian cue conditioning a subject learns that a neutral stimulus in the environment (conditioned stimulus, CS, e.g. a tone or picture) predicts the occurrence of an aversive outcome (unconditioned stimulus, US, e.g. an electrical shock). Once the subject learns the CS-US association, presentations of the CS alone begin to elicit threat-related defensive responses, such as freezing and increases in sympathetic arousal. Cue conditioning methodology is easily translatable from laboratory animals to humans, where simple sensory cues like tones and lights or more complex stimuli like faces and object categories can serve as CSs. Pavlovian threat conditioning continues to serve as a valuable and tractable model for psychopathologies characterized by acute or phasic threat responses triggered by discrete cues; exemplified by Obsessive Compulsive Disorder, Phobias, and symptom clusters of PTSD that involve defensive reactions to trauma-reminders [see ref.for review].
In Pavlovian context conditioning, the US is delivered while the subject is in a particular environment (i.e., the conditioning chamber) but is not signalled by a discrete CS. Notably, the term 'context' in associative learning research can be broadly defined, ranging from the sensory details of the environment (sights, smells, sounds, etc.), to internal states, to perceptions of time [bib_ref] Context and behavioral processes in extinction, Bouton [/bib_ref]. Context conditioning provides a more suitable model for psychopathologies characterized by sustained or 'free-floating' anxiety when there is no clear threat-eliciting stimulus in the environment [bib_ref] Phasic vs sustained fear in rats and humans: role of the extended..., Davis [/bib_ref] ; exemplified by Generalized Anxiety Disorder or symptom clusters of PTSD that involve hyper-arousal in the absence of threat-eliciting cues.
Context also interacts with cue conditioning to modulate expression of the CS-US associative memory. For example, a CS that predicts the US in one context might then be presented without the US in a separate context, i.e., extinction. Testing the CS in the first (i.e., acquisition), second (i.e., extinction context), or a novel context can reveal which CS association the subject retrieves. Neurobehavioral research has shown how context gates expression of CS-US associative memories [bib_ref] Context and behavioral processes in extinction, Bouton [/bib_ref] [bib_ref] The contextual brain: implications for fear conditioning, extinction and psychopathology, Maren [/bib_ref] ; for example, cue-elicited responses may be reduced at a later test in the extinction context (extinction recall) or may re-emerge in the extinction context (spontaneous recovery) or in the acquisition or novel context (renewal). Notably, the present article focuses largely on context conditioning paradigms where only the spatiotemporal environment signals the US in the absence of a discrete threat-eliciting cue. In human literature the term context conditioning has often been misapplied to describe paradigms where context modulates cue conditioning. We will explain the importance of distinguishing context conditioning and context modulated cue conditioning below.
Neurophysiological research in laboratory animals on context threat conditioning has focused predominately on the interplay between the amygdala, hippocampus, bed nucleus of the stria terminalis (BNST) and medial prefrontal cortex [bib_ref] Context and behavioral processes in extinction, Bouton [/bib_ref] [bib_ref] The contextual brain: implications for fear conditioning, extinction and psychopathology, Maren [/bib_ref] [bib_ref] Understanding contextual fear conditioning: insights from a two-process model, Rudy [/bib_ref] [bib_ref] Neural mechanisms of extinction learning and retrieval, Quirk [/bib_ref] [bib_ref] Mechanisms of fear extinction, Myers [/bib_ref]. While a comprehensive review of the neurobiology of context conditioning is beyond the scope of the present article, the amygdala is considered the critical site for the formation of CS-US associations and the expression of phasic learned defensive responses during aversive cue conditioning [bib_ref] Modality-specific retrograde amnesia of fear, Kim [/bib_ref] [bib_ref] Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning, Phillips [/bib_ref]. The hippocampus has a broad role in contextual processes -including spatial representations and navigation-contributing to the acquisition and storage of contextual threat information [bib_ref] Modality-specific retrograde amnesia of fear, Kim [/bib_ref] [bib_ref] Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning, Phillips [/bib_ref] [bib_ref] Context Fear Learning in the Absence of the Hippocampus, Wiltgen [/bib_ref] [bib_ref] Context representations, context functions, and the parahippocampal-hippocampal system, Rudy [/bib_ref]. The hippocampus also interacts with the bed nucleus of the stria terminalis (BNST) to sustain defensive responses to diffuse threats [bib_ref] Human Bed Nucleus of the Stria Terminalis Indexes Hypervigilant Threat Monitoring, Somerville [/bib_ref] [bib_ref] The Bed Nucleus of the Stria Terminalis Mediates Inter-individual Variations in Anxiety..., Duvarci [/bib_ref] , and interacts with the medial prefrontal cortex during the inhibition of threat responses in safe environments [bib_ref] The Role of Ventromedial Prefrontal Cortex in the Recovery of Extinguished Fear, Quirk [/bib_ref] [bib_ref] Gating of Fear in Prelimbic Cortex by Hippocampal and Amygdala Inputs, Sotres-Bayon [/bib_ref] [bib_ref] Switching on and off fear by distinct neuronal circuits, Herry [/bib_ref].
Despite the increasing number of studies on contextual processes in laboratory animals, there are to date few systematic investigations of context conditioning in humans . This is largely due to the challenge of creating a spatiotemporal context to pair with a US. In laboratory animals, context is manipulated by changing the animal's physical location (e.g., a different conditioning box) or space (e.g., change in the colours of the walls, texture or the floor and/or smell). The most obvious analogue to the conditioning chamber for human subjects is the laboratory testing room. Although the room is the predominant context, it is often not the target of context conditioning. In other words, unlike in laboratory animal research, in human conditioning studies the experimenter generally does not intend for the human subject to form an association between the physical testing room environment (composed of particular lighting, furniture, computers, research equipment, etc.) and the US.
One reason the testing room itself is not the target of context conditioning is that human conditioning research includes a within-subject control condition, the CS-(i.e. a stimulus not associated with an aversive outcome), to evaluate non-associative arousal induced by the US (i.e., sensitization). Thus, expression of threat-related behaviour in the shocked testing room (CTX+) would need to be compared to behaviour in another testing room (CTX−). Efforts have been made to create unique physical environments to investigate contextual modulation of cue conditioning [bib_ref] Reinstatement of conditioned fear in humans is context dependent and impaired in..., Labar [/bib_ref] [bib_ref] Evidence for recovery of fear following immediate extinction in rats and humans, Schiller [/bib_ref] [bib_ref] Conducting extinction in multiple contexts does not necessarily attenuate the renewal of..., Neumann [/bib_ref] [bib_ref] Delayed extinction attenuates conditioned fear renewal and spontaneous recovery in humans, Huff [/bib_ref] , but to our knowledge no studies have investigated context conditioning in physical locations, i.e. where the conditioned response is elicited by the context itself.
Investigation in humans on contextual influences on conditioning have instead manipulated context by presenting the CS on top of different static background images or embedded in movies. Such contextual CSs have included simple background colours [e.g. ref. [bib_ref] Modulation of auditory neural responses by a visual context in human fear..., Armony [/bib_ref] , static images of a scene (e.g., living room or office 29 ), and first person movies of different environments (e.g., an office and magazine store 30 ). Contextual CSs offer important advantages. For instance, the experimenter can counterbalance presentation of the threatening contextual CS and the within-subject control condition, and can control stimulus timing to service analysis of threat-related responses including fear-potentiated startle and tonic arousal indexed by changes in skin conductance levels. However, because contextual CSs are similar in many respects to CSs used in cue conditioning, it raises the question of whether these paradigms involve contextual learning processes, per se.
This distinction between context and contextual CSs is important because contexts can serve multiple functions in Pavlovian conditioning [bib_ref] Context, ambiguity, and unlearning: sources of relapse after behavioral extinction, Bouton [/bib_ref]. A context can be the target of conditioning (context conditioning) or modulate cue conditioning. When the context modulates the effectiveness of a cue, but does not elicit a response alone, it is referred to as an 'occasion setter'. As a practical matter, the testing room is very likely the prevailing physical Virtual reality technology can be used to optimize contextual stimuli and increase the chance that context conditioning engages contextual learning systems in the brain. Foremost, VR can induce a strong sense of 'presence' where people think, behave, feel, and have the sense of being in the virtual space rather than the real world [bib_ref] From presence to consciousness through virtual reality, Sanchez-Vives [/bib_ref]. A critical feature of VR is the headset (or head mounted display), which helps reduce sensory input from the outside environment and in addition allows head movements to be translated into visual rotations creating motion parallax for the participant, thereby increasing the feeling of being engrossed in a virtual environment and removed from the present physical location -referred to as 'immersion. ' To date, only a handful of context conditioning studies have incorporated VR headsets . VR has not yet been in wide use simply because early systems were costly and in some cases required large amounts of space e.g. . With the commercial release of VR headsets, the ability to augment context conditioning research is now within reach of nearly any research laboratory already investigating human conditioning with psychophysiological equipment. Accessible and validated protocols for commercially available VR systems will prove increasingly useful to the investigation of context conditioning in humans.
The goal of the present study was to develop and test a context conditioning protocol using the commercially available Oculus Rift (Oculus VR) head mounted display and the cross-platform game engine Unity (Unity Technologies, www.unity3D.com). We report the methods and results from this study, and describe obstacles and potential solutions to study learning, memory, and emotion in iVR. Additional details on the protocol and ancillary results are presented in the Supplemental Materials. The protocol and all code are made freely available to the scientific community (https://github.com/wemackey/Kroes_etal_VR). Our results indicate that the iVR context conditioning protocol was well tolerated and resulted in the acquisition of subjective threat, threat-conditioned defensive responses, and explicit threat memory for the aversive context. The development of a freely available context conditioning paradigm using a relatively affordable VR headset and free game engine with an extensive online community is a practical advancement that places the investigation of contextual processes in humans within reach of many laboratories.
# Materials and methods
Pilot studies. The context conditioning protocol was extensively piloted to optimize subject comfort and to test how to best maintain experimental control within immersive VR environments. One critical question during pilot testing was whether to allow volunteers to control first-person movement in a virtual environment themselves using a video game controller, or to remove control of movement from the subject and passively guide them through the environment. Pilot testing revealed that allowing subjects to control their own movement throughout the entire context conditioning task created motion-induced nausea in a number of volunteers. Participants with little or no video game experience had difficulty overall orienting to the controller settings during the task, since the VR headset occluded a view of their hands. In addition, some volunteers were also preoccupied with the navigational aspect of the VR environment at the cost of attending to the shock contingencies. This was revealed at debriefing, when a few of the subjects in the pilot experiment could not describe which room the shock occurred in, described being shocked in a room where they never received a shock, or misattributed the shock to an idiosyncratic behaviour, e.g., whatever they happened to be doing at the time, such as looking closely at an item in a room. For the main experiment reported here we therefore decided on allowing subjects to freely navigate and explore the environment for 2 minutes before the context conditioning task, but the subjects did not have control of navigation during the context conditioning task. This ensured that participants could initially in context, paradigm where specific cues signal the onset of the unconditioned stimulus dependent on the context in which they occur. Cue in CTX-ITI = Cue in context-inter trial interval, paradigm where specific cues signal the onset of the unconditioned stimulus and defensive responses during inter-trial intervals are taken as a index for context conditioned responses. Context manipulation: iVR = Immersive virtual reality, context manipulation is achieved using head-mounted virtual reality display where head movements are translated into changes in field of view. Test room = context manipulation is achieved by physically moving participants and testing them in different lab spaces. Full iVR = Full immersive Virtual Reality, context manipulation is achieved within room-sized cube (CAVE-like) where the environment is projected onto the walls, floor and ceiling and head movements are translated into changes in field of view. 2D movie = context manipulation is design in 3D but presented as a movie on a 2D computer screen or projected on a screen. 2D static = context manipulation is achieved by presenting a background image. N/A = not applicable, these studies do not manipulate context but take responses during inter-trial intervals as an index of context conditioned responses. *This study also measured heart rate during the task. **This study also measured EEG during the task. ***This study also measured fear ratings during the task. +This study assessed responses during inter-trial-intervals as a proxy for context conditioned responses. FPS = fear potentiated startle; SCR = skin conductance responses; SCL = skin conductance level; US exp. = unconditioned stimulus expectancy ratings; fMRI = functional magnetic resonance imaging; STAI-S = State-Trait Anxiety Inventory-S; PANAS -Positive Affect Negative Affect Scale. References: We apologize for any possible mistakes in our assessment of publications or omission of literature.
form a representation of the environments while having free movement and prevented navigation to be a source of distraction during the actual conditioning task. The limitation of movement control was effective as all but one participant in the main experiment could explicitly indicate the relationship between the rooms and shock correctly at the end of the study (see below). Further details on piloting are provided in the Supplemental Materials.
Participants. Volunteers for the final version of the task were twenty-two adult volunteers (10 female; Mean Age ± SD: 22.32 ± 4.7; range [bib_ref] Human Bed Nucleus of the Stria Terminalis Indexes Hypervigilant Threat Monitoring, Somerville [/bib_ref] [bib_ref] The Bed Nucleus of the Stria Terminalis Mediates Inter-individual Variations in Anxiety..., Duvarci [/bib_ref] [bib_ref] Lesions in the bed nucleus of the stria terminalis disrupt corticosterone and..., Sullivan [/bib_ref] [bib_ref] The Role of Ventromedial Prefrontal Cortex in the Recovery of Extinguished Fear, Quirk [/bib_ref] [bib_ref] Gating of Fear in Prelimbic Cortex by Hippocampal and Amygdala Inputs, Sotres-Bayon [/bib_ref] [bib_ref] Switching on and off fear by distinct neuronal circuits, Herry [/bib_ref] [bib_ref] Reinstatement of conditioned fear in humans is context dependent and impaired in..., Labar [/bib_ref] [bib_ref] Evidence for recovery of fear following immediate extinction in rats and humans, Schiller [/bib_ref] [bib_ref] Conducting extinction in multiple contexts does not necessarily attenuate the renewal of..., Neumann [/bib_ref] [bib_ref] Delayed extinction attenuates conditioned fear renewal and spontaneous recovery in humans, Huff [/bib_ref] [bib_ref] Modulation of auditory neural responses by a visual context in human fear..., Armony [/bib_ref] [bib_ref] Context modulation of memory for fear extinction in humans, Milad [/bib_ref] [bib_ref] Fear conditioning in virtual reality contexts: a new tool for the study..., Baas [/bib_ref] [bib_ref] Context, ambiguity, and unlearning: sources of relapse after behavioral extinction, Bouton [/bib_ref] [bib_ref] Conjunctive Representations in Learning and Memory: Principles of Cortical and Hippocampal Function, O'reilly [/bib_ref] [bib_ref] From presence to consciousness through virtual reality, Sanchez-Vives [/bib_ref] [bib_ref] Revealing Context-Specific Conditioned Fear Memories with Full Immersion Virtual Reality, Huff [/bib_ref]. Eligibility requirements were no psychiatric or neurological history, no medication (with the exception of paracetamol or ibuprofen) in the 72 hours prior to the experiment, no consumption of alcohol 24 hours before the experiment, normal or corrected-to-normal vision, normal hearing, and not easily prone to motion sickness. The study was approved by the University Committee on Activities Involving Human Subjects at New York University. All participants provided written informed consent. All methods were carried out in accordance with the Declaration of Helsinki.
Unconditioned Stimulus (US). The electrical shock was a 200 millisecond pulse delivered to the right wrist using disposable pre-gelled electrodes connected to a Grass Medical Instruments stimulator (Warwick, RI). Shocks were calibrated using an ascending staircase procedure starting with a low voltage setting near a perceptible threshold and increasing to a level deemed "maximally uncomfortable but not painful" by the participant, in keeping with prior threat conditioning protocols from our laboratory (e.g. refs 36, 37). VR and auditory equipment. Participants wore the consumer version of the Oculus Rift headset throughout the context conditioning task [fig_ref] Figure 1: iVR study design [/fig_ref]. The Oculus Rift displays stereoscopic 3D images at 106.19 degrees horizontal and 95.06 degrees vertical (i.e. 100 degrees diagonal) running at 90 Hz. The headset has a positional tracking system that allows subjects to have full 360° movement. The Oculus Rift allows for head-movements to be translated into rotations in the field of view creating motion parallax for the participant. We removed the headphone component of the Oculus rift headset and subjects wore Sennheiser HD280 headphones. These headphones fully covered the ear and therefore provided better control of sound presentation for the short static auditory bursts of white noise used to evoke eye-blink startle responses.
Eye-blink startle. The eye-blink startle response is a defensive reflex to the presentation of salient stimuli often measured by electromyography (EMG) of the muscles of the eye. Startle has been used in both cueand context conditioning studies in humans and provides a reliable and valid measure of conditioned learning. Startle responses can be elicited by the sudden presentation of brief salient stimuli, like loud noises, but the response tends to be enhanced under threat, referred to as 'fear-potentiated startle' . We measured EMG of the right orbicularis oculi muscle at 1000 Hz using two cup electrodes filled with electrolyte gel. A ground electrode was attached to the right hand. Startle probes were binaural bursts of 100 dB white noise presented for 50 milliseconds. Responses to the startle probe were quantified as the maximum EMG response 20-120 ms after the onset of the startle probe, subtracting a baseline measure of the mean EMG magnitude in a 500 ms period prior to the onset of the probe. Startle responses were transformed to T-Scores (z-score*10 + 50) as in prior studies of cue and context conditioning [bib_ref] Initial and sustained brain responses to contextual conditioned anxiety in humans, Andreatta [/bib_ref] [bib_ref] Contextual-specificity of short-delay extinction in humans: Renewal of fear-potentiated startle in a..., Alvarez [/bib_ref]. Before the context conditioning experiment began, subjects listened to 9 startle probes while viewing a blank grey screen (without the VR headset) to allow startle responses to habituate.
Skin conductance. Skin conductance was measured with pre-gelled snap electrodes (BIOPAC EL509) placed on the hypothenar eminence of the palmar surface of the non-dominant hand. Data were collected using BIOPAC MP-100 System (Goleta, CA), and analysed using an in-house analysis program written in Matlab (the MathWorks) using FieldTrip 42 . Data were low-pass filtered at 5 Hz. Responses to startle probes and upon transitions into the different contexts were determined as the peak-to-peak amplitude difference in skin conductance of the largest deflection in the latency window from 0-4.9 s after event onset (see results) to ensure that responses could not be contaminated by other events (e.g. the shock or following startle probes). The raw skin conductance responses were square root transformed, in keeping with previous studies [e.g. .
Virtual Contexts. The Virtual Reality environment was designed in Unity 5 (Unity Technologies, www.uni-ty3d.com). A C # script was used in Unity to send TTL pulses in order to trigger electrical shocks and record event timing in peripheral devices (i.e., BIOPAC ® Systems Inc.). The unity scripts and assets are available on : https:// github.com/wemackey/Kroes_etal_VR). The environment consisted of a virtual living room and kitchen/dining room connected via a hallway [fig_ref] Figure 1: iVR study design [/fig_ref]. One room had yellow walls, ceiling and floor and was decorated as a living room. The other room had blue walls, ceiling and floor and was decorated as a kitchen with dining area. These rooms were designed to be highly discriminable so as to prevent generalization of contextual threat learning [bib_ref] Generalization of Contextual Fear in Humans, Andreatta [/bib_ref]. Before context conditioning, we allowed subjects to freely explore the rooms with a game controller. This allowed subjects to encode a representation of the environment prior to conditioning, which animal research shows is critical to acquisition of threat to the context [see ref .
During the context conditioning task, participants were passively guided through the two rooms on a continuous predefined path. Subjects had free range of head movement and rotation of the field of view, but were asked to remain mostly still to reduce motion artefacts to the physiological equipment. The path started in the hallway then led through a room, back to the hallway, into a room, back to the hallway, etc. Subjects travelled through each room 10 times for 30 seconds each. Each trip through the hallway (20 total) lasted 15 seconds. We created unique paths so that subjects experienced subtly different trips through the rooms throughout the experiment. On 4 trips through the hallway, the path turned back 180 degrees and subjects returned back to the room they had just exited. The purpose of these 'return trips' was to prevent participants from predicting the next room with complete certainty. Such pseudo-randomization of CS trials is routine in differential conditioning research. Noise probes were presented in both rooms and the hallway. Shocks were only administered in one room (CTX+) but not in the other room (CTX−) or the hallway. We pseudo-randomized the order of the startle probes and shocks for different paths to mitigate temporal prediction of the US based on the startle probes (see Supplemental Information . Startle probes and US could occur pseudo-randomly from 5-25 seconds after entering a room with the limitation that there had to be 5 second between each event. Noise probes in the hallway occurred 5-10 second after entry.
Valence and Arousal Ratings. Valence and arousal ratings were obtained using self-assessment manikin scales 50 after freely navigating the rooms with a game controller but before context conditioning, and again after context conditioning. The arousal scale ranges 1 = extremely negative to 10 = extremely positive. The valence scale ranges 1 = extremely calm to 10 = extremely excited.
## Retrospective shock estimation and contingency awareness.
Participants were asked to estimate the number of shocks they had received in the blue and yellow room and to estimate the percentage of times that they received a shock when they were in the blue and yellow room [see ref ].
## Post-experimental episodic memory test. an exploratory test of episodic memory consisted of 18
multiple-choice questions for each room (36 questions total). Questions probed memory on whether particular items were in a room, and the number, colour, and position of certain items. For the yellow room for example, we asked: "What color was the rug below the circular table to the right as you entered the room? a) black, b) red, c) green, d) blue". iVR experience questionnaire. Participants indicated how they felt during the context conditioning task: "I felt no discomfort", "I was a tiny bit uncomfortable, but not too bad", "I was slightly uncomfortable", "I was moderately uncomfortable and slightly nauseous", "I was very uncomfortable and very nauseous". They also indicated their experience using Virtual Reality technology and playing video games in general.
## Inventories and anxiety questionnaires. participants completed the state-trait anxiety inventory 51 ,
Intolerance of Uncertainty Scale 52 , Berkman-Syme Social Network Index 53 , and Childhood Trauma Questionnaire -short form [bib_ref] Development and validation of a brief screening version of the Childhood Trauma..., Bernstein [/bib_ref] at the conclusion of the study.
Procedures. Upon arrival to the study (see [fig_ref] Figure 1: iVR study design [/fig_ref] for study timeline), participants were given a brief overview of the study that included information about the Oculus Rift headset and a general description of the conditioning task. Following informed consent, participants were explained how to use the video game controller and asked to freely navigate an 'infinite space' . They were then fitted with the Oculus Rift goggles and allowed to explore the rooms and hallway for 2 minutes to encourage pre-exposure to the contexts prior to context conditioning. Following free navigation the Oculus Rift goggles were removed and participants rated the rooms on dimensions of valence and arousal.
Shock electrodes were then attached and intensity was individually calibrated, followed by placement of EMG and SCR electrodes. Participants received headphones and were instructed that they would hear noise probes that would be loud but not uncomfortable and were presented with 9 startle probes to allow habituation of startle responses. Participants were then presented with 2D printed images of the rooms and the hallway and given the following instructions:
"We will now start the task. You will not be able to move yourself but will move on a track. When you are in either the blue or yellow room, you may receive electrical shocks. Note that there is a relationship between the rooms and the shocks. When you are in the hallway you will not receive any shocks. In addition you will hear noise bursts throughout the task. To make sure you understood the instructions, can you tell me what the two rooms are? Also, in which rooms can you get shocks? It is important for the study that you try to stay as still as possible during the study and sit straight up in your chair. Try to pay attention to the relationship between the rooms and the shocks. The task will start in the hallway".
We choose to use a version of the widely used semi-instructed conditioning procedure to make sure that participants paid attention to the rooms and shocks to aid differential conditioning. Critically, we did not inform the participants about the exact contingency between the rooms and shocks, which they thus had to learn from reinforcement. Results from our startle and SCR measures (see below) confirm that participants learned the relationship between the contexts and shocks from reinforcement, not instruction.
After free navigation, shock calibration, electrode and headphone placement, startle habituation, and instructions, participants were again fitted with the Oculus Rift goggles and the task commenced . Note, participants were explicitly told that there was a relationship between the rooms and the shocks but not which room would be associated with shocks, which they had to learn through experience, because at pilot testing some participants verbalized an illusory correlation between an idiosyncratic behaviour they happened to be doing at the time of the shock (e.g., an object they happened to be looking at). This raised a potential confound in the interpretation of context-conditioning per se. The iVR context conditioning task lasted for 15 minutes. After completion of the task, subjects completed the valence and arousal ratings, shock contingency estimate, episodic memory test, VR experience questionnaire, inventories and anxiety questionnaires, and compensated $20.
## Statistics. statistical analyses were performed in spss (ibm spss statistics inc.). dependent measures were
submitted to repeated measure ANOVAs and statistics were Greenhouse-Geisser or Huyn-Feldt corrected for non-sphericity when appropriate. Valence and Arousal ratings were subjected to a time (before, after) × context (CTX+, CTX−) 2 × 2 repeated measures ANOVA. Startle and SCR responses were subjected to a phase (early, late) x context (CTX+, CTX−, Hallway) 2 × 3 repeated measures ANOVAs. Significant findings from ANOVAs were followed up by paired-and independent samples t-tests. Means ± s.e.m are provided where relevant unless otherwise indicated.
# Results
We predicted that context conditioning in iVR would result in the acquisition of subjective and physiological threat responses and explicit threat memory. iVR tolerance. Participants on average tolerated the immersive Virtual Reality paradigm well. No participants dropped-out of the study or complained of motion sickness during the task. At the conclusion of the task, three participants (out of 22) indicated: "I felt no discomfort"; six indicated: "I was a tiny bit uncomfortable, but not too bad"; five indicated: "I was slightly uncomfortable"; four indicated: "I was moderately uncomfortable and slightly nauseous"; and four participants indicated: "I was very uncomfortable and slightly nauseous. " No subjects reported: "I was very uncomfortable and very nauseous, " which was the most extreme option.
Subjective valence and arousal ratings. Context conditioning resulted in the acquisition of conditioned subjective threat memory. Before and after the context conditioning task participants rated the CTX+ and CTX− room on valence (negative-positive) and arousal (calm-excited) on a 9-point Likert scale . Valence ratings changed from before to after context conditioning (time × context: F (1,21) = 50.540, p < 0.001, η 2 p = 0.706. See Supplementary Information for full results of ANOVAs, t-tests, and descriptive statistics). Following context conditioning participants rated the context in which they had received electrical stimulation (CTX+) as more negative than the context in which they had never received electrical stimulation (CTX−) [bib_ref] The Role of Ventromedial Prefrontal Cortex in the Recovery of Extinguished Fear, Quirk [/bib_ref] = −9.495, p < 0.001, Cohen's d: 2.66). This effect was driven by participants rating the CTX+ as more negative following the context conditioning task then they did before the task (t (21) = 8.549, p < 0.001, Cohen's d: 2.583). Arousal ratings also changed from before to after context conditioning (time × context: F (1,21) = 75.782, p < 0.001, η 2 p = 0.782). After conditioning participants rated the CTX+ context as more arousing than the CTX− context (t (21) = 8.805, p < 0.001, Cohen's d: 2.191) and this specifically stemmed from an increase in arousal ratings for the CTX+ . Context conditioning in iVR results in acquisition of subjective threat. Bar plots reflecting mean affect and arousal ratings before and after context conditioning for the threat (CTX+, red) and safe context (CTX−, blue). Context conditioning resulted in more negative affect ratings (a) and higher arousal ratings (b) for the threat context but did not affect ratings for the safe context. Error bars = s.e.m. Coloured geometrical shapes represent individual data-points. ***p < 0.001. context from before to after conditioning (t (21) = −9.383, p < 0.001, Cohen's d: 2.352). In sum, following conditioning participants reported the threat context to be more negative and more arousing than the control context.
## Eye-blink startle emg responses.
Context conditioning resulted in the acquisition of conditioned startle responses (phase × context: F (2,42) = 2.469, p = 0.097, η 2 p = 0.105) [fig_ref] Figure 3: Context conditioning in iVR results in acquisition conditioned defensive responses [/fig_ref]. Critically, during the late phase of conditioning participants showed enhanced startle responses in the CTX+ context compared to the CTX− context [bib_ref] The Role of Ventromedial Prefrontal Cortex in the Recovery of Extinguished Fear, Quirk [/bib_ref] We found no evidence that EMG results were moderated by STAI-T, IUS, or childhood trauma scores, but we note that these scores were relatively low and homogeneous in this healthy undergraduate population. Context conditioning using iVR thus resulted in acquisition of conditioned eye-blink startle EMG responses to the threat context (for trial-by-trial analyses see Supplementary Information).
## Skin conductance responses.
Although SCR is a common dependent measure of phasic responses in cue conditioning, it is less clear how to incorporate electrodermal measures of sympathetic arousal in context conditioning. One approach is to measure the tonic level of activity over the duration of the CTX+ as compared to the CTX−. However, startle probes occurred in each context and shocks were presented in CTX+ only, therefore complicating the analysis of tonic skin conductance levels. We therefore investigated SCR elicited by the startle probe and analysed SCRs upon the entrance of the CTX+, CTX−, and Hallway [fig_ref] Figure 3: Context conditioning in iVR results in acquisition conditioned defensive responses [/fig_ref]. Context conditioning resulted in the acquisition of conditioned SCRs to the startle probes (phase × context: F (2,42) = 2.908, p = 0.066, η 2 p = 0.122). During the late phase of conditioning participants showed greater skin conductance responses to the startle probes while in CTX+ compared to CTX− (t (21) = 2.706, p = 0.013, Cohen's d: 0.522) and compared to the hallway (t (21) = 3.361, p = 0.002, Cohen's d: 0.415), but no difference in responses while in CTX− compared to the hallway [bib_ref] The Role of Ventromedial Prefrontal Cortex in the Recovery of Extinguished Fear, Quirk [/bib_ref] = −0.455, p = 0.654, Cohen's d: 0.072). We thus found a potentiation of startle-evoked SCRs by learned threat.
The potentiation of SCR (or eye-blink startle EMG, for that matter) under threat reflects a combination of anticipatory responses to threat (i.e. the context) with defensive responses to an intrinsically aversive stimulus (i.e. the startle probe). To obtain an estimate of anticipatory responses in isolation we assessed skin conductance responses as participants transitioned between the rooms and the hallway [fig_ref] Figure 3: Context conditioning in iVR results in acquisition conditioned defensive responses [/fig_ref]. With context conditioning participants acquired conditioned SCRs at transition points (phase × context: F= 0.720, p = 0.033, η 2 p = 0.033) so that during the late phase participants showed greater responses upon transitioning into CTX+ than CTX− [bib_ref] The Role of Ventromedial Prefrontal Cortex in the Recovery of Extinguished Fear, Quirk [/bib_ref] Context conditioning using iVR thus resulted in acquisition of potentiated startle and skin conductance responses while in the threat context and acquisition of anticipatory skin conductance responses when transitioning into the threat context (for trial-by-trial analyses see . We found no evidence that STAI-T, IUS, or childhood trauma scores moderated SCR results.
Retrospective shock estimation and contingency awareness. Following context conditioning we asked participants to estimate the number of shocks they had received in CTX+ and CTX− and to estimate the percentage of times that being in each context resulted in shock, i.e. the reinforcement rate [fig_ref] Figure 4: Context conditioning results in acquisition explicit threat memory [/fig_ref]. Participants estimated having received more shocks in CTX+ than in CTX− (t(21) = 8.839, p < 0.001, Cohen's d: 2.574) and their estimation was no different from the actual number of shocks received in CTX+ (one-sample t-test: t(21) = 0.632, p = 0.535, Cohen's d: 0.135; actual number of shock = 8). Similarly, participants estimated a higher reinforcement rate for CTX+ than CTX− (t(21) = 8.063, p < 0.001, Cohen's d: 2.574) that was no different from the actual reinforcement rate of 60% for CTX+ (t(21) = 0.616, p = 0.545, Cohen's d: 1.131). Participants thus had explicit awareness of the association between the shocks and the threatening context, suggesting that the overall novelty of the iVR experience did not interfere with explicit knowledge of the CS-US contingencies.
Item memory. At the end of the experiment we tested episodic memory for particular items that had been present in each room. As arousal is broadly associated with enhancements of episodic memory [bib_ref] Mechanisms of emotional arousal and lasting declarative memory, Cahill [/bib_ref] , we reasoned that subjects might show differences in memory for items they encountered in the CTX+ versus CTX−. Memory performance overall was above chance level (four-alternative choice questionnaire = 25%) for CTX+ (t . Thus, we found no evidence that the context conditioning task had an effect on episodic item memory.
# Discussion
The goal of this study was to develop a reliable novel context conditioning paradigm using a commercially available iVR headset and a free cross-platform game engine. Our results show that our iVR context conditioning protocol was well tolerated and resulted in reliable acquisition of subjective threat (arousal and valence measures), threat-conditioned defensive responses (EMG startle responses, and skin conductance responses to startle probes and in anticipation to transitioning into the threatening context) as well as explicit threat memory (knowledge of where and approximately how many shocks were given). These results add to a small body of literature of context conditioning in VR (see , and show iVR to be an effective and accessible tool to investigate contextual processes in humans [see ref .
Development of readily available iVR context conditioning protocols provide a critical step toward bridging a long-standing translational gap between rodent and human research on the role of contextual processes. In rodents, context conditioning protocols provides a wealth of insight into contextual processes in learned threat [bib_ref] Context and behavioral processes in extinction, Bouton [/bib_ref] [bib_ref] The contextual brain: implications for fear conditioning, extinction and psychopathology, Maren [/bib_ref] [bib_ref] Understanding contextual fear conditioning: insights from a two-process model, Rudy [/bib_ref] [bib_ref] Neural mechanisms of extinction learning and retrieval, Quirk [/bib_ref] [bib_ref] Mechanisms of fear extinction, Myers [/bib_ref]. The use of iVR now allows investigators to 'place' human participants in different environments while maintaining experimental control, akin to context conditioning research with rodents. An advantage of iVR context conditioning protocols is that these can easily be adjusted to address a variety of research questions; for example the role of context representations in extinction, generalization [bib_ref] Effects of context preexposure and delay until anxiety retrieval on generalization of..., Andreatta [/bib_ref] , and renewal of threat responses [bib_ref] Reinstatement of contextual anxiety in humans: Effects of state anxiety, Glotzbach-Schoon [/bib_ref] , the role of context as an occasion setter, or the contribution of context to avoidance [bib_ref] Contextual fear conditioning predicts subsequent avoidance behaviour in a virtual reality environment, Glotzbach [/bib_ref] To understand the neural mechanisms underlying contextual threat learning in humans, future studies can augment iVR context conditioning protocols for combination with electroencephalography, near infrared spectroscopy, or transcranial magnetic stimulation. However, the ability of the former methods to study the deep brain structures critical to contextual threat processes -like the hippocampal complex -is limited. Functional magnetic resonance imaging (fMRI) can be used to study such deep brain structures, but commercially available iVR head-mounted displays are currently not MR compatible and MR compatible VR displays are costly. Furthermore, head-movement in fMRI induces motion artefacts and therefore one of the defining immersive functionalities of iVR displays cannot be utilized during fMRI. One solution is to pre-expose participants to iVR contexts with motion prior to fMRI and study effects of contextual representations without motion during fMRI. Ongoing development of eye-tracking iVR head-mounted displays will also be useful to reduce the need for head motion to induce the sense of immersion and can also be utilized to study neural processes.
A second gap bridged by the development of iVR protocols is between laboratory studies and real-world experiences implicated in the development of emotional disorders. Future protocols should assess includes factors of emotional experiences that affect learning, such as threat intensity and egocentric distance to threat stimuli In the current study we report data from 22 healthy young participants (a sample size consistent with the extant human fear conditioning literature), and we therefore note the appropriate caution while discussing these results and hope to see attempts to replicate from other laboratories and in other populations. Importantly, as the iVR protocol was well tolerated it should be possible to assess contextual conditioning in a variety of populations including psychiatric patients. VR is increasingly being used as a treatment method for psychiatric disorders, such as posttraumatic stress disorder [bib_ref] Virtual reality exposure therapy in anxiety disorders: a systematic review of process-andoutcome..., Meyerbröker [/bib_ref] [bib_ref] Virtual Reality Exposure Therapy for Post-Traumatic Stress Disorder and Other Anxiety Disorders, Gerardi [/bib_ref] [bib_ref] Efficacy of Virtual Reality Exposure Therapy in the Treatment of PTSD: A..., Gonçalves [/bib_ref]. VR exposure therapy is an effective and validated form of treatment that is especially beneficial in situations where in vivo exposure is impractical, impossible, or unlikely to be tolerated [bib_ref] Can virtual reality exposure therapy gains be generalized to reallife? A meta-analysis..., Morina [/bib_ref] [bib_ref] Affective outcomes of virtual reality exposure therapy for anxiety and specific phobias:..., Parsons [/bib_ref]. Yet, as most of the research on VR exposure is rightfully focused on clinical outcomes, it remains unclear how virtual experiences precisely engage the learning mechanisms underlying threat and safety learning. One recent investigation [bib_ref] Targeting memory reconsolidation to prevent the return of fear in patients with..., Maples-Keller [/bib_ref] provides an example of incorporating recent advances in the neurobiology of learning and memory to a VR exposure situation. In this study participants with a fear of flying underwent VR exposure treatment 10 minutes after a phobia-reminder, a time window that has been shown in humans and animals to prevent the return of fear responses [bib_ref] Extinction-Reconsolidation Boundaries: Key to Persistent Attenuation of Fear Memories, Monfils [/bib_ref] [bib_ref] Preventing the return of fear in humans using reconsolidation update mechanisms, Schiller [/bib_ref]. Although the results were mixed, it provides an example of bringing knowledge from SCIeNtIfIC RePoRtS | 7: 8640 | DOI:10.1038/s41598-017-08184-7 the domain of associative learning theory and the neurobiology of learning and memory to optimize VR treatment methods for anxiety-and stress-related disorders.
A limitation to the present study is that we did not allow volunteers to actively control their first-person movement in the virtual environment. We did so to limit the occurrence of motion sickness, to prevent problems novices had using a game controller, and to reduce distraction. However, this could have limited the full immersive experience. Unfortunately we did not include a subjective measure of immersion so we cannot assess the effect of our manipulations on the immersive experience of participants, a limitation future studies should address. Research in rodents suggest that self-movement can affect processes that underlie the formation of spatial representations [see e.g. ref . We did include a pre-exposure phase during which volunteers could control first-person movement to increase the immersive experience and to allow the initial formation of a representation of the spatial environment. In future studies, more extensive pre-exposure might circumvent navigation and distraction issues and could potentially allow self-control of movement throughout the paradigm. Furthermore, as commercial VR technology advances the risk of motion sickness will be reduced. In addition, with the emergence of devices that allow hand and limb movement and directed sound to be integrated into the iVR environment we expect that the immersive experience will increase and that movement can also be taken as a readout of human threat memory [see e.g. refs 59, 72 and 73].
As fully immersive VR technology advances and becomes more accessible, it will gain use as an important tool to the study of psychology and cognitive neuroscience. One area that stands to gain massively is the study of contextual processes -an area that has lagged behind laboratory animal research. We are convinced that the availability of a validated iVR context conditioning protocol that is made freely available to the scientific community is a critical step toward bridging a translational gap between rodent and human research on the role of contextual processes in threat learning as well as providing a more ecologically valid experimental approach to studying contextual contributions to psychopathology, which may ultimately lead to more optimal treatment strategies for anxiety-and stress-related disorders. As computer technology advances to replicate the sensation of realistic environments, there are increasing opportunities to investigate how context modulates human learning, memory, and emotions.
[fig] Figure 1: iVR study design. (a) iVR experimental set-up showing a participant (not an actual participant) wearing the Oculus Rift head mounted display while the context conditioning paradigm is presented with Unity on a standard desktop and electromyography and skin conductance responses are simultaneously recorded. (b) Time-line of experimental design. (c) 2D depiction of the two rooms and the hallway in the iVR environment (note that control buttons at the top and top right of the screen were not visible to the participants). [/fig]
[fig] Figure 3: Context conditioning in iVR results in acquisition conditioned defensive responses. Bar plots reflecting mean startle and skin conductance responses in the first half (Early) and second half (Late) of the context-conditioning task for the threat (CTX+, red), safe context (CTX−, blue), and neutral context (Hallway, green). (a) Context conditioning resulted in greater electromyography responses (i.e. eye blink magnitude) to startle probes when participants traversed the threat context. (b) Context conditioning resulted in greater skin conductance responses (i.e. sweating) to startle probes when participants traversed the threat context. (c) Context conditioning resulted in greater skin conductance responses (i.e. sweating) when participants transitioned into the threat context. Error bars = s.e.m. Coloured geometrical shapes represent individual datapoints. *p < 0.05, **p < 0.01, ***p < 0.001. [/fig]
[fig] Figure 4: Context conditioning results in acquisition explicit threat memory. Bar plots reflecting mean estimated number of received shocks (a) and mean estimated reinforcement rate (b) for the threat (CTX+, red), safe context (CTX−, blue) tested at the end of the experiment. Dashed grey line indicates actual number of administered shocks (a: 8 shocks in CTX+ only) and actual reinforcement rate (b) 60% of CTX+ trials featured delivery of shock). Following iVR conditioning, participants accurately estimated having received more shocks in the CTX+ than CTX− and associated the CTX+ with a higher reinforcement rate then the CTX−. (c) Bar plots reflecting mean memory scores on four-alternative choice memory questionnaire testing memory for items that had been present in the CTX+ and CTX−. Dashed line indicates chance level (25%). Participants remembered items from both context above change there were no difference in item memory between contexts. Error bars = s.e.m. Coloured geometrical shapes represent individual data-points. ***p < 0.001.SCIeNtIfIC RePoRtS | 7: 8640 | DOI:10.1038/s41598-017-08184-7 [/fig]
[fig] 21: = 3.864, p = 0.001, Cohen's d: 0.824) and CTX− (t(21) = 2.187, p = 0.040, Cohen's d: 0.466). However, there was no difference in memory for objects that had been present in the CTX+ compared to the CTX− (t(21) = 0.598, p = 0.557, Cohen's d: 0.112; CTX+: 36.363, 2.941, CTX−: 34.343, 4.273) [/fig]
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Moonlighting chromatin: when DNA escapes nuclear control
Extracellular chromatin, for example in the form of neutrophil extracellular traps (NETs), is an important element that propels the pathological progression of a plethora of diseases. DNA drives the interferon system, serves as autoantigen, and forms the extracellular scaffold for proteins of the innate immune system. An insufficient clearance of extruded chromatin after the release of DNA from the nucleus into the extracellular milieu can perform a secret task of moonlighting in immune-inflammatory and occlusive disorders. Here, we discuss (I) the cellular events involved in the extracellular release of chromatin and NET formation, (II) the devastating consequence of a dysregulated NET formation, and (III) the imbalance between NET formation and clearance. We include the role of NET formation in the occlusion of vessels and ducts, in lung disease, in autoimmune diseases, in chronic oral disorders, in cancer, in the formation of adhesions, and in traumatic spinal cord injury. To develop effective therapies, it is of utmost importance to target pathways that cause decondensation of chromatin during exaggerated NET formation and aggregation. Alternatively, therapies that support the clearance of extracellular chromatin are conceivable.Cell Death & Differentiation; https://doi.
# Introduction
DNA is a polymeric macromolecule that displays distinct molecular and functional properties depending on its location [bib_ref] Cell-free DNA as a biomarker in autoimmune rheumatic diseases, Duvvuri [/bib_ref] [bib_ref] Self-DNA at the epicenter of SLE: immunogenic forms, regulation, and effects, Soni [/bib_ref] [bib_ref] The origin and properties of extracellular DNA: from PAMP to DAMP, Pisetsky [/bib_ref]. Inside the nucleus, DNA serves as the essential molecule of heredity, encoding information for gene structure and regulation. Nuclear DNA is bound to histones in the form of nucleosomes, constituting a material known as chromatin [bib_ref] The nucleosome: from structure to function through physics, Onufriev [/bib_ref] [bib_ref] Mechanisms of chromatin remodeling and repurposing during extracellular translocation, Pisetsky [/bib_ref]. Once outside the cell, DNA can expand in space and display other functional activities to drive inflammation and thrombosis [bib_ref] The role of extracellular DNA (exDNA) in cellular processes, Fernandez-Dominguez [/bib_ref]. Moonlighting is an extra activity or occupation, sometimes performed in secret. If heredity and gene regulation are DNA's main functions in the nucleus, immunity is DNA's main function in the extracellular space, whether tissue or blood.
The structural bases of the intracellular and extracellular activities of DNA differ. The nuclear functions of DNA result from gene sequences and base modifications, while the extracellular functions result primarily from the charged phosphodiester backbone and its extended polymeric structure. The structures of DNA, both sequence and backbone, facilitate the binding of proteins and provide the basis for a multitude of intermolecular interactions. The translocation of DNA from the inside to the outside of the cell is the key mechanism that reveals the full diversity of DNA's biological activities [bib_ref] Mechanisms of chromatin remodeling and repurposing during extracellular translocation, Pisetsky [/bib_ref].
As demonstrated in many model systems, the translocation of DNA outside the cell can occur with cell death, stress and injury, with cell death being the predominant source of extracellular DNA [bib_ref] Modeling nuclear molecule release during in vitro cell death, Beyer [/bib_ref] [bib_ref] Types of necroinflammation, the effect of cell death modalities on sterile inflammation, Mazlo [/bib_ref] [bib_ref] Release mechanisms of major DAMPs, Murao [/bib_ref]. With cell death, DNA is a byproduct that is often considered as debris. This DNA is subject to rapid removal. With persistence and heightened levels, however, DNA can become noxious or "dangerous" as it can enter cells and interact with nucleic acid sensors; these sensors are part of an internal host defense system which can be triggered by foreign DNA from bacteria or viruses as well as self-DNA arising from cell stress or impaired nuclease activity [bib_ref] Signaling through nucleic acid sensors and their roles in inflammatory diseases, Okude [/bib_ref].
In addition to inadvertent or programmed cell death, extracellular DNA and chromatin can occur in the context of neutrophil extracellular trap (NET) formation [bib_ref] To NET or not to NET:current opinions and state of the science..., Boeltz [/bib_ref]. NET formation is an elaborate program of polymorphonuclear granulocytes that involves the movement of DNA from the nucleus to the cytoplasm where it mixes with the contents of granules to form NETs. The latter play diverse and important roles in inflammation. The principal components of NETs, DNA and histones, are ancient and can even be found in archaea. From the point of view of evolution, it is noteworthy that extracellular chromatin decorated with histones and other antimicrobial proteins also occurs in invertebrates such as crabs, mussels and sea anemones [bib_ref] Invertebrate extracellular phagocyte traps show that chromatin is an ancient defence weapon, Robb [/bib_ref] , fish [bib_ref] beta-Glucan protects neutrophil extracellular traps against degradation by Aeromonas hydrophila in carp..., Brogden [/bib_ref] [bib_ref] Bacterial infection induces pyroptotic signaling-mediated neutrophil extracellular traps (NETs) formation in turbot..., Zhao [/bib_ref] , birds [bib_ref] Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils, Chuammitri [/bib_ref] , as well as protozoans and plants.
Also, in mammals, extracellular DNA traps can originate not only from neutrophils [bib_ref] Neutrophil extracellular traps kill bacteria, Brinkmann [/bib_ref] but also from other immune cells (eosinophils, dendritic cells, monocytes, macrophages, mast cells, basophils, T cells, and B cells); DNA traps can also arise from nonimmune cells (endothelial cells, platelets, and cardiomyocytes) [bib_ref] Extracellular DNA Traps: origin, function and implications for anti-cancer therapies, Mamtimin [/bib_ref]. The evolutionary conservation of DNA traps suggests that the evolution of DNA has involved both hereditary and gene regulation as well as the potential weaponization against invading pathogens [bib_ref] Neutrophil extracellular traps: is immunity the second function of chromatin?, Brinkmann [/bib_ref].
Depending on whether extracellular DNA or chromatin arises from cell death or NET formation, the molecular properties of the DNA, as well as the identity of associated macromolecules (e.g., histones, enzymes) will differ. The release of DNA from dead and dying cells can be studied in both in vitro as well as in vivo models, although in vivo models allow better assessment of potential mechanisms of clearance and degradation as well as interplay of dead cells with phagocytic cells [bib_ref] The expression of plasma nucleosomes in mice undergoing in vivo apoptosis, Jiang [/bib_ref] [bib_ref] Role of macrophages in the generation of circulating blood nucleosomes from dead..., Jiang [/bib_ref] [bib_ref] Release of DNA from dead and dying lymphocyte and monocyte cell lines..., Choi [/bib_ref] [bib_ref] The extracellular release of DNA and HMGB1 from Jurkat T cells during..., Beyer [/bib_ref]. In vivo models to study DNA translocation can involve the transfer of dead and dying cells to a recipient animal or the in vivo induction of apoptosis or necrosis. Other models involve infection or the stimulation of inflammation that can lead to cell death as well as NET formation. In models tested thus far, extracellular DNA shows a major peak of approximately 166 basesthe size of a mononucleosomeno matter whether induction of death was by apoptosis or necroptosis [bib_ref] The expression of plasma nucleosomes in mice undergoing in vivo apoptosis, Jiang [/bib_ref] [bib_ref] Role of macrophages in the generation of circulating blood nucleosomes from dead..., Jiang [/bib_ref]. This size range is the same as that observed in studies on the molecular properties of DNA in the blood [bib_ref] Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies, Lo [/bib_ref].
As demonstrated in in vivo models, the translocation of DNA into the blood depends on macrophages and can be modulated by glucocorticoids as well as sex hormones [bib_ref] Role of macrophages in the generation of circulating blood nucleosomes from dead..., Jiang [/bib_ref] [bib_ref] The generation of extracellular DNA in SLE: the role of death and..., Pisetsky [/bib_ref] [bib_ref] The role of macrophages in the in vitro generation of extracellular DNA..., Choi [/bib_ref] [bib_ref] The effect of dexamethasone on the generation of plasma DNA from dead..., Jiang [/bib_ref]. Thus, the occurrence of DNA in the blood is the culmination of complex processes that are subject to strict regulation, including nucleolytic digestion. As these processes proceed, the size of extracellular DNA changes since DNA, when released during NET formation, for example, can show very high molecular weight (thousands to tens of thousands of bases) while, in the blood, most of the DNA is less than 200 bases [bib_ref] Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies, Lo [/bib_ref].
In addition to a soluble form, extracellular DNA can exist as a particle [bib_ref] Bioactive DNA from extracellular vesicles and particles, Malkin [/bib_ref]. This particulate form of DNA resides in microparticles which are released from cells during apoptosis and likely correspond to blebs on the cell surfaces [bib_ref] The content of DNA and RNA in microparticles released by Jurkat and..., Reich Cf 3rd [/bib_ref] [bib_ref] Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis, Schiller [/bib_ref]. This DNA is accessible and antigenically active and can be bound by monoclonal anti-DNA antibodies as well as sera from patients with systemic lupus erythematosus (SLE) to form large immune complexes [bib_ref] Microparticles in the blood of patients with SLE: Size, content of mitochondria..., Mobarrez [/bib_ref] [bib_ref] Microparticles in the blood of patients with systemic lupus erythematosus (SLE): phenotypic..., Mobarrez [/bib_ref] [bib_ref] The properties of microparticles from RAW 264.7 macrophage cells undergoing in vitro..., Spencer [/bib_ref] [bib_ref] Effect of lipopolysaccharide administration on the number, phenotype and content of nuclear..., Soop [/bib_ref] [bib_ref] Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic..., Ullal [/bib_ref]. Mitochondria represent a further source for extracellular DNA in a particle form that can bind anti-DNA antibodies [bib_ref] The binding of SLE autoantibodies to mitochondria, Pisetsky [/bib_ref]. Recognition of the various physicochemical forms of DNA circulating in the blood is important since their detection may differ depending on the use of sera or plasma as well as the conditions for isolation and analysis.
In this conceptualization, extracellular DNA or chromatin is an ensemble of molecules that vary in their origin from different cell populations; mechanisms of translocation (e.g., apoptosis, necrosis, NET formation); different physicochemical forms (i.e., high vs low molecular weight, soluble vs particulate); and the array of associated macromolecules. Rather than debris or a simple byproduct of cell death, extracellular DNA represents a multifunctional complex that displays activities to drive the pathogenesis of many diseases. Importantly, extracellular DNA and chromatin can provide a structure to organize and promote the activity of other mediators and thereby intensify inflammation and drive thrombosis.
The pro-inflammatory and pro-thrombotic activity of extracellular DNA occurs prominently during the process of NET formation, a unique element in host defense based on the elaboration of extracellular DNA in a high molecular weight form that can serve as a scaffold decorated by other intracellular molecules. This review will focus on the extracellular release of DNA during the process of NET formation and the many roles that NETs can play in disease.
## The process of net formation and degradation
Depending on the cellular viability, NET formation has been classified as suicidal or vital NET formation [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref] [bib_ref] Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps, Yousefi [/bib_ref] [bib_ref] A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response..., Pilsczek [/bib_ref] [bib_ref] Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways, Dworski [/bib_ref]. Since the nuclear genome (~3.2 billion bp) is 200,000 times larger than the mitochondrial genome (16,569 bp) [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] , nuclear DNA can indisputably form the backbone of the structure of NETs in suicidal NET formation [fig_ref] Figure 1: Mechanisms of Neutrophil NET formation [/fig_ref] [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref]. However, mitochondrial DNA and over 20 other components are also associated with NETs [bib_ref] Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute..., Lood [/bib_ref] [bib_ref] New insights into neutrophil extracellular traps: mechanisms of formation and role in..., Yang [/bib_ref]. The nucleus is the source for extracellular DNA NETs in suicidal NET formation. In the nucleus, chromatin is enclosed by the nuclear envelope, which consists of outer and inner nuclear lipid membranes (ONM and INM) and the underneath nuclear lamina [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref] [bib_ref] Filaments made from A-and B-type lamins differ in structure and organization, Goldberg [/bib_ref]. The latter is a filamentous structure consisting of A-type (A, C) or B-type (B 1 , B 2 ) lamins [bib_ref] Filaments made from A-and B-type lamins differ in structure and organization, Goldberg [/bib_ref]. A-type lamins are assembled as thick filament bundles, which affect the mechanical properties of the nuclei. In contrast, B-type lamins are assembled as a thin but highly organized meshwork which is crucial to the integrity and elasticity of the nuclear envelope [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref] [bib_ref] Filaments made from A-and B-type lamins differ in structure and organization, Goldberg [/bib_ref].
The nuclear envelope is the first physical barrier for chromatin extranuclear extrusion. Nuclear envelope rupture occurs when the nuclear lamina is either cleaved proteolytically or disassembled by phosphorylation [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [fig_ref] Figure 1: Mechanisms of Neutrophil NET formation [/fig_ref]. Lamin B is proteolytically cleaved by caspase-3 during apoptosis [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref]. However, NET formation is a caspase-independent process [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref] [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref] [bib_ref] Neutrophil extracellular trap cell death requires both autophagy and superoxide generation, Remijsen [/bib_ref] , during which caspase-3 remains inactive [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref]. Recent studies indicate that protein kinase C-α (PKC-α)-mediated lamin B phosphorylation and disassembly is responsible for nuclear envelope rupture [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref] [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref]. In addition, cyclin dependent kinase 4/6 (CDK4/6) controls NET formation through modulation of lamin A/C phosphorylation, resulting in nuclear envelope rupture [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Cell-cycle proteins control production of neutrophil extracellular traps, Amulic [/bib_ref]. Mice with deficiency of CDK4/6 or PKCα [bib_ref] Cell-cycle proteins control production of neutrophil extracellular traps, Amulic [/bib_ref] [bib_ref] Pkcα Deficiency Protected mice from UVB Induced-Skin Inflammation through Attenuation of Neutrophil..., Li [/bib_ref] , or overexpression of lamin B [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref] , display impaired NET formation in vivo. Thus, kinase-mediated nuclear lamina phosphorylation-disassembly [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref] [bib_ref] Cell-cycle proteins control production of neutrophil extracellular traps, Amulic [/bib_ref] , but not proteolytic cleavage [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] , is responsible for nuclear envelope rupture during NET formation. PKCα and CDK4/6 are located in the cytoplasm of resting neutrophils, and their nuclear translocation requires a functional actin cytoskeleton in the early stage of neutrophil activation [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref] [bib_ref] Functional actin cytoskeleton is required in early stage of NETosis induction, Liu [/bib_ref]. Genetic [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref] [bib_ref] ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation, Stojkov [/bib_ref] or pharmacologic [bib_ref] Chromatin swelling drives neutrophil extracellular trap release, Neubert [/bib_ref] inhibition of actin assembly or its upstream regulatory molecules, Rho kinase [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref] or Wiskott-Aldrich syndrome protein [bib_ref] ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation, Stojkov [/bib_ref] , impair NET formation. This argues for a crucial role of the actin cytoskeleton in NET formation [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Functional actin cytoskeleton is required in early stage of NETosis induction, Liu [/bib_ref].
Nuclear DNA is tightly packaged as chromatin by histones. The extranuclear extrusion of chromatin requires its decondensation, which is mediated through histone citrullination by peptidyl arginine deiminase 4 (PAD4) [bib_ref] Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation, Wang [/bib_ref] and/or histone cleavage by neutrophil elastase (NE) [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref] [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref] [fig_ref] Figure 1: Mechanisms of Neutrophil NET formation [/fig_ref]. In resting neutrophils, both PAD4 and NE are located in cytoplasmic granules [bib_ref] Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst..., Zhou [/bib_ref] [bib_ref] Protein arginine deiminase 4 (PAD4): Current understanding and future therapeutic potential, Jones [/bib_ref]. PAD4 has a nuclear localization sequence (NLS) which mediates nuclear translocation of cytoplasmic PAD4 [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref]. Since NE does not have a NLS, it is unclear how NE is imported into the nucleus [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] , however, the actin cytoskeleton might be involved in nuclear translocation of NE [bib_ref] A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis, Metzler [/bib_ref]. Also, CDK4/6-or PKCα-mediated nuclear envelope rupture may contribute to NE nuclear translocation which can be blocked by inhibition of these kinases [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Cell-cycle proteins control production of neutrophil extracellular traps, Amulic [/bib_ref]. Furthermore, gasdermin D (GSDMD) pores may be involved in NE release from granules and its nuclear translocation. NE in turn may also process GSDMD for its maturation and pore formation in nuclear, granular, and plasma membranes [bib_ref] Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps, Chen [/bib_ref] [bib_ref] Gasdermin D plays a vital role in the generation of neutrophil extracellular..., Sollberger [/bib_ref].
The plasma membrane is the second physical barrier for extracellular release of nuclear DNA. The cortical actin cytoskeleton is attached underneath the plasma membrane and strengthens its integrity [bib_ref] Cortical actin and the plasma membrane: inextricably intertwined, Koster [/bib_ref] [bib_ref] NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and..., Thiam [/bib_ref]. A recent study found that dynamic changes of actin polymerization in early stage, and actin depolymerization in late-stage, are accompanied by corresponding changes of Rho kinase activities [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref]. The aforementioned dynamic changes explain the role of the actin cytoskeleton in the early-stage nuclear translocation of lamin kinase PKCα and CDK4/6 [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref] [bib_ref] Functional actin cytoskeleton is required in early stage of NETosis induction, Liu [/bib_ref] , and involvement of actin depolymerization in plasma membrane rupture in later stages of NET formation [fig_ref] Figure 1: Mechanisms of Neutrophil NET formation [/fig_ref] [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref] [bib_ref] NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and..., Thiam [/bib_ref]. Disassembly of cortical cytoskeleton weakens the plasma membrane. This together with expanding forces from chromatin swelling [bib_ref] Chromatin swelling drives neutrophil extracellular trap release, Neubert [/bib_ref] , contributes to plasma membrane rupture and extracellular NET release.
Based on emerging evidence [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B..., Li [/bib_ref] [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref] [bib_ref] Cell-cycle proteins control production of neutrophil extracellular traps, Amulic [/bib_ref] [bib_ref] Chromatin swelling drives neutrophil extracellular trap release, Neubert [/bib_ref] [bib_ref] NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and..., Thiam [/bib_ref] , rupture of the nuclear envelope, nuclear chromatin decondensation, and the plasma membrane breakdown, are the key and necessary cellular events for nuclear chromatin extracellular release in suicidal NET formation [fig_ref] Figure 1: Mechanisms of Neutrophil NET formation [/fig_ref]. The signaling pathways that regulate key b Nuclear envelope rupture is modulated by nuclear translocation of PKCα or CDK4/6 which mediate nuclear lamina disassembly (electron microscopy images of b1 well organized nuclear lamina, or b2 disassembled nuclear lamina [bib_ref] The nuclear lamina is a meshwork of intermediate-type filaments, Aebi [/bib_ref]. c Rupture of the plasma membrane is achieved by disassembly of cortical cytoskeleton (c1, electron microscopy image of actin cortex [bib_ref] Simultaneous stabilization of actin cytoskeleton in multiple nephron-specific cells protects the kidney..., Mukherjee [/bib_ref]. (α, β) Representative confocal microscopy images of an untreated neutrophil (α) and a PMA-treated neutrophil with ruptured nuclear envelope and extracellular NETs in which nuclear DNA forms the backbone of NETs that are decorated with the disassembled lamin B (β), stainings of lamin B and DNA with fluorescent-labeled anti-lamin B1 and DAPI. Vital NET formation: Vital NET formation has been described as either derived through nuclear blebbing, or released from mitochondria. cellular morphological changes might be candidate targets for therapeutics in NET-related diseases. Since NET formation has been described [bib_ref] Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate..., Takei [/bib_ref] , and detailed in seminal experiments [bib_ref] Neutrophil extracellular traps kill bacteria, Brinkmann [/bib_ref] , the involvement of various signaling pathways has been reported [bib_ref] To NET or not to NET:current opinions and state of the science..., Boeltz [/bib_ref] [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref]. Reactive oxygen species (ROS) are crucial for NET formation as they activate several downstream effectors. ROS modulate the release of granule myeloperoxidase (MPO) and NE [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref] [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref] , and regulate cytoskeletal dynamics [bib_ref] ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation, Stojkov [/bib_ref] , which is involved in NET formation [bib_ref] Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin..., Li [/bib_ref] [bib_ref] ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation, Stojkov [/bib_ref] [bib_ref] Chromatin swelling drives neutrophil extracellular trap release, Neubert [/bib_ref] [bib_ref] NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and..., Thiam [/bib_ref]. Activated neutrophils generate ROS through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX2) or mitochondrial dysfunction [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref] [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref] [bib_ref] Neutrophil extracellular trap cell death requires both autophagy and superoxide generation, Remijsen [/bib_ref] [bib_ref] SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium..., Douda [/bib_ref]. Depending on the stimuli, NOX-dependent, and -independent pathways have been reported to drive NET formation [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref] [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref] [bib_ref] Neutrophil extracellular trap cell death requires both autophagy and superoxide generation, Remijsen [/bib_ref] [bib_ref] SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium..., Douda [/bib_ref].
In NOX-dependent pathways, stimuli (like, PMA, LPS, PAF) activate NOX2 that drives NET formation through ROS generation [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref] [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref] [bib_ref] Neutrophil extracellular trap cell death requires both autophagy and superoxide generation, Remijsen [/bib_ref] [bib_ref] NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits, Belambri [/bib_ref] , while genetic mutation or pharmacological inhibition of NOX2 attenuates NET formation [bib_ref] Neutrophil extracellular traps in immunity and disease, Papayannopoulos [/bib_ref] [bib_ref] Novel cell death program leads to neutrophil extracellular traps, Fuchs [/bib_ref] [bib_ref] Neutrophil extracellular trap cell death requires both autophagy and superoxide generation, Remijsen [/bib_ref]. However, requirements for NOX are stimulus dependent, and NOX activity is not required for calcium ionophore-induced NET formation [bib_ref] Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ..., Parker [/bib_ref]. Calcium ionophores induce calcium influx, which activates the mitochondrial SK3 channel, resulting in mitochondrial ROS production for NOX-independent NET formation [bib_ref] SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium..., Douda [/bib_ref]. A recent study found that calcium ionophores activate calpain, which may with involvement of calcium-dependent PAD4 mediate interdomain proteolysis of the nuclear lamina and high mobility group box 1 protein (HMGB1); the latter an architectural chromatin binding protein [bib_ref] Citrullination licenses calpain to decondense nuclei in neutrophil extracellular trap formation, Gosswein [/bib_ref]. The collective activity of PAD4 and calpain may contribute to the destruction of the nuclear lamina and, thus enable chromatin decondensation in calcium-mediated NET formation [bib_ref] Citrullination licenses calpain to decondense nuclei in neutrophil extracellular trap formation, Gosswein [/bib_ref]. However, more detailed studies are needed for understanding the role of calpain in nuclear lamina disintegration. Intracellular calcium mobilization is also involved in regulation of actin cytoskeleton dynamics. These are important in nuclear translocation of PKCα and CDK4/6 for nuclear lamina disassembly [bib_ref] Functional actin cytoskeleton is required in early stage of NETosis induction, Liu [/bib_ref] and NE for chromatin decondensation [bib_ref] A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis, Metzler [/bib_ref].
In contrast to suicidal NET formation, "vital NET formation" has also been reported as extracellular release of either mitochondrial [bib_ref] Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps, Yousefi [/bib_ref] or nuclear [bib_ref] A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response..., Pilsczek [/bib_ref] DNA, without loss of plasma membrane integrity [fig_ref] Figure 1: Mechanisms of Neutrophil NET formation [/fig_ref]. Vital NET formation was initially described in neutrophils that were first primed by GM-CSF and then consequently stimulated with LPS/C5a, resulting in rapid release of NETs which DNA is solely from mitochondria [bib_ref] Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps, Yousefi [/bib_ref]. An intact cytoskeleton is required for vital NET formation with involvement of ROS [bib_ref] In vivo evidence for extracellular DNA trap formation, Yousefi [/bib_ref]. Extracellular release of mitochondrial DNA from viable cells has been observed not only in granulocytes [bib_ref] In vivo evidence for extracellular DNA trap formation, Yousefi [/bib_ref] , but also in lymphocytes [bib_ref] Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG..., Ingelsson [/bib_ref] and amoebae [bib_ref] Social amoebae trap and kill bacteria by casting DNA nets, Zhang [/bib_ref]. This phenomenon has been considered an intrinsic innate immune response by either directly killing bacteria [bib_ref] Social amoebae trap and kill bacteria by casting DNA nets, Zhang [/bib_ref] , or indirectly by inducing anti-viral interferons [bib_ref] In vivo evidence for extracellular DNA trap formation, Yousefi [/bib_ref] [bib_ref] Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG..., Ingelsson [/bib_ref]. Interestingly, mitochondrial DNA release has also been observed in viable fibroblasts [bib_ref] Extracellular mitochondrial DNA is generated by fibroblasts and predicts death in idiopathic..., Ryu [/bib_ref] or chondrocytes [bib_ref] is released by viable chondrocytes after induction of mitochondrial dysfunction, Keller [/bib_ref] , which might be an acute-phase response to mitochondrial stress or dysfunction. The latter findings, however, raise the question if extracellular release of mitochondrial DNA from viable cells is a broader phenomenon not limited to immune cells [bib_ref] Mitochondria Can Cross Cell Boundaries: An overview of the biological relevance, pathophysiological..., Valenti [/bib_ref]. More studies are needed to understand this important and interesting phenomenon comparing extracellular release of mitochondrial DNA from viable immune cells vs non-immune cells.
In addition to mitochondrial DNA release, another study reported that exposure of neutrophils to Staphylococcus aureus can induce rapid NET formation without cell membrane breakdown [bib_ref] A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response..., Pilsczek [/bib_ref]. Upon stimulation, the multilobular nucleus rapidly became rounded, followed by nuclear blebbing of chromatin containing vesicles, which deliver and release their contents into the extracellular space for NET formation [bib_ref] A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response..., Pilsczek [/bib_ref]. The entire process occurs in 5-60 min in a ROS-independent manner [bib_ref] A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response..., Pilsczek [/bib_ref] and requires chromatin decondensation [bib_ref] Classical ROS-dependent and early/rapid ROSindependent release of Neutrophil Extracellular Traps triggered by..., Rochael [/bib_ref]. Although the mechanism that regulates the nuclear blebbing in vital NET formation is unclear, histone modification during chromatin decondensation may contribute to nuclear blebbing, known to be determined by alteration of chromatin compaction and histone modification [bib_ref] Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins, Stephens [/bib_ref]. The budded nuclear vesicles may rupture over time and release their enclosed DNA into the cytoplasm, and eventually extrude into the extracellular space as described for suicidal NET formation [bib_ref] A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response..., Pilsczek [/bib_ref]. Two studies found that parasites may induce rapid vital NET formation at 10-30 mins of neutrophil-parasite interaction, and suicidal NET formation when they are co-incubated for a longer time. The latter condition results in increased total NET formation [bib_ref] Classical ROS-dependent and early/rapid ROSindependent release of Neutrophil Extracellular Traps triggered by..., Rochael [/bib_ref] [bib_ref] Besnoitia besnoiti bradyzoite stages induce suicidal-and rapid vital-NETosis, Zhou [/bib_ref]. One may speculate that vital/rapid NET formation might be the early event of suicidal NET formation before the neutrophils lose their viability. More studies are needed to address the relationship between vital and suicidal NET formation with extracellular release of chromatin.
All in all, the coexistence between the suicidal lytic and vital NET formation remains uncertain [bib_ref] Recent progress in the mechanistic understanding of NET formation in neutrophils, Liu [/bib_ref] [bib_ref] New insights into neutrophil extracellular traps: mechanisms of formation and role in..., Yang [/bib_ref]. Most importantly, the diversity of NET formation signaling pathways makes it difficult to identify unified targets for therapeutic purposes of NETrelated diseases. To control NET formation, there is a need for identifying the pathways in different clinical settings to allow specific targeting. Another option is to target and improve the degradation of NETs for a fine-tuning of the balance between NET formation and degradation. NETs are reportedly degraded by macrophages; preprocessing of NETs with DNase1 and opsonization with C1q facilitates this process [bib_ref] Macrophage clearance of neutrophil extracellular traps is a silent process, Farrera [/bib_ref]. Macrophages take up NETs through micropinocytosis, but how exactly the degradation is achieved warrants further research [bib_ref] Macrophage clearance of neutrophil extracellular traps is a silent process, Farrera [/bib_ref] [bib_ref] Intra-and extracellular degradation of neutrophil extracellular traps by macrophages and dendritic cells, Lazzaretto [/bib_ref]. Dendritic cells (DCs) are able to take up NETs, albeit to a much lesser extent than macrophages, and to secrete DNase1L3 for extracellular digestion of NETs [bib_ref] Intra-and extracellular degradation of neutrophil extracellular traps by macrophages and dendritic cells, Lazzaretto [/bib_ref]. Recently, it was also described that 13-series (T-series) resolvins reduce NET formation by enhancing NET uptake by human macrophages in a phospho-AMP-activated protein kinase (AMPK)-dependent manner [bib_ref] Resolvin T-series reduce neutrophil extracellular traps, Chiang [/bib_ref]. One should be cautious in enhancing NET degradation since degraded NETs reportedly foster the growth of Actinobacillus pleuropneumoniae, causing severe porcine pneumonia; [bib_ref] Degraded neutrophil extracellular traps promote the growth of Actinobacillus pleuropneumoniae, De Buhr [/bib_ref] this might also be true for further pathogens. The degradation of NETs by circulating DNases leads to the release of so-called NET degradation products (NDPs) such as cell-free DNA (complexed with MPO or NE) and histones. These NDPs themselves have toxic effects like fixation of complement (cell-free DNA) [bib_ref] Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate..., Leffler [/bib_ref] , induction of oxidative tissue damage (MPO) [bib_ref] Role of myeloperoxidase and oxidant formation in the extracellular environment in inflammation-induced..., Hawkins [/bib_ref] , promotion of thrombosis by local proteolysis of the tissue factor pathway inhibitor [bib_ref] Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases, Massberg [/bib_ref] , or activation of platelets and cytotoxicity for epithelial cells (histones) [bib_ref] Extracellular histones promote thrombin generation through plateletdependent mechanisms: involvement of platelet TLR2..., Semeraro [/bib_ref] [bib_ref] Histones induce rapid and profound thrombocytopenia in mice, Fuchs [/bib_ref] [bib_ref] Extracellular histones are major mediators of death in sepsis, Xu [/bib_ref] [bib_ref] Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant..., Saffarzadeh [/bib_ref].
## The composition of nets net-borne enzymes
During the process of NET formation or cell death, chromatin escapes the nuclear control with an abundance of various proteins. In a first assessment of the neutrophil proteome, a total of 251 major cellular proteins in different compartments were identified by gel-LC-MS/MS [bib_ref] Proteomic analysis of total cellular proteins of human neutrophils, Tomazella [/bib_ref]. This proteome included the azurophilic granule proteins NE and MPO as well as PAD4, which catalyzes the deimination of arginine to citrulline and mediates chromatin decondensation [bib_ref] Direct cost of illness for spinal cord injury: a systematic review, Malekzadeh [/bib_ref] [bib_ref] Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps, Papayannopoulos [/bib_ref] [bib_ref] Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation, Wang [/bib_ref]. In a recent proteome analysis by Petretto et al., 330 NET-associated proteins were identified; many with posttranslational modifications [bib_ref] Neutrophil extracellular traps (NET) induced by different stimuli: A comparative proteomic analysis, Petretto [/bib_ref]. Of these, 74 were detected in all NETs but others differed dependent on the inducer of NET formation. Interestingly, the cellular origin of these NET-associated proteins seemed independent of the respective inducers with most proteins originating from the cytoplasm/cytoskeleton, followed by organelle-and membranederived proteins. The identification of only four to six different proteins associated with NETs from patients with SLE compared to rheumatoid arthritis (RA) determined that the nature of the stimulant is more important for the NET proteome composition than the underlying disease profile [bib_ref] Caught in a Trap? Proteomic analysis of neutrophil extracellular traps in rheumatoid..., Chapman [/bib_ref].
## Aggregation of nets
The binding of NE and other antimicrobial proteins to extruded chromatin of NETs mediate the digestion and elimination of microbial pathogens at the site of insult. At these areas of high cell densities, neutrophils tend to aggregate and form enzymatically stable clumps called aggregated NETs or aggNETs [bib_ref] Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines, Schauer [/bib_ref]. NETs and aggNETs immobilize, neutralize and/or kill bacteria [bib_ref] Neutrophil extracellular traps kill bacteria, Brinkmann [/bib_ref] , fungi [bib_ref] Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms, Urban [/bib_ref] , viruses [bib_ref] Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1, Saitoh [/bib_ref] , parasites [bib_ref] Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps, Abi Abdallah [/bib_ref] , and inhibit their dissemination [bib_ref] DNase Sda1 provides selection pressure for a switch to invasive group A..., Walker [/bib_ref] [bib_ref] Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response..., Branzk [/bib_ref] [bib_ref] Net effects of NETs: new concepts, Dainichi [/bib_ref]. In addition, aggNETs also contribute to the resolution of inflammation. The externalized chromatin fibers are decorated with a plethora of cytoplasmic and granular proteases. Consequently, aggNETs can not only scavenge inflammatory mediators, but also degrade these molecules [bib_ref] Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines..., Hahn [/bib_ref]. The proteolysis of toxic molecules like highly cationic histones, which had entered the extracellular space during NET formation, protects surrounding tissues from chronic damage and allows re-establishment of tissue homeostasis [bib_ref] Aggregated NETs sequester and detoxify extracellular histones, Knopf [/bib_ref]. AggNETs also sequester and degrade inflammatory cytokines and chemokines. This process prevents further recruitment of neutrophils and supports the resolution of inflammation [bib_ref] Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines, Schauer [/bib_ref]. Despite possessing beneficial effects, NETs are also involved in the induction of pro-thrombotic events. NETbound histones interact directly with T-cells resulting in Th17 differentiation [bib_ref] Neutrophil extracellular traps and their histones promote Th17 cell differentiation directly via..., Wilson [/bib_ref] , and activate platelets, thereby stimulating thrombogenesis [bib_ref] Extracellular DNA traps promote thrombosis, Fuchs [/bib_ref]. Impaired NET aggregation and clearance can drive the development of autoimmunity and become detrimental. Therefore, a dysregulated immune response and an imbalance between NET formation and degradation can lead to devastating diseases as summarized in and in the following paragraphs.
## Methods to detect nets in tissue samples
The detection of tissue-borne NETs is crucial to identify dysregulated NET formation and clearance in infections, sepsis, autoimmune diseases, thrombosis, metabolic disorders, and cancer. Currently, the most general technique to visualize NETs in tissues is immunostaining of paraffin-embedded tissues followed by immunofluorescence microscopy. After deparaffinization of tissue sections, heat-induced epitope retrieval (HIER) buffer is used for rehydration, breaking the methylene bridges and making epitopes such as NE, MPO, and citrullinated histone H3 accessible for binding of antibodies. The antibody-stained tissues are counterstained with DNA intercalating dyes that have detected extended NETs in many tissue sections with reliable signal intensity [bib_ref] Immunodetection of NETs in paraffin-embedded tissue, Brinkmann [/bib_ref]. Recently, a protocol for multiplex staining demonstrated the detection of NETs in paraffin-embedded human biopsies of phlegmonous appendicitis, lung abscess and nonsmall cell lung cancer [bib_ref] Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially..., De Andrea [/bib_ref]. Over the past two decades, numerous technological advances have been made in immunofluorescence microscopy. Recent achievements allow greater insights into the morphology and high-resolution analysis to detect subtle changes in the tissues. High resolution stimulated emission depletion (STED) microscopy was used to detect citrullinated NETs frequently recurring in tissue biopsies from patients with colon cancer. Using anti-DNA antibodies directed to extracellular chromatin, the authors distinguished between condensed and compacted chromatin inside neutrophils with a healthy Illustrative representation of the neutrophil extracellular trap (NET) formation and degradation cascade. 1. Neutrophils, via a transendothelial mechanism, are recruited to the site of an incident within hours. There they form NETs by releasing nuclear chromatin or mitochondrial DNA decorated with potent antimicrobial granular proteins. 2. Monocyte and activated macrophages secrete neutrophil chemoattractants that lead to a rapid influx of a large number of neutrophils. At these high densities, neutrophils aggregate, forming so-called aggregated NETs (aggNETs). These foster the resolution of inflammation by degrading small soluble mediators of inflammation. 3. Within days to weeks, after NETs enabled a successful entrapment of pathogens and restricted their dissemination, intracellular DNases break down the DNA backbone of NETs. These remnants can then be cleared by phagocytic cells (e.g., macrophages) to promote clearance and thereby restore homeostasis. 4. Ineffective clearance of NETs leads to their extended and prolonged ripening by forming stable aggNET-fibrin coaggregates, in which fibrin polymerizes in the scaffold formed by NETs. In these aggregates fibrin can be citrullinated and, consequently, resists degradation by plasmin. In addition, the NETs incorporated in these aggregates were protected from DNases. A reduced clearance then may lead to long-term secondary inflammation and formation of stones and tophi.
appearance and ejected decondensed DNA displaying the typical characteristic of NETs [bib_ref] Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer, Stehr [/bib_ref].
Label-free emission recorded in immunofluorescence microscopy of tissue biopsies from patients with COVID-19 revealed widespread immune thrombosis in a devastated pulmonary vasculature due to native endogenous fluorophores. Contrary to standard detection of NETs via immunofluorescence using antibodies, a recently published article reported the detection of NETs in inflammatory diseases using a fluorogenic peptide. The authors developed a highly specific, triple-quenched, tribranched fluorescent human neutrophil elastase sensor. This sensor offers a more than 20-fold increase in fluorescence intensity upon enzymatic cleavage and extends the current detection methods from antibodies to the first molecular probe detectors [bib_ref] A fluorogenic peptide-based smartprobe for the detection of neutrophil extracellular traps and..., Rios [/bib_ref].
Additionally, several live-cell imaging techniques directly monitor the neutrophils' release of NETs at the cellular level in various complex tissue and organs affected by infection and autoimmunity. Two-photon microscopy enables real-time detection of NETs in Aspergillus fumigatus infected murine lung lobules using the SYTOX dye without organ fixation [bib_ref] Production of extracellular traps against Aspergillus fumigatus in vitro and in infected..., Bruns [/bib_ref]. Intravital microscopy showed activated platelets resulting in the formation of NETs in liver sinusoids [bib_ref] Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood, Clark [/bib_ref] , and using SYTOX dye, spinning disk confocal intravital microscopy visualized the formation of NETs, over two hours on murine skin infected with Staphylococcus aureus [bib_ref] How neutrophil extracellular traps become visible, De Buhr [/bib_ref] [bib_ref] NETosis: how vital is it?, Yipp [/bib_ref] [bib_ref] Infectioninduced NETosis is a dynamic process involving neutrophil multitasking in vivo, Yipp [/bib_ref]. Cell-permeable and impermeable DNA dyes allow the direct visualization of intact neutrophils and of NETs in various organs, respectively [bib_ref] Infectioninduced NETosis is a dynamic process involving neutrophil multitasking in vivo, Yipp [/bib_ref] [bib_ref] Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis, Cools-Lartigue [/bib_ref] [bib_ref] In vivo imaging of neutrophil extracellular traps (NETs): visualization methods and outcomes, Alasmari [/bib_ref]. Intravital microscopy with a laser scanning microscope characterized NETs in blood vessels of different organs [bib_ref] How neutrophil extracellular traps become visible, De Buhr [/bib_ref] [bib_ref] In vivo characterization of neutrophil extracellular traps in various organs of a..., Tanaka [/bib_ref].
To date, classical antibody-based immunofluorescence methods are still the most commonly used to detect neutrophils and NETs in tissue sections. However, many promising new methods like live-cell imaging and STED microscopy have recently been developed to directly monitor neutrophils and NETs in vivo and with ultrahigh resolution, respectively.
## Extracellular chromatin moonlighting diseases immunothrombosis
In the last decade, it has become increasingly clear that neutrophils and especially NETs are intertwined into the processes of thrombus formation and maturation in diverse pathological settings [bib_ref] Neutrophil extracellular traps: villains and targets in arterial, venous, and cancer-associated thrombosis, Thalin [/bib_ref]. The term immunothrombosis has been coined to highlight the interaction of the cellular innate immune system with pathological thrombosis [bib_ref] Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in..., Von Bruhl [/bib_ref]. The evolutionary advantage of the activation of thrombosis by players of the innate immune system lies in physically trapping infectious agents in occluded vessels to limit spread via the circulation and thus contain an inflammatory focus [bib_ref] Thrombosis as an intravascular effector of innate immunity, Engelmann [/bib_ref]. Exaggerated immunothrombosis, however, is central in the exacerbation of several pathological settings including coagulopathy in sepsis [bib_ref] Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood, Clark [/bib_ref] , necroinflammation [bib_ref] Histones and neutrophil extracellular traps enhance tubular necrosis and remote organ injury..., Nakazawa [/bib_ref] , and severe COVID-19 [bib_ref] Vascular occlusion by neutrophil extracellular traps in COVID-19, Leppkes [/bib_ref] [bib_ref] Endothelial dysfunction and immunothrombosis as key pathogenic mechanisms in COVID-19, Bonaventura [/bib_ref]. As shown in pancreatitis [bib_ref] Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis, Leppkes [/bib_ref] , not only blood vessels, but also glandular ducts can be occluded by NETs. Apart from physical trapping of microbes, immunothrombosis also fulfils beneficial hemostatic tasks in the setting of mucosal damage in acute flares of ulcerative colitis [bib_ref] Neutrophils prevent rectal bleeding in ulcerative colitis by peptidyl-arginine deiminase-4-dependent immunothrombosis, Leppkes [/bib_ref]. Here, the absence of PAD4 is associated with increased mucosal blood loss. The interactions of neutrophils and components of NETs with classical players of thrombosis are numerous; these players include platelets, serine proteases of the coagulation cascade, fibrinolysis and the fibrin mesh itself. Neutrophils and NETs interact with these players via membrane-bound receptors, degranulated effector proteins, chromatin of NETs, including NET-bound nuclear and granular proteins [bib_ref] Thrombosis: tangled up in NETs, Martinod [/bib_ref] and extracellular vesicles [bib_ref] Role of neutrophils, platelets, and extracellular vesicles and their interactions in COVID-19-associated..., Caillon [/bib_ref]. Activated platelets can induce formation of NETs [bib_ref] Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy..., Maugeri [/bib_ref]. More specifically, deletion of HMGB1 in platelets reduced NET formation and associated organ damage in various experimental models [bib_ref] Plateletderived HMGB1 is a critical mediator of thrombosis, Vogel [/bib_ref]. Vice versa, platelets may bind to and aggregate on extracellular chromatin of NETs [bib_ref] Extracellular DNA traps promote thrombosis, Fuchs [/bib_ref].
The aggNETs provide a scaffold for thrombus formation and are able to occlude vessels and ducts [bib_ref] Extracellular DNA traps promote thrombosis, Fuchs [/bib_ref] [bib_ref] Neutrophils: back in the thrombosis spotlight, Noubouossie [/bib_ref]. The obstruction of the microvasculature in organs and a consequent inhibition of the blood flow in the capillaries, together with NET-driven endothelial dysfunction, may precipitate organ failure and mortality [bib_ref] Endothelial cellactivating antibodies in COVID-19, Shi [/bib_ref] [bib_ref] Neutrophil extracellular trap-driven occlusive diseases, Yaykasli [/bib_ref]. Thereby, aggNETs can contribute to the pathogenesis of various diseases. As already mentioned above, NETs occlude the vessels in patients with COVID-19 [bib_ref] Vascular occlusion by neutrophil extracellular traps in COVID-19, Leppkes [/bib_ref]. Furthermore, NETs-associated occlusions have been reported for coronary vessels in acute myocardial infarction and artherosclerosis [bib_ref] Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi..., De Boer [/bib_ref] [bib_ref] Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in..., Knight [/bib_ref] and for cerebral vessels in ischemic stroke [bib_ref] Neutrophil extracellular traps in ischemic stroke thrombi, Laridan [/bib_ref].
In addition to cellular interactions which may foster NET formation and pathological thrombosis, soluble mediators are studied. PAD4 becomes a focus of attention since it reportedly links inflammation and thrombosis. Injection of recombinant human PAD4 in vivo induced the formation of von Willebrand factor (vWF)-platelet strings in mesenteric venules. These strings are naturally degraded by ADAMTS13, a metalloproteinase, but citrullination of ADAMTS13 dramatically reduces the endogenous enzymatic activity [bib_ref] Plasma peptidylarginine deiminase IV promotes VWF-platelet string formation and accelerates thrombosis after..., Sorvillo [/bib_ref]. In line with these findings, studies have shown that a class of serpins with an arginine residue in the P1 position (including antithrombin, C1INH, 1-antiplasmin, PAI1/PAI2) is inhibited by citrullination [bib_ref] The rheumatoid arthritis-associated citrullinome, Tilvawala [/bib_ref] [bib_ref] The role of SERPIN citrullination in thrombosis, Tilvawala [/bib_ref] , thus unleashing the proteolytic power of the serine proteases thrombin, plasmin and tissue plasminogen activator in the thrombo-inflammatory microenvironment. PAD4-guided thrombin activation may then further facilitate thrombus maturation by FXIII-mediated cross-linking [bib_ref] Neutrophils prevent rectal bleeding in ulcerative colitis by peptidyl-arginine deiminase-4-dependent immunothrombosis, Leppkes [/bib_ref]. Citrullination has also recently been identified as a major posttranslational modifier that impacts proteolysis [bib_ref] Citrullination licenses calpain to decondense nuclei in neutrophil extracellular trap formation, Gosswein [/bib_ref]. Future studies will unravel the translational potential of targeting immunothrombosis in clinical settings. These studies could explain the puzzling failure of classical anticoagulants to prevent thrombus formation under certain septic conditions and in disseminated intravascular coagulopathy. Pan-PAD inhibitors and PAD4-specific inhibitors are already central tools in the research of NETs and immunothrombosis and will surely be further studied as therapeutic options in general.
## Nets in lung diseases
Chronic respiratory diseases affect the airways and other structures of the lung. In 2017, 544.9 million people worldwide were affected by a chronic lung disease, such as asthma or chronic obstructive pulmonary disease (COPD), making them the third leading cause of death behind cardiovascular diseases and cancer [bib_ref] Prevalence and attributable health burden of chronic respiratory diseases, 1990-2017: a systematic..., Collaborators [/bib_ref]. Even though asthma was always considered to be an eosinophilic disease, recent reports also highlight the role of neutrophils in this disease [bib_ref] Neutrophilic asthma is associated with increased airway bacterial burden and disordered community..., Yang [/bib_ref] [bib_ref] Sputum colour can identify patients with neutrophilic inflammation in asthma, Pabreja [/bib_ref] [bib_ref] Th-17 regulatory cytokines IL-21, IL-23, and IL-6 enhance neutrophil production of IL-17..., Halwani [/bib_ref]. Similar to this subset of patients with neutrophilic asthma, patients with COPD show high neutrophilic airway inflammation; higher levels of blood neutrophil counts have been correlated with mortality in these patients [bib_ref] Local and systemic cellular inflammation and cytokine release in chronic obstructive pulmonary..., Moermans [/bib_ref] [bib_ref] Blood neutrophil counts are associated with exacerbation frequency and mortality in COPD, Lonergan [/bib_ref]. In both lung diseases, NETs were found in the airways of patients and were associated with inflammation [bib_ref] Neutrophil extracellular traps are associated with inflammation in chronic airway disease, Wright [/bib_ref] , and in the case of COPD, also with airflow limitation [bib_ref] Neutrophil extracellular trap (NET) formation characterises stable and exacerbated COPD and correlates..., Grabcanovic-Musija [/bib_ref].
Next to chronic respiratory diseases, acute lung injury and acute respiratory distress syndrome (ARDS) are further major causes of morbidity and mortality, especially in the critically ill patients. In these disorders, acute lung inflammation, as indicated by excessive transepithelial neutrophil migration and the release of pro-inflammatory and cytotoxic mediators, disrupts the endothelial and epithelial barriers of the lungs [bib_ref] Acute lung injury: epidemiology, pathogenesis, and treatment, Johnson [/bib_ref] [bib_ref] Contribution of neutrophils to acute lung injury, Grommes [/bib_ref]. Increased plasma levels of NETs have been associated with ARDS severity and mortality and lower plasma levels of DNase1 were associated with the development of sepsisinduced ARDS. This indicates that a balance in NET formation and degradation is crucial to prevent lung injury [bib_ref] Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury, Lefrancais [/bib_ref]. In this context, disulfiram, an aldehyde dehydrogenase inhibitor, was recently shown to inhibit NET formation and to protect from acute lung injury in a mouse model [bib_ref] Disulfiram inhibits neutrophil extracellular trap formation and protects rodents from acute lung..., Adrover [/bib_ref].
Cystic fibrosis (CF) is characterized by impaired mucus hydration and clearance due to mutations in the CFTR gene leading to chronic pulmonary infection and (neutrophilic) inflammation [bib_ref] Cystic fibrosis, Shteinberg [/bib_ref]. The sputum of patients with CF is heavily loaded with NETs and NET-related proteins. The activity of NE and the presence of MPO are correlated with disease progression, severity and reduction in lung function [bib_ref] Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron..., Manzenreiter [/bib_ref] [bib_ref] Pulmonary function is negatively correlated with sputum inflammatory markers and cough clearability..., Kim [/bib_ref] [bib_ref] Elastase activity on sputum neutrophils correlates with severity of lung disease in..., Dittrich [/bib_ref] [bib_ref] Neutrophil extracellular traps in chronic lung disease: implications for pathogenesis and therapy, Keir [/bib_ref]. Interestingly, it was shown that NE has a higher enzymatic activity within the extracellular DNA of sputum from patients with CF [bib_ref] Protease FRET Reporters Targeting Neutrophil Extracellular Traps, Guerra [/bib_ref].
Despite the seemingly negative influence of neutrophils and NET formation on disease severity in the above-mentioned lung diseases, it is also becoming increasingly clear that it is not the NET formation per se that is responsible for worse disease outcomes but rather an imbalance in NET formation and degradation. It was, for example, shown in a murine model of pathogen-induced lung injury that a complete PAD4 deficiency reduced NET formation and, therefore, lung injury but was counterbalanced by an increased bacterial load and inflammation [bib_ref] Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury, Lefrancais [/bib_ref]. Additionally, neutrophils seem to be not only responsible for tissue disruption and early lung damage but also for orchestrating later repair. Here they promote epithelial proliferation and release proteases, needed for the processing of the collagen scar [bib_ref] The emerging role of neutrophils in repair after acute lung injury, Blazquez-Prieto [/bib_ref].
## Nets in autoimmune diseases
Autoimmunity is defined as loss of self-tolerance, meaning that, cellular or humoral immunity or both, respond against endogenous macromolecules and cells. If this response injures cells or tissues, it is usually referred to as autoimmune disease [bib_ref] Definition of human autoimmunity-autoantibodies versus autoimmune disease, Lleo [/bib_ref]. Despite their importance in pathogen clearance, NETs contribute to the development and pathogenesis of various autoimmune diseases such as RA, SLE, Anti-neutrophil cytoplasmic antibodyassociated vasculitis (AAV), anti-phospholipid Syndrome (APS), psoriasis, and others [bib_ref] Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses, Fousert [/bib_ref] [bib_ref] Autoimmune, rheumatic, chronic inflammatory diseases: Neutrophil extracellular traps on parade, Podolska [/bib_ref].
Disruption of the balance between NET formation and degradation by DNases in favor of the formation results in accumulation of the released chromatin and the associated proteins into the extracellular matrix, the interstitium and into the lumina of vessels and ducts. Here these released nuclear constituents can serve as sources of autoantigens that may drive the development of autoantibodies and immune complexes, especially if the material carries post-translational modifications like oxidation [bib_ref] Autoimmunity and oxidatively modified autoantigens, Kurien [/bib_ref] , citrullination [bib_ref] PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling, Desai [/bib_ref] , carbamylation [bib_ref] PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling, Desai [/bib_ref] , or neoepitopes generated after proteolytic cleavage [bib_ref] Autoantigens as substrates for apoptotic proteases: implications for the pathogenesis of systemic..., Rosen [/bib_ref]. These autoantibodies are directed against a plethora of highly variable disease-specific targets, like double-stranded (ds)DNA in SLE [bib_ref] Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers..., Baumann [/bib_ref] , citrullinated proteins in RA [bib_ref] NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in..., Khandpur [/bib_ref] , anti-lysosome-associated membrane protein 2 and anti-MPO in AAV [bib_ref] Neutrophil extracellular trap formation is associated with autophagy-related signalling in ANCAassociated vasculitis, Tang [/bib_ref] [bib_ref] Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, O'sullivan [/bib_ref] , or phospholipids in APS. These autoantibodies can also alter the persistence and immunogenic potential of NETs themselves. Binding of autoantibodies to the chromatin structures stabilizes NETs and prevents their degradation by DNases [bib_ref] Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis, Hakkim [/bib_ref] [bib_ref] Autoantibodies stabilize neutrophil extracellular traps in COVID-19, Zuo [/bib_ref]. Anti-dsDNA-NET complexes of patients with SLE stimulate type I interferon secretion by mononuclear phagocytes, and NF-κB activity in endothelial cells in an Fc-gamma dependent manner. Thus, they enhance inflammatory immune responses and foster the progression of disease and autoimmunity [bib_ref] Autoantibody-dependent amplification of inflammation in SLE, Lou [/bib_ref]. NET formation in capillaries and aggNET formation in larger vessels activate endothelial cells and the coagulation cascade, and promote platelet aggregation. Together with the autoantibodies, NETs build immune complexes and foster thrombogenesis. The occlusion of vessels, especially those of the microvascular bed, can precipitate organ damage, and can even be fatal [bib_ref] At the Bench: Neutrophil extracellular traps (NETs) highlight novel aspects of innate..., Grayson [/bib_ref].
NETs additionally modulate immune responses through interaction with other immune cells and humoral components; NETassociated proteins, especially histones, serve as damageassociated molecular patterns [bib_ref] PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling, Desai [/bib_ref]. NETs activate the inflammasome [bib_ref] Neutrophil extracellular trap-associated protein activation of the NLRP3 inflammasome is enhanced in..., Kahlenberg [/bib_ref] , the complement system via classical, alternative and lectin pathways [bib_ref] Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate..., Leffler [/bib_ref] [bib_ref] Neutrophil extracellular trap formation is associated with autophagy-related signalling in ANCAassociated vasculitis, Tang [/bib_ref] , and the coagulation cascade [bib_ref] At the Bench: Neutrophil extracellular traps (NETs) highlight novel aspects of innate..., Grayson [/bib_ref]. The formation of NETs by splenic neutrophils induces immunoglobulin class switch and, thus, can shape B cell responses [bib_ref] B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the..., Puga [/bib_ref]. In SLE, specific LL37-DNA complexes trigger selfreactive memory B cells for autoantibody production [bib_ref] Netting neutrophils activate autoreactive B cells in lupus, Gestermann [/bib_ref]. NETs lower the activation threshold of T cells [bib_ref] T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune..., Tillack [/bib_ref] , and directly activate production of type I interferons by plasmacytoid dendritic cells (pDCs), the hallmark cytokines of SLE [bib_ref] Netting neutrophils are major inducers of type I IFN production in pediatric..., Garcia-Romo [/bib_ref].
A self-reinforcing loop of dysregulated NET formation and inflammation is created, as some NET-mediated responses drive further neutrophil attraction and NET formation [bib_ref] Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses, Fousert [/bib_ref]. In AAV with microscopic polyangiitis and SLE, this loop is exacerbated by reduced DNase1 activities and the consecutive accumulation and aggregation of NET remnants [bib_ref] Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCAassociated microscopic polyangiitis, Nakazawa [/bib_ref]. Low DNase activities can occur by genetic deficiency [bib_ref] Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus, Al-Mayouf [/bib_ref] , consumption of the enzyme, circulating inhibitors [bib_ref] Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis, Hakkim [/bib_ref] [bib_ref] Extracellular actin in health and disease, Sudakov [/bib_ref] or autoantibodies impairing the activity of DNase1L3 as observed in patients with sporadic SLE [bib_ref] Autoantibody-mediated impairment of DNASE1L3 activity in sporadic systemic lupus erythematosus, Hartl [/bib_ref]. Hence, prevention of NET formation by inhibitors or supporting NET degradation by addition of DNases represent therapeutic approaches for the treatment of NET-driven chronic autoimmune diseases [bib_ref] At the Bench: Neutrophil extracellular traps (NETs) highlight novel aspects of innate..., Grayson [/bib_ref] [bib_ref] Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps, Park [/bib_ref].
## Obstruction of exocrine ducts and stone diseases
The original defensive function of NETs can contribute to the development of obstructive and subsequently inflammatory diseases when ducts of exocrine glands are affected. Neutrophils physiologically patrol the ducts of exocrine organs [bib_ref] Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis, Leppkes [/bib_ref] [bib_ref] Neutrophil extracellular traps initiate gallstone formation, Munoz [/bib_ref] [bib_ref] Neutrophils as main players of immune response towards nondegradable nanoparticles, Bilyy [/bib_ref]. The factors that trigger NET and aggNET formation within the ducts are various, ranging from changes in ion concentrations or pH, to crystal precipitations, bacteria or foreign bodies [bib_ref] Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines, Schauer [/bib_ref] [bib_ref] Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis, Leppkes [/bib_ref] [bib_ref] Neutrophil extracellular traps initiate gallstone formation, Munoz [/bib_ref] [bib_ref] Menage-a-Trois: The Ratio of Bicarbonate to CO2 and the pH regulate the..., Maueroder [/bib_ref] [bib_ref] Neutrophil extracellular traps promote the development and growth of human salivary stones, Schapher [/bib_ref].
Irrespective of the cause, aggNET formation results in reduction of the excretory flow and further accumulation of occlusive material within the NETs. Secretory stasis, which develops gradually, can in turn create an environment in which calculi can form. This process initiates a vicious circle of further obstruction of the ducts, flow rate reduction and inflammation of the adjacent gland, as reported for the pancreas [bib_ref] Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis, Leppkes [/bib_ref] [bib_ref] Neutrophil extracellular traps induce trypsin activation, inflammation, and tissue damage in mice..., Merza [/bib_ref] , the gall bladder [bib_ref] Neutrophil extracellular traps initiate gallstone formation, Munoz [/bib_ref] , tooth-supporting tissues [bib_ref] NETs are double-edged swords with the potential to aggravate or resolve periodontal..., Vitkov [/bib_ref] [bib_ref] The role of neutrophil extracellular traps in periodontitis, Wang [/bib_ref] [bib_ref] Neutrophils orchestrate the periodontal pocket, Vitkov [/bib_ref] , ocular [bib_ref] Aggregated neutrophil extracellular traps occlude Meibomian glands during ocular surface inflammation, Mahajan [/bib_ref] [bib_ref] Neutrophil extracellular traps: current perspectives in the eye, Estua-Acosta [/bib_ref] and salivary glands [bib_ref] Neutrophil extracellular traps promote the development and growth of human salivary stones, Schapher [/bib_ref].
By incorporating crystals, pathogens, cellular debris and viable immune cells, aggNETs serve as a glue that increasingly condenses the material to facilitate tophus and calculus formation, as observed in gouty arthritis [bib_ref] PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling, Desai [/bib_ref] [bib_ref] How neutrophil extracellular traps orchestrate the local immune response in gout, Maueroder [/bib_ref] , and stone diseases like cholelithiasis [bib_ref] To NET or not to NET:current opinions and state of the science..., Boeltz [/bib_ref] , and sialolithiasis [bib_ref] Neutrophil extracellular traps promote the development and growth of human salivary stones, Schapher [/bib_ref] , and meibomian gland disorders.
Gouty arthritis develops as uric acid precipitates in the form of monosodium urate (MSU) crystals in the joints, causing acute inflammation [bib_ref] Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines, Schauer [/bib_ref] [bib_ref] Bonding the foe-NETting neutrophils immobilize the pro-inflammatory monosodium urate crystals, Schorn [/bib_ref]. The crystals are taken up by resident macrophages [bib_ref] Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: a novel link..., Rajamaki [/bib_ref] , followed by NALP3 inflammasome activation [bib_ref] Gout-associated uric acid crystals activate the NALP3 inflammasome, Martinon [/bib_ref] [bib_ref] NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals, Duewell [/bib_ref] , pro-inflammatory cytokine secretion, and abundant neutrophil recruitment [bib_ref] Missing in action-The meaning of cell death in tissue damage and inflammation, Munoz [/bib_ref] [bib_ref] Sodium overload and water influx activate the NALP3 inflammasome, Schorn [/bib_ref]. The latter bind to the crystals and induce NET formation [bib_ref] Crystal-induced neutrophil activation, Popa-Nita [/bib_ref]. During this process, the neutrophils release pro-inflammatory mediators, like tumor necrosis factor α (TNFα) [bib_ref] Neutrophil-mediated inhibition of proinflammatory cytokine responses, Gresnigt [/bib_ref] and interleukin-6 (IL-6), and the neutrophil attractant CXCL8 as well as the elicitor of neutrophil extravasation, CCL3, and CXCL10, which plays a critical role in oxidative stress induced inflammation [bib_ref] How neutrophil extracellular traps orchestrate the local immune response in gout, Maueroder [/bib_ref] [bib_ref] C-C motif chemokine CCL3 and canonical neutrophil attractants promote neutrophil extravasation through..., Reichel [/bib_ref] [bib_ref] CCL7 and CXCL10 orchestrate oxidative stress-induced neutrophilic lung inflammation, Michalec [/bib_ref]. In the presence of high neutrophil counts, the NETs are not sufficiently degraded by DNases. The NETs tend to co-aggregate with the crystals and form tophi that may reach several cm in size. When the pro-inflammatory boost has terminated, the tophus-borne proteases facilitate the resolution of inflammation by degrading certain cytokines and chemokines [bib_ref] Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines, Schauer [/bib_ref] [bib_ref] Missing in action-The meaning of cell death in tissue damage and inflammation, Munoz [/bib_ref]. In vitro, MSU crystals also induce ROS-dependent NET formation [bib_ref] Bonding the foe-NETting neutrophils immobilize the pro-inflammatory monosodium urate crystals, Schorn [/bib_ref]. Cholesterol crystals activate the complement system, and generate C3a and C5a that facilitate further neutrophil influx [bib_ref] Cholesterol crystals induce complement-dependent inflammasome activation and cytokine release, Samstad [/bib_ref]. In contact with the crystals NETs are generated and promote growth of gall stones. Various kinds of crystals, among other sterile stimuli, are potent inducers of neutrophil activation and NET formation [bib_ref] Particles of different sizes and shapes induce neutrophil necroptosis followed by the..., Desai [/bib_ref].
Likewise, sialoliths are formed in patients with sialadenitis. The presence of leucocytes in saliva, along with high concentrations of bicarbonate ions and calcium-based crystals, trigger NET formation, a step that contributes to the development of sialoliths [bib_ref] Neutrophil extracellular traps promote the development and growth of human salivary stones, Schapher [/bib_ref]. The aggregation of NETs then leads to its growth, a common final path in sialolithogenesis. Sialoliths reflect the mechanism of lithogenesis by their layered structure of alternating organic and inorganic components, resulting in an appositional growth of a stone. Once a macroscopic sialolith is formed, salivary gland ducts may be occluded and lose their function [bib_ref] Neutrophil extracellular traps promote the development and growth of human salivary stones, Schapher [/bib_ref] ; chronic inflammations and autoimmunity may occur. Further candidates for NETdriven pathologies are stones in kidney, pancreas, prostate, as well as calcinosis cutis.
NETs in the periodontal crevice precipitate periodontitis The first encounter between neutrophils and dental biofilms occurs within the space delineated by gingival and oral tooth surfaces. This space, referred to as gingival crevice, is filled with a transudate from blood plasma referred to as periodontal crevicular fluid. The latter is characterized by excessive NET formation in periodontitis [bib_ref] NETs are double-edged swords with the potential to aggravate or resolve periodontal..., Vitkov [/bib_ref] [bib_ref] Neutrophils orchestrate the periodontal pocket, Vitkov [/bib_ref]. The crevice contains abundant outer membrane vesicles originating from the dental biofilm that are endocytosed by crevicular neutrophils. These vesicles orchestrate bacterial colonisation, delivery of virulence factors, and pathogenesis. Bacteria use outer membrane vesicles to modulate the host's immune response, which eventually allows the bacteria to evade the immunity of the host [bib_ref] Host immunity and cellular responses to bacterial outer membrane vesicles, Tiku [/bib_ref].
When the pre-activated neutrophils that infiltrate the gingival epithelium enter the periodontal crevice [bib_ref] Connection between periodontitis-induced lowgrade endotoxemia and systemic diseases: neutrophils as protagonists and..., Vitkov [/bib_ref] , they encounter and endocytose a multitude of lipopolysaccharide-filled outer membrane vesicles [bib_ref] Host immunity and cellular responses to bacterial outer membrane vesicles, Tiku [/bib_ref] [bib_ref] Bacterial outer membrane vesicles mediate cytosolic localization of LPS and Caspase-11 activation, Vanaja [/bib_ref]. These vesicles, containing components of the bacteria's outer membrane, had been translocated from the early endosomal compartments into the neutrophils' cytosols, the caspase-4/11/GSDMD signaling pathway is activated and NETs are formed [bib_ref] Neutrophil extracellular traps in host defense, Burgener [/bib_ref] [bib_ref] Caspase-11 contributes to pulmonary host defense against Klebsiella pneumoniae and local activation..., Perlee [/bib_ref] [bib_ref] Beyond inflammasomes: emerging function of gasdermins during apoptosis and NETosis, Chen [/bib_ref] [bib_ref] Innate immunity to intracellular LPS, Rathinam [/bib_ref]. Indeed, caspase-4/11-deficient neutrophils form fewer NETs when compared with wild type controls [bib_ref] Neutrophil extracellular traps in host defense, Burgener [/bib_ref]. Surprisingly, the toll-like receptors (TLR) 2, 3, 4, 7, and 9 are not required for NET formation by neutrophils stimulated with outer membrane vesicles from oral pathogens in vitro [bib_ref] Modulation of neutrophil extracellular trap and reactive oxygen species release by periodontal..., Hirschfeld [/bib_ref]. TLR4 is not necessary for caspase-4/11-mediated NET formation [bib_ref] Innate immunity to intracellular LPS, Rathinam [/bib_ref]. Chloroquine (inhibitor of TLR3, 7, and 9) and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (inhibitor of TLR2 and 4) did not affect NET formation when activated with supernatant of oral pathogens in vitro [bib_ref] Modulation of neutrophil extracellular trap and reactive oxygen species release by periodontal..., Hirschfeld [/bib_ref]. Virtually all crevicular neutrophils appear to be in some stage of NET formation [bib_ref] NETs are double-edged swords with the potential to aggravate or resolve periodontal..., Vitkov [/bib_ref]. Despite the ability of caspase 4/11 to induce NETs independently of NE, MPO and PAD4 [bib_ref] Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps, Chen [/bib_ref] , NE translocation and H3 citrullination was observed in virtually all crevicular neutrophils [bib_ref] NETs are double-edged swords with the potential to aggravate or resolve periodontal..., Vitkov [/bib_ref] and suggests their involvement in crevicular NET formation.
## Net formation in ischemic disease
Many steps of NET formation depend on a sufficient supply with oxygen in tissues [bib_ref] Singlet oxygen is essential for neutrophil extracellular trap formation, Nishinaka [/bib_ref]. Also, changes of pH in the microenvironment influence neutrophil capabilities of NET formation [bib_ref] Menage-a-Trois: The Ratio of Bicarbonate to CO2 and the pH regulate the..., Maueroder [/bib_ref]. Tissue alkalosis favors the release of NETs whereas more acidic conditions lead to reduced NET formation [bib_ref] Menage-a-Trois: The Ratio of Bicarbonate to CO2 and the pH regulate the..., Maueroder [/bib_ref]. Commonly, tissue hypoxia is accompanied by acidification of the microenvironment due to metabolic changes.
In human tissues, hypoxia-inducible factor-1α (HIF-1α) regulates cellular responses to low oxygen [bib_ref] Hypoxia and the regulation of myeloid cell metabolic imprinting: consequences for the..., Sadiku [/bib_ref] [bib_ref] Metabolic reprograming shapes neutrophil functions in severe COVID-19, Borella [/bib_ref]. Under hypoxia, HIF-1α is stabilized and translocated to the nucleus where it induces the transcription of hypoxia-regulated genes [bib_ref] Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity, Walmsley [/bib_ref]. In neutrophils, translocated nuclear HIF-1α upregulates the transcription of NF-kB, prolongs their viability [bib_ref] Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity, Walmsley [/bib_ref] , and promotes degranulation and chemotaxis during hypoxia [bib_ref] Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in..., Mcgovern [/bib_ref] [bib_ref] The impact of hypoxia on neutrophil degranulation and consequences for the host, Lodge [/bib_ref].
In human acute myocardial infarction, an ischemic disease, infiltrating neutrophils show high nuclear HIF-1α content and remain primarily viable [bib_ref] Hypoxia promotes neutrophil survival after acute myocardial infarction, Dolling [/bib_ref]. In contrast, neutrophils with low nuclear HIF-1α protein levels form NETs [bib_ref] Hypoxia promotes neutrophil survival after acute myocardial infarction, Dolling [/bib_ref]. By staying viable in hypoxic tissue neutrophils remain capable of phagocytosis and clear cell debris as an important step of wound healing [bib_ref] Neutrophils from patients with heterozygous germline mutations in the von Hippel Lindau..., Walmsley [/bib_ref].
## The role of nets in cancer
The increased occurrence of NETs in tumor indicates a worse prognosis for cancer patients. NETs are associated with a high histopathological tumor grade, disease progression, metastasis and reduced disease-free and cancer-related survival in various cancer entities [bib_ref] Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer, Stehr [/bib_ref] [bib_ref] A proposed role for neutrophil extracellular traps in cancer immunoediting, Berger-Achituv [/bib_ref] [bib_ref] Lipopolysaccharides increase the risk of colorectal cancer recurrence and metastasis due to..., Wang [/bib_ref] [bib_ref] DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25, Yang [/bib_ref]. The presence of NETs in cancer patients is often indirectly detected through high serum levels of MPO-DNA complexes [bib_ref] Lipopolysaccharides increase the risk of colorectal cancer recurrence and metastasis due to..., Wang [/bib_ref] [bib_ref] Neutrophil extracellular traps drive mitochondrial homeostasis in tumors to augment growth, Yazdani [/bib_ref] [bib_ref] Neutrophil extracellular traps promote the development and progression of liver metastases after..., Tohme [/bib_ref] and to a lesser extent directly in the tumor tissues [bib_ref] Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially..., De Andrea [/bib_ref] [bib_ref] Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer, Stehr [/bib_ref]. A recent study showed that citrullinated NETs in human colon cancer tissues were correlated to the stages 3/4 [bib_ref] Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer, Stehr [/bib_ref].
The functions of NETs in tumorigenesis have been extensively evaluated in murine models. Interestingly, surgical stress and increased LPS levels after postoperative infections were found to induce NET formation concomitantly with an increased occurrence of metastases [bib_ref] Lipopolysaccharides increase the risk of colorectal cancer recurrence and metastasis due to..., Wang [/bib_ref] [bib_ref] Neutrophil extracellular traps promote the development and progression of liver metastases after..., Tohme [/bib_ref]. NETs may directly support metastasis formation by trapping tumor cells at the distant site through the coiled-coil domain containing protein 25 (CCDC25), which can bind to NETs and is expressed on certain cancer cells (colorectal, breast, prostate, liver) [bib_ref] DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25, Yang [/bib_ref]. Moreover, many key processes involved in cancerogenesis and metastasis are co-regulated by NETs. These processes include the establishment of an immune evasive micromilieu, the activation of dormant tumor cells, tumor cell extravasation, angiogenesis and vascular permeability [bib_ref] CXCR1 and CXCR2 chemokine receptor agonists produced by tumors induce neutrophil extracellular..., Teijeira [/bib_ref] [bib_ref] IL-8 mediates a positive loop connecting increased neutrophil extracellular traps (NETs) and..., Yang [/bib_ref]. Moreover, NETs promote a mesenchymal, prometastatic phenotype in breast [bib_ref] Neutrophil Extracellular Traps (NETs) Promote Pro-Metastatic Phenotype in Human Breast Cancer Cells..., Martins-Cardoso [/bib_ref] , colorectal [bib_ref] Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer, Stehr [/bib_ref] , gastric [bib_ref] Neutrophil extracellular traps promote gastric cancer metastasis by inducing epithelialmesenchymal transition, Zhu [/bib_ref] , and pancreatic cancer [bib_ref] Neutrophil extracellular DNA traps promote pancreatic cancer cells migration and invasion by..., Jin [/bib_ref] cell lines by inducing epithelialmesenchymal transition (EMT) associated with an increased migration and invasion of the tumor cells. Specifically, the protein content of NETs seems to be necessary for the EMT induction [bib_ref] Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer, Stehr [/bib_ref]. Altogether, the present knowledge indicates that NETs may be active components in the progression of cancer and putative targets of therapy and prevention.
## Nets in ocular diseases
NETs serve important functions in ocular antimicrobial responses [bib_ref] Frontline Science: Aggregated neutrophil extracellular traps prevent inflammation on the neutrophil-rich ocular..., Mahajan [/bib_ref] [bib_ref] Neutrophil extracellular traps may have a dual role in Pseudomonas aeruginosa keratitis, Zhu [/bib_ref]. However, NETs are also involved in pathologies of the eye. Severe cases of chronic Dry Eye Disease (DED) in graftversus-host disease have been associated with NETs [bib_ref] Neutrophil extracellular traps (NETs) contribute to pathological changes of ocular graft-vs.-host disease..., An [/bib_ref] and hyperosmolar stress is thought to induce NET formation on the ocular surface of patients with DED [bib_ref] Hyperosmolar stress induces neutrophil extracellular trap formation: implications for dry eye disease, Tibrewal [/bib_ref]. NETs are involved in molecular pathological alterations in patients with corneal injuries [bib_ref] Neutrophil extracellular traps may have a dual role in Pseudomonas aeruginosa keratitis, Zhu [/bib_ref] [bib_ref] Anti-inflammatory and anti-fibrotic effects of human amniotic membrane mesenchymal stem cells and..., Navas [/bib_ref]. The choroidal and retinal compartments can also be affected by NETs. Patients with Behcet's disease, a subtype of non-infectious uveitis, showed an increased formation of NETs possibly responsible for the extended vasculitis in this disease [bib_ref] Neutrophils contribute to vasculitis by increased release of neutrophil extracellular traps in..., Safi [/bib_ref]. In vivo and in vitro data of diabetic retinopathy, a major reason for irreversible blindness worldwide, suggest that high blood glucose levels can induce NET formation [bib_ref] Hyperglycemia induces neutrophil extracellular traps formation through an NADPH Oxidase-dependent pathway in..., Wang [/bib_ref] [bib_ref] Diabetes primes neutrophils to undergo NETosis, which impairs wound healing, Wong [/bib_ref].
## Perspective
In addition to the aforementioned diseases, further moonlighting tasks of extracellular chromatin in the form of NETs come to light. There is increasing evidence that NETs contribute to peritoneal adhesions and traumatic spinal cord injury as discussed shortly in the following paragraphs.
## Nets in adhesions
Peritoneal adhesions are a common consequence of serosal repair after almost all abdominal interventions. Adhesions are associated with serious complications such as intestinal obstruction, pelvic pain, and infertility [bib_ref] Adhesiolysis-related morbidity in abdominal surgery, Ten Broek [/bib_ref]. As a result, the quality of life of millions of patients throughout the world is affected. In the US, complications from adhesions cost more than two billion dollar per year and are responsible for more than 5% of hospital readmissions in surgery [bib_ref] Intraurethral irradiation of prostatic cancer using afterloading technique, Fritjofsson [/bib_ref].
Recently, it has been reported that formation and aggregation of NETs worsened primary and secondary intention wound healing. NETs intensified and prolonged the inflammatory phase [bib_ref] Therapeutic targeting of neutrophil extracellular traps improves primary and secondary intention wound..., Heuer [/bib_ref]. Activated neutrophils have been found in burn patients even months after the initiating thermal injury [bib_ref] The pathogenesis of burn wound conversion, Singh [/bib_ref]. DNases reportedly accelerated dermal wound healing in mice and in diabetic patients [bib_ref] Diabetes primes neutrophils to undergo NETosis, which impairs wound healing, Wong [/bib_ref] [bib_ref] Therapeutic targeting of neutrophil extracellular traps improves primary and secondary intention wound..., Heuer [/bib_ref]. Contrary to the notion that inflammation is essential for wound healing, areas with low levels of neutrophils, macrophages and T cells, like oral wounds, healed faster with almost no scarring [bib_ref] Inflammation in surgical wound healing: friend or foe?, Szpaderska [/bib_ref].
Despite its clinical impact, the pathomechanisms of adhesions remain poorly understood. Adhesion formation is a form of peritoneal healing which consists of hemostasis, angiogenesis, and tissue remodeling [bib_ref] Response to pathological processes in the peritoneal cavity-sepsis, tumours, adhesions, and ascites, Stommel [/bib_ref]. The most important factor appears to be the inflammation orchestrated by the innate immune system [bib_ref] Innate immune responses to trauma, Huber-Lang [/bib_ref]. Within hours, neutrophils are recruited to sites of bacterial infection or sterile tissue injury where they start phagocytosis, degranulation, and NET formation [bib_ref] Neutrophil extracellular traps kill bacteria, Brinkmann [/bib_ref] [bib_ref] Response to pathological processes in the peritoneal cavity-sepsis, tumours, adhesions, and ascites, Stommel [/bib_ref].
A recent preprint describes that abundant fibrin-associated NET deposits form murine as well as human adhesions. The digestion of extracellular DNA with DNases abolished the formation of adhesions induced after surgical procedures. These data suggest that NETs form the first scaffold and that fibrin attachment stabilizes the primary structure to eventually form mechanically robust adhesions. Mice with a targeted deletion of PAD4 (Padi4 -/-) or mice treated with DNases showed significantly reduced adhesions.
## Nets in spinal trauma
Traumatic spinal cord injury (tSCI) can result in permanent paralysis of patients. Beside individual sequelae and psychosocial trauma, the socioeconomic burden is substantial [bib_ref] Direct cost of illness for spinal cord injury: a systematic review, Malekzadeh [/bib_ref]. A recent multicenter cohort study showed that tSCI patients are among the most resource intense patient group of $11.193 per admission in Canada [bib_ref] Patient-level resource use for injury admissions in Canada: A multicentre retrospective cohort..., Porgo [/bib_ref]. Treatment strategies include surgical intervention, type and timing of anticoagulation, as well as the use of corticosteroids. The long-term use of the latter has a number of adverse effects. Evidence-based strategies and novel pharmacologic treatment options are lacking [bib_ref] Acute spinal cord injury: Pathophysiology and pharmacological intervention (Review), Zhang [/bib_ref] [bib_ref] A clinical practice guideline for the management of acute spinal cord injury:..., Fehlings [/bib_ref] [bib_ref] Efficacy and safety of methylprednisolone sodium succinate in acute spinal cord injury:..., Fehlings [/bib_ref].
The pathophysiology of tSCI consists of two distinct phases. A primary mechanical injury disrupts axons, neuronal cells, surrounding glia cells, and the blood-brain barrier. Neutrophils accumulate locally within minutes after spinal injury and initiate a phase of secondary damage via the release of NETs, aggravating cell damage and severity of neurological deficits after the initial trauma [bib_ref] Neutrophil extracellular traps exacerbate secondary injury via promoting neuroinflammation and bloodspinal cord..., Feng [/bib_ref]. Secondary injuries include vascular damage, disturbed hemostasis, edema formation, and particularly inflammation [bib_ref] Acute spinal cord injury: Pathophysiology and pharmacological intervention (Review), Zhang [/bib_ref].
A murine model showed that a decrease of neutrophil infiltration by selective inhibition of phosphodiesterase 4 (PDE4-I 0.5 or 1.0 mg/kg s.c. bolus; IC486051) decreased MPO, key markers of oxidative stress, and leukocyte infiltration. This resulted in cellular protection, locomotor improvements (by the Basso-Beattie-Bresnahan scale (BBBS)), and reduced neuropathic pain [bib_ref] A selective phosphodiesterase-4 inhibitor reduces leukocyte infiltration, oxidative processes, and tissue damage..., Bao [/bib_ref]. MPO, a hallmark enzyme of NETs, increased the levels of the pro-inflammatory cytokines IL-6, IL-1ß and TNFα [bib_ref] Post-traumatic inflammation following spinal cord injury, Hausmann [/bib_ref]. Indeed, in a murine model of spinal tSCI, the lack of MPO reduced neutrophil infiltration, pro-inflammatory cytokine expression and apoptosis. Consequently, the motor recovery of the MPO knockout mice was improved (by BBBS). The findings indicate that MPO precipitates secondary injury and exacerbates tissue damage after tSCI, mainly via MPO-derived HOCl mediated apoptotic cell death [bib_ref] Myeloperoxidase exacerbates secondary injury by generating highly reactive oxygen species and mediating..., Kubota [/bib_ref].
Interestingly, intravenous DNase1 treatment (5 mg/kg) of rats one hour after tSCI decreased pro-inflammatory cytokine levels in favor of the anti-inflammatory IL-10. The treatment attenuated the NETs exacerbate secondary injury and promote inflammation in spinal cord injury. Neutrophils infiltrate the lesion core within hours and release NETs which lead to local tissue damage disrupting the blood-spinal cord barrier (BSCB). Resulting tissue hypoxia promotes neuronal apoptosis. Inhibition of peptidylarginine deiminase 4 (PAD4) or degradation of NETs via DNase1 could alleviate damage and promote functional recovery after tSCI.
NET-induced neuroinflammation and the tSCI-associated edema; it reduced glial and fibrotic scarring as quantified 28 days after injury [bib_ref] Neutrophil extracellular traps exacerbate secondary injury via promoting neuroinflammation and bloodspinal cord..., Feng [/bib_ref].
Considering these findings, anti-NET-therapies (PAD4 or DNase1) should be evaluated in further mechanistic studies . The aim of these studies is to transfer in vitro data and the pre-clinical findings of the animal models from bench-to-bedside into patients with tSCI. Topical applications of DNases could be greatly beneficial during surgical decompression of the spine, minimizing secondary injury and scarring. Pharmacological stunning of neutrophils during the extended surgical window or treatment of patients with heparin, which has been shown to dismantle NETs and to limit NET formation [bib_ref] Extracellular DNA traps promote thrombosis, Fuchs [/bib_ref] [bib_ref] Low molecular weight heparins prevent the induction of autophagy of activated neutrophils..., Manfredi [/bib_ref] , are further treatment possibilities.
# Conclusion
Chromatin extrusion and the formation of NETs have been the subject of intense investigation since its discovery in 2004. The characteristic feature of these DNA traps is to limit the spread of invading pathogens, kill or suppress them and preserve tissue integrity. NET formation is evolutionarily preserved and must be considered advantageous (Graphical Abstract). Generally, NETs are beneficial. However, there is also a downside to NETs. In their fight against invaders, infiltrating neutrophils and excessive NET formation trigger many pathological processes. NETs can be considered offenders in immunothrombosis, autoimmune diseases, gout, obstruction of exocrine ducts and stone formation, periodontitis, adhesions, spinal trauma, cancer and ocular disorders (Graphical Abstract). Given the involvement of NETs in various pathologies, several state-of-the-art technologies have identified moonlighting extranuclear DNA-protein complexes. As a result, many interventions, especially regarding different forms of DNase and heparin, have been shown to accelerate the disruption and clearance of NETs. However, effective therapies are required for the future that can block pathways leading to the aberrant formation of NETs and preserve the ejected DNA's protective role.
[fig] Figure 1: Mechanisms of Neutrophil NET formation. Suicidal NET formation: a Chromatin decondensation is mediated by PAD4 and/or NE. [/fig]
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Epidemiologic Parameters for COVID-19: A Systematic Review and Meta-Analysis
Background:The World Health Organization (WHO) declared the coronavirus disease 2019 outbreak to be a public health emergency and international concern and recognized it as a pandemic. This study aimed to estimate the epidemiologic parameters of the COVID-19 pandemic for clinical and epidemiological help.Methods: In this systematic review and meta-analysis study, 4 electronic databases, including Web of Science, PubMed, Scopus, and Google Scholar were searched for the literature published from early December 2019 up to 23 March 2020. After screening, we selected 76 articles based on epidemiological parameters, including basic reproduction number, serial interval, incubation period, doubling time, growth rate, case-fatality rate, and the onset of symptom to hospitalization as eligibility criteria. For the estimation of overall pooled epidemiologic parameters, fixed and random effect models with 95% CI were used based on the value of between-study heterogeneity (I2).Results: A total of 76 observational studies were included in the analysis. The pooled estimate for R0 was 2.99 (95% CI, 2.71-3.27) for COVID-19. The overall R0 was 3.23, 1.19, 3.6, and 2.35 for China, Singapore, Iran, and Japan, respectively. The overall serial interval, doubling time, and incubation period were 4.45 (95% CI, 4.03-4.87), 4.14 (95% CI, 2.67-5.62), and 4.24 (95% CI, 3.03-5.44) days for COVID-19. In addition, the overall estimation for the growth rate and the case fatality rate for COVID-19 was 0.38% and 3.29%, respectively.Conclusion: The epidemiological characteristics of COVID-19 as an emerging disease may be revealed by computing the pooled estimate of the epidemiological parameters, opening the door for health policymakers to consider additional control measures.
2 Cov2) or COVID-19 occurred in Wuhan, China, in December 2019 with a human outbreak (2).
The World Health Organization (WHO) declared the outbreak to be a public health emergency and international concern and recognized it as a pandemic on March 11, 2020 (3). COVID-19 has spread widely in the world and is prevalent in different countries such as China, Italy, United States, France, Spain, Iran, and Germany, with 2,833,697 cases and 197,354 deaths and 807,469 recovered until April 24 2020 worldwide (4). The main rout of transmission of COVID-19 is based on human-to-human transmission via either respiratory droplets, saliva, or close contacts with infected people or aerosol generation procedures during the clinical care of COVID-19 patients (5).
Most COVID-19 infected people (80.9%) are with mild to moderate respiratory syndromes, old people or patients with underlying diseases such as diabetes, cardiovascular disease, cancer, immune deficiency, and respiratory diseases are more at risk to develop the severe (13.8%) and critical (4.7%) form of the disease (6, 7).
Knowledge regarding epidemiological characteristics and parameters of the infectious diseases such as incubation period (time from exposure to the agent until the first symptoms develop), serial interval (duration between symptom onset of a primary case and symptom onset of its secondary cases), basic reproduction number (R 0 ) (the transmission potential of a disease), and other epidemiologic parameters is important for modelling and estimating epidemic trends and also implementing and evaluating preventive procedures .
With regard to COVID-19 pandemic parameters, there are many reports from different countries in the world. For example, about 25.6% to 51.7% of patients have been reported to be asymptomatic or with mild symptoms (12) and 25% to 30% of them have been admitted to the intensive care unit for medical care (13). The case-fatality rate was reported in China and other countries among old patients to be 6% (range: 4%-11%) and 2.3% in all ages . Furthermore, the median incubation period was reported as 5 to 6 days (2-14 ranges) from the WHO, while in China the incubation period was reported up to 24 days (15, 16). Also, according to different mathematical models, R 0 was reported about 6.47 (range, 1. in China, 2.6 in South Korea, and 4.7 in Iran (17-19).
Thus, according to the reports from different countries about epidemiological characteristics of the COVID-19 pandemic, different methods and values of parameters have been observed. Thus, to estimate and forecast the spread of the disease efficiently, we need acceptable and real values for each parameter. The present study was conducted to provide a systematic assessment and estimation of parameters related to COVID-19. This evaluation will help researchers with better prediction and estimation of current epidemic trends.
# Methods
This is a systematic review and meta-analysis to determine the epidemiologic parameters for COVID-19.
## Search strategy
To find relevant studies, a comprehensive literature search of the Web of Science, Medline (PubMed), Scopus, and Google Scholar was performed for observational studies published electronically from early December 2019 up to 23 March 2020.
Two researchers independently searched studies. In the search strategy, English keywords (MeSH termas) and probable combination of them were used. Epidemiologic parameters in infectious diseases are combination of some specific keywords and definitions such as basic reproduction number (R 0 ), serial interval, incubation period, doubling time, growth rate, case-fatality rate, mortality rate, and onset of symptom to hospitalization. These keywords with the Boolean operators ('OR 'and 'AND') were combined in search process.
The terms of search strategies were according to the following keywords: ("novel coronavirus" OR "2019-nCov" OR "COVID-19" OR "SARS-CoV-2") AND ("basic reproduction number" OR "basic reproductive rate" OR "case fatality rate" OR "case fatality ratio" OR "mortality rate" OR "doubling time" OR "growth rate" OR "incubation period" OR "onset of symptom to hospitalization"). Moreover, for comprehensive assessment of available evidences, grey literatures such as web-based nonpeer review studies were searched in this topic as well.
## Study selection
We included studies in accordance with the PRISMA guidelines and standard meta-analysis methods. All of the extracted articles were screened independently by 2 researchers. The abstracts and full texts of the articles were reviewed, duplicate studies were excluded, and relevant articles were selected for data extraction.
## Inclusion and exclusion criteria
The COVID-19 epidemiologic parameters of interest were provided by all epidemiological study designs (observational studies), including peer-reviewed and nonpeerreviewed articles. In addition, irrelevant studies, letters, news, and studies that did not report epidemiologic parameters were excluded.
## Screening and data extraction
All articles were reviewed independently by 4 researchers and information was extracted using a designed checklist (Appendix 1). Extracted items were the first author, year and month of publication, duration of the study, location of the study, type of parameters, point estimate, or mean/median and its confidence interval for epidemiological parameters, and the review status of articles (peerreviewed or not).
## Quality assessment of studies
To assess the quality of the included peer-reviewed and nonpeer-reviewed articles, 2 authors separately assessed the quality of the studies using the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) checklist as a scale for assessing the quality of observational studies. The STROBE includes 22 questions 3 about methodology, aim of study, study design, and frame of original article. Finally, we scored the quality of the study as high if its rating was at least 70% (score of 16 out of 22), medium if its rating was at least 55% (12 out of 22), and poor if its rating was less than 55% (lower 12 out of 22). After that, studies with high and medium quality were included in the analysis. Given that there is a possibility of error in nonpeer review studies, we have analyzed this group of studies separately, regardless of the quality score of these studies.
# Statistical analysis
The "Metan" command was used to apply a fixed or random effects model based on Cochran's Q-test results or a large Higgins and Thompson's I 2 value. Forest plots were used for graphical description of the results. Cumulative meta-analysis was used to examine the R0 trend during different months. However, due to the small number of months in this study, this part was removed from the analysis and results.
In studies that mortality rate was reported, because the denominator was confirmed cases, it was considered a CFR. In addition, for studies that reported the median and interquartile range (IQR), the median was considered equivalent to the mean and the IQR was converted to standard deviation using the "IQR/1.35" formula. Finally, publication bias was examined using the Begg and Egger test. Stata 14 was used for all statistical analyses. Satistical significance was set at P < 0.05.
# Results
Having assessed the quality of relevant studies, 76 observational studies up to March 23, 2020, were included in this study [fig_ref] Figure 1: Flow diagram of the study selection process including publications for the epidemiologic... [/fig_ref]. The majority of studies were done in Wuhan, China. Detailed information of the eligible studies and their characteristics are presented in Appendix 1 (12, 17, 18, 20-92).
## -the overall basic reproductive number (r 0 ) by coun-try and peer review status
Total: The overall R 0 was 2.99 (95% CI, 2.71-3.27) for COVID-19 [fig_ref] Table 1: Overall Estimation of Epidemiologic Parameters for COVID-19 [/fig_ref].
Country: The overall R 0 was 3.23, 1.19, 3.6, and 2.35 for China, Singapore, Iran, and Japan, respectively [fig_ref] Table 1: Overall Estimation of Epidemiologic Parameters for COVID-19 [/fig_ref].
Peer Review Status: The overall R 0 was 2.75 and 3.08 for peer-reviewed and nonpeer-reviewed articles, respectively [fig_ref] Table 1: Overall Estimation of Epidemiologic Parameters for COVID-19 [/fig_ref].
## -overall serial interval (si) by country and peer review status
Total: The overall SI was 4.45 days (95% CI, 4.03-4.87) for COVID-19.
Country: Using the random effect model, the overall SI was 4.46 and 4.64 days for China and Singapore, respectively [fig_ref] Figure 2: Overall serial interval [/fig_ref].
Peer Review Status: The overall SI was 5.3 and 4.39 days for peer-reviewed and nonpeer-reviewed articles, respectively [fig_ref] Figure 3: Overall serial interval [/fig_ref].
## -overall doubling time by peer-review status
Total: The overall doubling time was 4.14 days (95% CI, 2.67-5.62) for COVID-19.
Peer-review Status: The overall doubling time was 3.33 and 4.64 days for peer-reviewed and non-peer reviewed articles, respectively [fig_ref] Figure 4: Overall doubling time for COVID-19 by peer review status NOTE [/fig_ref].
## -overall incubation period by peer-review status
Total: The overall incubation period was 4.24 days (95% CI, 3.03-5.44) for COVID-19.
Peer-review Status: The overall incubation period was 4.03 and 5.82 days for peer-reviewed and nonpeerreviewed articles, respectively [fig_ref] Table 1: Overall Estimation of Epidemiologic Parameters for COVID-19 [/fig_ref].
## -overall estimation for other epidemiologic parameters
The overall estimation for the growth rate and the case fatality rate for COVID-19 was 0.38% and 3.29%, respec- [fig_ref] Figure 5: Overall case fatality rate [/fig_ref]. In addition, the overall time from symptom onset to hospitalization was 5.09 days for COVID-19 [fig_ref] Table 1: Overall Estimation of Epidemiologic Parameters for COVID-19 [/fig_ref].
## -trend of r0 for covid-19
Based on the cumulative meta-analysis, the trend of R 0 had been increasing at first and, then, decreasing in March.
-
## Assessment of publication bias
The Begg and/or Egger tests indicated no publication bias in the parameters of R 0 , serial interval, doubling time, and incubation period (P > 0.05).
# Discussion
In this secondary analysis, we aimed to calculate the pooled estimate of some epidemiological parameters of COVID-19; namely, basic reproductive number (R 0 ), serial interval, doubling time, incubation period, growth rate, CFR, and time from symptom onset to hospitalization. Overall, the estimates were 2.99, 4.45 days, 4.14 days, 4.24 days, 0.38%, 3.29%, and 5.09 days in the same order. The pooled estimated values may differ from the pooled reported values from other studies. This variation is ex- 5 pected because factors such as place of sampling, the sample size, surveillance system, and quality of reported data from countries in emergency condition, and type of data analysis may affect these values. For example, R 0 variations to some extent might be due to different methods calculations, including exponential growth method, maximum likelihood, and Bayesian time-dependent method [bib_ref] Real time bayesian estimation of the epidemic potential of emerging infectious diseases, Bettencourt [/bib_ref] [bib_ref] The R0 package: a toolbox to estimate reproduction numbers for epidemic outbreaks, Obadia [/bib_ref]. The pooled estimated R 0 in this study was nearly accordant with the pooled estimation found by Alimohamadi et al in 2020. (R 0 = 3.32 (95% CI, 2.81 to 3.82) (96).
According to our results, the pooled estimate of CFR 3.29% (95% CI, 2.78-3.81) is lower than SARS-CoV [bib_ref] Epidemiological determinants of spread of causal agent of severe acute respiratory syndrome..., Donnelly [/bib_ref] and MERS-CoV [bib_ref] Estimation of MERS-Coronavirus Reproductive Number and Case Fatality Rate for the Spring..., Majumder [/bib_ref]. Health control policies, medical standard, and detection rate could affect CFR (35). Moreover, the CFR estimate in the early phase of the epidemic might be biased (overestimated). Usually in the early phase, some subclinical cases and patients with mild symptoms may not be detected (detection bias) [bib_ref] Methods for estimating the case fatality ratio for a novel, emerging infectious..., Ghani [/bib_ref] [bib_ref] Case fatality rate of coronavirus disease 2019 (COVID-19) in Iran-a term of..., Rahmanian [/bib_ref].
The pooled estimate of incubation period in 22 studies was 4.24 days (95% CI, 3.03, 5.44), while in a study of Jie Li et al the pooled mean incubation period in 7 studies 6 was 5.3 days (95% CI, 4.5-6.0) [bib_ref] Epidemiology of COVID-19: A systematic review and meta-analysis of clinical characteristics, risk..., Li [/bib_ref]. A valid and precise estimate of incubation period has a pivotal role for duration of quarantine [bib_ref] Incubation period of 2019 novel coronavirus (2019-nCoV) infections among travellers from Wuhan,..., Backer [/bib_ref]. Indeed, understanding the incubation period is beneficial for surveillance and control methods, as well as modeling and monitoring operations [bib_ref] The Incubation Period of Coronavirus Disease 2019 (COVID-19) From Publicly Reported Confirmed..., Lauer [/bib_ref]. Our estimate for overall doubling time-time for a given quantity to double in size or number at a constant growth rate-was 4.14 days (95% CI, 2.67, 5.62). This estimation was in accordance with the study of Zhang et al in 2020 [bib_ref] Meta-analysis of several epidemic characteristics of COVID-19, Zhang [/bib_ref]. The doubling time has an important implication for predicting epidemic. Generally, social distancing, quarantine, and active surveillance are needed to reduce transmission and extend the doubling time. Moreover, the authors tried to estimate pooled measures for the growth rate and the serial interval. These 2 epidemiological parameters are used to estimate the reproduction number. In this study, the serial interval was calculated as 4.45 (95% CI, 4.03-4.87). In addition, the pooled serial interval of COVID-19 obtained in this study was shorter than the pooled serial interval in study of Rai et al (5.19 (95% CI, 4.37, 6.02) [bib_ref] Estimates of serial interval for COVID-19: A systematic review and meta-analysis, Rai [/bib_ref].
As a limitation, all 76 studies (except for 1, Mirjam E Kretzschmar et al)have been conducted in Asia, particularly in Wuhan, China. Some epidemiological parameters in Europe, Africa, and the United States could be different based on control strategies. Hence, distribution of these epidemiological parameters could be more global. Future studies to calculate more generalized pooled estimates, using studies all over the world is recommended.
# Conclusion
The epidemiological characteristics of COVID-19 as an emerging disease may be revealed by calculating the pooled estimate of the disease's epidemiological parameters, paving the way for health policymakers to consider additional control measures.
# Acknowledgment
The authors would like to appreciate all those researchers who helped in conducting this study. Authors' Contributions N.I. was involved in design, data analysis, and participated as a reviewer on the topic. Also, she designed tools for the data extraction. N.T. performed an independent systematic literature search, wrote the first manuscript version, and participated as a reviewer on the topic. Y.M. wrote the first manuscript version and participated as a reviewer on the topic. S.S.GH. performed an independent systematic literature search and participated as a reviewer on the topic. KH.R. participated in project administration. S.S.H.N. as a supervisor, directed every step of the review, revised the results, and versions of the manuscript. All authors read and approved the final version of manuscript.
# Ethical approval
## Conflict of interests
The authors declare that they have no competing interests.
[fig] Figure 1: Flow diagram of the study selection process including publications for the epidemiologic parameters for COVID-19 [/fig]
[fig] Figure 2: Overall serial interval (SI) for COVID-19 by country NOTE: Weights are from random [/fig]
[fig] Figure 3: Overall serial interval (SI) for COVID-19 by peer review status [/fig]
[fig] Figure 4: Overall doubling time for COVID-19 by peer review status NOTE: Weights are from random [/fig]
[fig] References 1: Reusken CBEM, Raj VS, Koopmans MP, Haagmans BL. Cross host transmission in the emergence of MERS coronavirus. Curr Opin Virol. 2016;16:55-62. [/fig]
[fig] Figure 5: Overall case fatality rate (CRF) for COVID-19 NOTE: Weights are from random effects analysis Overall (I-squared = 99.9%, [/fig]
[table] Table 1: Overall Estimation of Epidemiologic Parameters for COVID-19 [/table]
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Aplasia Cutis Congenita of the Scalp with a Familial Pattern: A Case Report
Aplasia Cutis Conginita (ACC) is a condition characterized by congenital absence of skin, usually on the scalp. ACC can occur as an isolated condition or in the presence of other congenital anomalies. Here we describe a case of a 16 days old baby girl with an isolated ACC of the scalp. Her elder two siblings have been diagnosed with ACC with concomitant cardiac or limb anomalies. The patient was managed conservatively until the defect has scarred 6 months later.
# Introduction
Cutis aplasia or Aplasia Cutis Congenita (ACC) is an uncommon and rare congenital abnormality involving variant layers of the skin, mostly as a solitary lesions involving the midline over the skull vertex; and less commonly, underlying periosteum and bone. [bib_ref] Aplasia cutis cerebri with partial acrania-total reconstruction in a severe case and..., Bajpai [/bib_ref] Other sites may occur as well on the chest, abdomen or limbs. [bib_ref] Aplasia cutis congenita: a case of scalp defect repair using two opposing..., O'neill [/bib_ref] [bib_ref] Aplasia cutis congenita: a clinical review and associated defects, Blunt [/bib_ref] Of lesions on the scalp, 20% can involve the cranium, exposing the underlying dura membrane. ACC could also be found in other congenital anomalies; since it was first described in 1767 by Cordon, around 500 similar cases have been reported so far. [bib_ref] Extrait d'une lettre au sujet de trois enfants de la même mère..., Cordon [/bib_ref] Different anomalies were classified into 9 groups based on the number and the presence or absence of other anomalies [fig_ref] Table 1: Classification for ACC [/fig_ref]. [bib_ref] Aplasia cutis congenita: a clinical review and proposal for classification, Frieden [/bib_ref] The lesions in those cases are quite variable, ranging from only local absence of skin to a complete absence of epidermis, subcutaneous tissue, bone, or in some cases the dura. [bib_ref] Congenital midline scalp and skull defects, Lassman [/bib_ref] [bib_ref] Aplasia cutis congenital of the scalp, Martı´nez-Lage [/bib_ref] The incidence of ACC is estimated as 1 per 10,000 live births. [bib_ref] Aplasia cutis cerebri with partial acrania-total reconstruction in a severe case and..., Bajpai [/bib_ref] This failure of formation is frequently more observed in females. The etiology remains unclear so far; however, both genetic and environmental causes have been implicated, including vascular blood supply, a sudden arrest of midline embryological development, failure in neural tube closure, and syphilis have at one time contributed as the cause. [bib_ref] Aplasia cutis cerebri with partial acrania-total reconstruction in a severe case and..., Bajpai [/bib_ref] [bib_ref] Total reconstruction of aplasia cutis congenita involving scalp, skull and dura, Argenta [/bib_ref] Rupture of amniotic membrane in an early time, forming amniotic bands, may also be from the cause. [bib_ref] Aplasia cutis congenita: a clinical review and proposal for classification, Frieden [/bib_ref] A number of teratogenic drugs such as methimazole, is a thiomedazole derivative used as an anti-thyroid agent, have shown to be involved. [bib_ref] Hyperthyroidism during pregnancy, Inoue [/bib_ref] [bib_ref] Aplasia cutis congenita with skull defect in a monozygotic twin after exposure..., Iwayama [/bib_ref] [bib_ref] Aplasia cutis congenital after methimazole exposure in utero successfully treated with basic..., Abe [/bib_ref] [bib_ref] Aplasia cutis congenita following in utero methimazole exposure, Baid [/bib_ref] There are similar cases, classified as being of an autosomal dominant inheritance. 14 Establishing a diagnosis is usually based on the findings of the clinical examination, typically presenting as a hairless, smooth skin defect covered up by atrophic tissue or a dark-colored eschar. Superficial defects presenting as an ulcer are usually treated conservatively. Extensive or deep defects may require reconstruction of the scalp area or the use of bone transplants. However, hairless or scarcely haired scars mandate excision of the lesion and covering it with local flap from the scalp. 9,15-20
## Case report
A 16 days old newborn female from Saudi Arabia was presented to the clinic with a skin defect localized on the scalp since birth. The baby did not suffer from any ailments, and her medical history was unremarkable. Her mother, 32 years old, denied any history of illnesses during her pregnancy, infection or drug intake taking including Non-Steroidal Anti-Inflammatory Drugs (NSAID) or methimazole. She completed 38 weeks of gestation, and delivered her baby via a normal vaginal delivery.
The newborn did not sustain any birth injury and did not suffer from any other abnormalities or feeding difficulties. She did not require any intensive care, and went home from hospital with her mother. Upon local examination, the defect was solitary, localized with an irregular shape and approximately 6×6 cm in size [fig_ref] Figure 1: The newborn presented with skin defect of the scalp with an overlying... [/fig_ref]. The lesion involved the epidermis and the upper dermis only. Neurosurgical team was involved in the care of this patient. A CT Scan of the head was performed, and no deep tissue involvement was noted.
Reconstruction solutions were offered to the parents but they insisted on non-surgical intervention. Therefore, the patient was treated with non-invasive debridement of the lesion and local therapy, including gentle water cleansing and the application of topical antibiotic ointment. 6 months later, the patient has returned for a follow up. Scar tissue has formed over the defect [fig_ref] Figure 2: Six months follow up shoes scar formation over the affected area [/fig_ref]. Family history revealed that none of her parents had the same condition; however, two of her sisters did, and were diagnosed with cutis aplasia. The elder one is currently 4 years of age, with right unilateral terminal reduction of the first and second toes [fig_ref] Figure 3: Unilateral terminal reduction of the right first and second tow [/fig_ref]. The other sister was born prematurely and died shortly after birth due to cardiac anomalies.
# Discussion
ACC occurs as a solitary defect, it can happen alone or in the presence of syndromic congenital anomalies. The involvement of the scalp area may lead to the understanding of the etiology. Upon our review to the literature available, cases were often characterized by an entire absence of skin and subcutaneous tissues. Histologically, we found that most of the lacking tissues belonged to epithelial ectoderm. The condition could be associated with Chromosomal defects. [bib_ref] Placental morphology in spontaneous human abortuses with normal and abnormal karyotypes, Honore [/bib_ref] Some researches showed the association with gestational conditions such as an intrauterine vascular ischemia, amniotic adherences, and viral infections. [bib_ref] Congenital skin defects and fetus papyraceous, Mannino [/bib_ref] [bib_ref] Aplasia cutis congenital and associated disorders: an update, Evers [/bib_ref] A rise of alpha-fetoprotein levels and a distinct amniotic fluid acetylcholine sterase band were found in recent article as markers for ACC. [bib_ref] Aplasia cutis congenita, elevated alphafetoprotein, and a distinct amniotic fluid acetylcholinesterase electrophoretic..., Dror [/bib_ref] Also a number of drugs have been linked to ACC. For example, the use of cocaine during pregnancy can lead to vasoconstriction of the placenta or disruption of the fetus vascularity, causing the cranial defects and anomalies of the central nervous system (CNS). [bib_ref] Adams-Oliver syndrome revisited, Whitley [/bib_ref] Methimazole, a drug used for the treatment of hyperthyroidism, may show some skin affection.
Benzodiazepines use is also linked with ACC. [bib_ref] Aplasia cutis congenital of the scalp, Martı´nez-Lage [/bib_ref] Surgical treatment requires careful preoperative planning. [bib_ref] Diastematomyelia: A clinical review of the naturel history and treatment, Goldberg [/bib_ref] Minimal superficial lesions are treated conservatively to heal gradually by re-epithelialization and result with a hypertrophic or atrophic scar. Tissue expander insertion may be necessary in extensive lesions reaching the scalp; whereas the one deep enough to reach brain, bone and meningeal transplants may be indicated. [bib_ref] Total reconstruction of aplasia cutis congenita involving scalp, skull and dura, Argenta [/bib_ref] [bib_ref] Scalp aplasia cutis congenita: closure by the L-shaped flap, Attalla [/bib_ref] Deep defects overlying the sagittal sinus are indicators for urgent surgical intervention to prevent potentially lethal infections or hemorrhage. [bib_ref] Congenital defect of the scalp: report of a case with fatal termination, Peer [/bib_ref] [bib_ref] Congenital scalp defects: aplasia cutis congenita, Kosnik [/bib_ref] [bib_ref] Aplasia cutis congenita of the scalp, Sargent [/bib_ref] Grafting, [bib_ref] Aplasia cutis congenita of neck and shoulder requiring a skin graft: a..., Bailie [/bib_ref] biological dressings use temporarily, [bib_ref] What syndrome is this?, Lambert [/bib_ref] and silver sulfadiazine dressings while waiting for the processes of skin and bony ingrowth have been published with variable degree of success. [bib_ref] Aplasia cutis congenita: a clinical review and associated defects, Blunk [/bib_ref]
[fig] Figure 1: The newborn presented with skin defect of the scalp with an overlying crust. [/fig]
[fig] Figure 2: Six months follow up shoes scar formation over the affected area. [/fig]
[fig] Figure 3: Unilateral terminal reduction of the right first and second tow. [/fig]
[table] Table 1: Classification for ACC [/table]
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Prevalence and predictors of metabolic abnormalities in Chinese women with PCOS: a cross- sectional study
Background: Polycystic ovary syndrome (PCOS) is a common condition estimated to affect 5.61% of Chinese women of reproductive age, but little is known about the prevalence and predictors in Chinese PCOS patients. This study aimed to determine the prevalence and predictors of the metabolic abnormalities in Chinese women with and without PCOS. Methods: A large-scale national epidemiological investigation was conducted in reproductive age women (19 to 45 years) across China. 833 reproductive aged PCOS women, who participated in the healthcare screening, were recruited from ten provinces in China. Clinical history, ultrasonographic exam (ovarian follicle), hormonal and metabolic parameters were the main outcome measures. Results: The prevalence of metabolic syndrome (MetS) as compared in PCOS and non-PCOS women from community were 18.2% vs 14.7%, and IR (insulin resistance) were 14.2% vs 9.3% (p < 0.001) respectively. After adjusting for age, the indicators (central obesity, hypertension, fasting insulin, SHBG, dyslipinaemia) for metabolic disturbances were significantly higher in PCOS than in non-PCOS groups. Using multivariate logistic regression, central obesity and FAI were risk factors, while SHBG was a protective factor on the occurrence of Mets and IR in PCOS women (OR: 1.132, 1.105 and 0.995). Conclusions: The risk factors of the metabolic syndrome and insulin resistance were BMI and FAI for PCOS women, respectively. The decrease of SHBG level was also a risk factor for insulin resistance in both PCOS and metabolic disturbance.BackgroundPolycystic ovary syndrome (PCOS) is a common condition estimated to affect 5.6% of Chinese women of reproductive age[1,2]. PCOS is associated with reproductive and metabolic disturbances[3]. Based on the Adult Treatment Panel III criteria [4], the prevalence of metabolic syndrome (MetS) has been previously reported to be 1.6% [5], 8.2% [6] and 43% [7] in Czech, Italian and US women with PCOS, and 24.9% in Chinese Hongkong women[8]. The prevalence of MetS in PCOS women showed a marked variation between countries and ethnic groups, probably due to differences in diet, lifestyle and genetic factors. In addition, it was also associated with the investigated population. A meta-analysis supported a greater prevalence of glucose intolerance (IGT), Types 2 diabetes (DM2) and the metabolic syndrome in women with PCOS as compared with women without PCOS[9]. The odds of metabolic disturbance were two to four times as high in PCOS women[9]. The predisposition of PCOS women to various metabolic disturbances, including obesity, IGT, atherogenic dyslipidaemia and hypertension, increased in the long-term risk of DM2 and cardiovascular disease (CVD), which indicated that PCOS carried significant public health implication[10]. Recent evidence also indicated more frequent CVD death in women with PCOS [bib_ref] Health care-related economic burden of the polycystic ovary syndrome during the reproductive..., Azziz [/bib_ref]. An economic evaluation estimated that 40% of the economic costs of PCOS can be attributed to DM2 in the USA, highlighting the need for prevention of long term complications through appropriate screening, diagnosis and intervention for PCOS [bib_ref] Health care-related economic burden of the polycystic ovary syndrome during the reproductive..., Azziz [/bib_ref].
Insulin resistance (IR) is the most likely the pathogenic link between PCOS and MetS. The co-morbidities associated with IR are common to both conditions. All surrogate markers of reduced insulin sensitivity have consistently been found in women with concomitant PCOS and MetS compared to those without MetS, even after controlling for Body Mass Index (BMI) [bib_ref] Screening women with polycystic ovary syndrome for metabolic syndrome, Dokras [/bib_ref]. Some evidence suggested that women with PCOS had a greater predisposition to obesity.
The increased risk of MetS and IR in women with PCOS has raised further interest in identifying the predictors for MetS and IR in these women. The objective of this study was to investigate the prevalence of the metabolic syndrome in PCOS women in China, and detect the predictive risk factors in metabolic disturbances in order to find an effective tool to screen for potential CVD risk factor in Chinese women with PCOS.
# Methods
Anthropometric and metabolic measures were performed for women who participated in the epidemiological study of PCOS around China. All participants were systematically evaluated and written informed consent was obtained from all participants. The study route was in the flowchart, .
The diagnosis of PCOS was based on Rotterdam-PCOS criteria. According to these criteria, PCOS were diagnosed if at least two of the following criteria were present: oligo/amenorrhoea, clinical or biochemical hyperandrogenism and PCO on ultrasonography. Other etiology that could mimic PCOS, like Cushing syndrome, late onset adrenal hyperplasia or androgen producing neoplasm had to be excluded.
Oligomenorrhea and amenorrhea were defined as having fewer than 8 menstrual cycles per year, or the absence of 3 to 6 consecutive menstrual cycles per year.
Clinical hyperandrogenism was defined as the presence of hirsutism (Ferriman-Galwey score ≥6, no oral contraceptive pills were used within three months). Biochemical hyperandrogenism was present if testosterone >2.8 nmol/L or androgen > 10.8 nmol/L, which were the normal range of 95% percentile in the population in our laboratory. PCO was defined as the presence of at least one ovary with 12 or more follicles measuring 2-9 mm in diameter.
MetS was defined according to modified NCEP ATP III guidelines 2005 [bib_ref] Metabolic syndrome-A new world-wide definition. A consensus statement from the international diabetes..., Alberti [/bib_ref]. MetS was diagnosed if at least three of the following five measures were present: (i) waist circumference ≥ 80 cm, (ii)serumtriglyceride ≥ 1.7 mmol/l, (iii) serum high-density lipoprotein (HDL)-cholesterol < 1.3 mmol/l or the use of lipid lowering medication, (iv) blood pressure ≥130/85 mmHg or the use of antihypertensive (v) fasting plasma glucose ≥5.6 mmol/l. IR was evaluated by using the homeostatic model assessment (HOMA-IR: (fasting insulin × fasting glucose)/ 22.5) and 2.69 was selected as the cut-off point [bib_ref] The diagnostic significance of homeostasis model assessment of insulin resistance in metabolic..., Xing [/bib_ref].
A large-scale national epidemiological investigation was conducted in women of reproductive age (19 to 45 years) from the top ten provinces and municipalities in China [bib_ref] Prevalence of polycystic ovary syndrome in women in China: a large community-based..., Li [/bib_ref]. Ten geographically distributed provinces/municipalities contained the major residential population from 30 provinces of Route diagram of the present prevalence study. the whole China. All selected participants were chosen from both rural and urban communities (1:1). Ten provinces were chosen from all 30 provinces according to geographically distributed, then used a multi-layer, stratified sampling method from city or district to communities. A total of 5163 women underwent the investigation from 2008 to 2009. Only 3565 participants completed the panel of physical examination, ultrasound and laboratory data for the classification of PCOS. Twenty interviewers, gynaecologists and ultrasonographcis were trained to administrate the standardized questionnaire and conduct, physical examination and ultrasound following the same Standard Operating Procedure. MetS and IR, the main issue, were considered for analysis. Other disorders such as nonclassical congenital and renal hyperplasia, thyroid dysfunction, and hyperprolactinemia were excluded by examining medical histories and hormones tests, and we deemed the abnormality for prolactin (PRL ≥25 ng/ml) and thyroid dysfunction (TSH <0.5 mU/ Lor >4.78 mU/L), according to the reference value. Other exclusionary criteria included unsolved medical conditions, pregnancy and the use of medications known or suspected to affect fertility or metabolic function with 90 days of the study entry was prohibited. This study was approved by the Ethics Committee of Peking University Third Hospital and conducted based on guidelines of the institutional review boards in each of the participating centres. All subjects provided written informed consent.
## Physical examination, ultrasound and laboratory assessment
Blood pressure was obtained in sitting patients after a five-minute rest. Waist circumference was measured in the standing position, halfway between the lower ribs and the superior anterior iliac spine of the pelvic. The hip circumference was measured at the level of the pubic symphysis. Hirsutism was established by using the modified Ferriman-Galwey score [bib_ref] Hirsutism: implications, etiology, and management, Hatch [/bib_ref]. Transvaginal ultrasonography was performed by the investigators to detect PCO. Height and body weight were measured, and BMI value were calculated. The BMI values were classified following the established criterion, that is normal (23 kg/m 2 ≤ BMI < 25 kg/m 2 ), overweight (25 kg/m 2 ≤ BMI 30 < kg/m 2 ) and obese (BMI ≥ 30 kg/m 2 ) obese in Asian.
## Laboratory tests
All blood samples were collected in the morning after fasting for at least 8 hours. Fasting plasma glucose (FPG) was measured by using finger stick blood glucose method (Roche ACCU-CHEK). Venous blood sample were collected and were immediately centrifuged, then the serum was separated and frozen at −20°C until assayed. All blood samples' test was done in Peking University Third Hospital. Fasting insulin (FI), SHBG, total testosterone (TT), androstenedione (A), PRL and TSH were assessed by chemiluminescence under the Immulite 1000 (DPC, USA). Manufacturer's instructions were followed for preparation, set-up, dilutions, adjustments, assay, and quality control procedures. For all measurements, the inter-assay coefficient of variation was less than 10%, while the intra-assay variation was less than 15%. Fasting cholesterol, triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured by dry slide enzymatic colorimetric assay. The free androgen index (FAI) was calculated using the formula [TT (nmol/L) × 100/SHBG (nmol/L)].
# Statistical analysis
All analyses were performed by using Statistical Product and Service Solutions (SPSS) (version 13.0, spss inc., Chicago, IL, USA). Continuous variables were presented as mean ± SD or median (interquartile range), and analysed using independent sample t-test for normally distributed data or Mann-Whitney U-test for skewed data. Categorical variables were expressed as proportion (percentage) and analysed by X 2 or Fisher's exact tests as appropriate. Multivariate logistic regression was used to examine independent predictors of MetS and IR. The results were expressed as age and BMI adjusted odds ratio (OR) with 95% confidence interval (CI) or two sided pvalue. Baseline characteristics of the different PCOS phenotype and controls were evaluated by analysis of variance. Proportion was compared using the chi-square test. Univariate logistic regression analysis was applied to qualify the association between several clinical and laboratory variables, and the presence of the metabolic disturbances. Variables that appear to be associated were further analysed using multivariate logistic regression analysis with backwards stepwise selection. Statistical significance was considered present if the P-value was <0.05.
# Results
## Prevalence of the metabolic syndrome and insulin resistance in different phenotype pcos in community
The prevalence of the metabolic syndrome and insulin resistance were 19.1% (159/833) and 14.2% (118/833) in women with PCOS. [fig_ref] Table 1: Prevalence of MetS and IR in different phenotypes in PCOS women [/fig_ref] showed the prevalence of MetS in different PCOS phenotype subgroups was similar [fig_ref] Table 1: Prevalence of MetS and IR in different phenotypes in PCOS women [/fig_ref].
## Clinical and biochemical characteristics of the pcos and non-pcos
Of the 3,565 women screened, 833 were diagnosed as PCOS according to Rotterdam-PCOS criteria, and 2,732 were non-PCOS population. Compared women without PCOS, those with PCOS were younger, but there was no difference in central obesity as measured by Waist to hip ratio (WHR). However, since age and central obesity were closely correlated, the indicators for central obesity in PCOS women were significantly higher than in non-PCOS when adjusted for ages.
In addition, T, A, FAI were significantly higher, while SHBG was lower in PCOS than non-PCOS women. After controlling for age and BMI, that was no difference in blood pressure, cholesterol, HDL, LDL, FPG, FI, HOMA-IR between the two groups, with the exclusion of TG which was higher in PCOS.
## Prevalence of the metabolic syndrome and insulin resistance in pcos and non-pcos women
The prevalence of MetS (ATPIII2005) was 19.1% (159/833) and 14.7% (401/2732) (P < 0.001) respectively, in PCOS women and non-PCOS women. In addition, 34.1, 18.6, 13.0, 4.8, 1.3% of women with PCOS had at least one, two, three, four or five components of Mets (ATPIII2005), respectively. The frequency of each component of metabolic syndrome population, in decreasing order, was reduced HDL-C (85.9%), central obesity (84.8%), increased TG (63.4%), increased fasting glucose (55%), and hypertension (45.7%).
The prevalence of IR was 14.2% (118/833) and 9.3% (254/2732) respectively, with P <0.001. After adjusted for age and BMI, the prevalence of IR and MetS between the two groups was still significantly different [fig_ref] Table 2: Comparison of women with and without PCOS [/fig_ref].
We performed stratified analysis, by age and weight category, since age and BMI were well known confounding factors for MetS [fig_ref] Figure 2: Age-stratified prevalence of MetS in PCOS [/fig_ref] and [fig_ref] Figure 3: BMI-stratified prevalence of MetS in PCOS [/fig_ref]. As was expected, the prevalence of MetS increased with increasing age and was significantly higher in PCOS than non-PCOS women in all age groups. IR was also significantly higher with age in PCOS. When compared the prevalence of MetS after stratification into subgroups of normal-weight or overweight/obese, we found the prevalence of MetS Comments: Data were presented as means ± SD, proportion (%) or median (inter-quartile range), and analysed by independent sample t-test, X 2 /fisher's exact tests, or Mann-Whitney U-test as appropriated. BP, blood pressure; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipopreotein cholesterol; FPG, fasting plasma glucose; FI, fasting insulin.
was dramatically increased with increasing BMI, and obese II group had 13-16 fold risk of MetS than normal weight woman, irrespective of the presence of PCOS. In the four groups, the prevalent differences of PCOS and non-PCOS women were not significant except obese I group (p = 0.006) [fig_ref] Figure 3: BMI-stratified prevalence of MetS in PCOS [/fig_ref].
## Characteristics of pcos women with and without metabolic syndrome
As shown in [fig_ref] Table 3: Comparison of PCOS women with MetS and without MetS FI, fasting insulin [/fig_ref] , women with the metabolic syndrome were older and more obese. They also had lower SHBG concentrations, higher FAI and reduced insulin sensitivity as manifested by greater HOMA-IR and higher fasting insulin concentrations than those without MetS. There were no significant differences in serum total testosterone or androgen between those PCOS women with and without MetS. The proportion of HA, IM, PCOM were not significantly different between PCOS woman with MetS and without MetS.
## Predictors of mets and ir by using multivariate logistic regression analysis
Multivariate logistic regression was also performed to examine the independent predictors of Mets and IR in all women with and without PCOS. For women with PCOS, BMI
# Discussion
In the present study, a large-scale national epidemiological investigation was conducted in reproductive age women (19 to 45 years) across China, and total 833 reproductive aged PCOS women who were accordance with the established criterions participated from ten provinces in China. Compared with non-PCOS patients, we found that obesity and FAI were both risk factors for PCOS women, while SHBG acted as a protective factor role on the occurrence of Mets and IR in PCOS women.
## Prevalence of metabolic disturbances in pcos and non-pcos women
The consequences of the polycystic ovary syndrome extended beyond the reproductive axis; as women with PCOS are at substantial risk for the metabolic syndrome. Also, previous studies indicated that 30-40 percent women with PCOS have impaired glucose tolerance, and as many as 10 percent have type 2 diabetes by their fourth decade [bib_ref] Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary..., Ehrmann [/bib_ref] [bib_ref] Prevalence and predictors of risk for type 2 diabetes mellitus and impaired..., Legro [/bib_ref].
In agreement with the previous studies, we demonstrated that a higher prevalence of MetS (ATPIII2005) and IR in women with PCOS than those without PCOS. After controlling for the age and BMI, the difference was still significant, indicating PCOS women had a higher risk of metabolic disturbance compared to the controls.
The prevalence of MetS and IR were significantly higher in PCOS women than non-PCOS women in general population of China. The PCOS phenotype had no effect on the occurrence of MetS. However, previous study concluded that obesity was strongly associated with MetS in adolescent women, whether they had PCOS or not [bib_ref] Prevalence of metabolic syndrome and related characteristics in obese adolescents with and..., Rossi [/bib_ref]. Obesity appeared to be closely associated with PCOS. For example, in the United States, more than half of the patients with PCOS are either overweight or obese [bib_ref] The prevalence and features of the polycystic ovary syndrome in an unselected..., Azziz [/bib_ref]. Our data also showed the PCOS women are more obese than control, but the percentage of overweight/obesity was not significantly different between PCOS (33.9%) women and Non-PCOS women (34.4%). Our study demonstrated that the metabolic disturbance was occurred more often in overweight and obese women. It was interesting that SHBG level was significantly lower in PCOS women compared with non-PCOS women, as well as SHBG level was also significantly lower in MetS than in non-MetS. Since the SHBG had correlation to the IR [bib_ref] Clinical manifestations and insulin resistance (IR) in polycystic ovary syndrome (PCOS) among..., Wijeyaratne [/bib_ref]. This study indicated that SHBG was a well-established marker of insulin resistance in diabetics [bib_ref] Hyperinsulinemia in postpubertal girls with a history of premature pubarche and functional..., Ibanez [/bib_ref] , and low levels had been reported in adolescent girls with premature pubarche (who were known to be at risk for PCOS and insulin resistance) [bib_ref] Molecular and cellular aspects of the insulin-like growth factor I receptor, Leroith [/bib_ref]. Our results regarding the SHBG were protective factor, which was not reported by other research. We interpreted that the variation in SHBG levels might mostly result from the effects of IR/hyperinsulinemia upon hepatic SHBG production. Thus a low level of SHBG might serve as another marker of insulin resistance, and a key factor during the pathogenesis of both PCOS and MetS.
## Relationship of hyperandroginaemia and metabolic abnormalities in pcos
The relationship between hyperandrogenaemia and metabolic abnormalities is controversial. Apridonidze et al. described a higher prevalence of hyperandrogenaemia in women with concomitant PCOS and MetS [bib_ref] Prevalence and characteristics of the metabolic syndrome in women with polycystic ovary..., Apridonidze [/bib_ref] , others study concluded that DHEAS correlated inversely with arterial structure, suggesting possible cardio-protective effects of endogenous DHEAS in women with PCOS [bib_ref] Vascular dysfunction and metabolic parameters in polycystic ovary syndrome, Meyer [/bib_ref]. However, our data, similar studies from Dokras et al. [bib_ref] Screening women with polycystic ovary syndrome for metabolic syndrome, Dokras [/bib_ref] and L.P. Cheung et al. [bib_ref] Goggins WB: Cardiovascular risks and metabolic syndrome in Hong Kong Chinese women..., Cheung [/bib_ref] , all failed to demonstrate any significant differences in serum concentrations in total testosterone and androgen between those PCOS women with or without MetS. Therefore, it appeared that hyperandrogenaemia, by itself, may not directly contribute to the development of MetS in women with PCOS.
Considering the higher prevalence of insulin resistance in the PCOS women, we analyzed the high risk factors including BMI, testosterone, androgen, SHBG and FAI as the predictors of IR in PCOS women.
Currently, it was shown that lipid abnormalities and hyperinsulinaemia persisted despite suppression of androgens with GnRH agonists in hirsute hyperandrogenic women [bib_ref] Lipid and apolipoprotein abnormalities in hirsute women. I. The association with insulin..., Wild [/bib_ref]. The recognition of the role of insulin resistance, rather than hyperandrogenism, as the main culprit in the pathogenesis of MetS in PCOS had an important therapeutic implication. Due to the higher prevalence of MetS and IR in the PCOS women, and serious complications such as CVD and DM2, it was also important to treat these diseases beyond the fertility aspects.
The strength of the study was the large scale investigation, and all the women participated were recruited from the general population. AS compared with other studies on MetS in PCOS, most PCOS women were recruited from the hospital or clinics, which might contribute to potential selection bias and over estimated the risk of MetS and IR in the PCOS cohort. However, there were also several limitations of this study. We measured total testosterone concentration and FAI, which might be a less sensitive marker for hyperandrogenism than free testosterone levels. . In the present study, being the first large scale study in general population, might provide valuable insight towards better understanding of IR and MetS profiles in PCOS women in China.
# Conclusion
In conclusion, the prevalence of MetS and IR were significantly higher in PCOS women than non-PCOS women in Chinese community, and their risk factors were BMI and SHBG. Moreover FAI was also one of risk effects on the insulin resistance in PCOS women since the hyperandroginaemia. The present study facilitated the understand for PCOS pathological characteristics, and was helpful to seek the new target for treatment.
[fig] Figure 2: Age-stratified prevalence of MetS in PCOS. Diagnosis of MetS based on the modified Adult Treatment Penal III (ATP2005). [/fig]
[fig] Figure 3: BMI-stratified prevalence of MetS in PCOS. Diagnosis of MetS based on the modified Adult Treatment Penal III (ATP2005). [/fig]
[table] Table 1: Prevalence of MetS and IR in different phenotypes in PCOS women [/table]
[table] Table 2: Comparison of women with and without PCOS [/table]
[table] Table 3: Comparison of PCOS women with MetS and without MetS FI, fasting insulin: IM: irregular menstruation HA:hyperandrogenism PCOM: pco morpholity. [/table]
[table] Table 4: Predictors of MetS and IR by using multivariate logistic regression analysis [/table]
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Tipping points induced by parameter drift in an excitable ocean model
Numerous systems in the climate sciences and elsewhere are excitable, exhibiting coexistence of and transitions between a basic and an excited state. We examine the role of tipping between two such states in an excitable low-order ocean model. Ensemble simulations are used to obtain the model's pullback attractor (PBA) and its properties, as a function of a forcing parameter γ and of the steepness δ of a climatological drift in the forcing. The tipping time t tp is defined as the time at which the transition to relaxation oscillations (ROs) arises: at constant forcing this occurs at γ = γ c . As the steepness δ decreases, t tp is delayed and the corresponding forcing amplitude decreases, while remaining always above γ c . With periodic perturbations, that amplitude depends solely on δ over a significant range of parameters: this provides an example of rate-induced tipping in an excitable system. Nonlinear resonance occurs for periods comparable to the RO time scale. Coexisting PBAs and total independence from initial states are found for subsets of parameter space. In the broader context of climate dynamics, the parameter drift herein stands for the role of anthropogenic forcing. ; this ES corresponds to τ = 1 300 yr but with a very small periodic perturbation superimposed on the forcing G(t). (a) Evolution of G(t), with β = 0.01 and T = 5 yr, acting on the interval 1 000 ≤ t ≤ 1 050 yr; the periodic perturbation is highlighted by the oval. (b) Evolution of the model's Ψ 3 (t) component: orange lines -for the G(t) in panel (a); cyan lines: for the same G(t) but with β ≡ 0. The latter ES belongs to Exp1 and it corresponds to the filled circle P 4 of Supplementary discussion
## Supplementary information on: "the model and the experimental setup"
The ramp function R τ (t) present in Eq.
(2) and shown in is defined as:
[formula] t < t 1 : R = 0; t 1 < t < t 2 : R = 1 2 1 + cos π t 1 − t τ + 1 ; t 2 < t : R = 1. (S1) [/formula]
The behavior of the model's autonomous version, with α = β = 0 in Eq.
(2), is described by the bifurcation diagram of : it shows the minimum and maximum values attained by Ψ 1 after spinup vs. the control parameter γ. The diagram was obtained in [62] by using a "poor man's continuation" method ([7] and references therein), i.e., by performing many forward time integrations with different values of γ, all initialized from rest, to attain the corresponding asymptotic solutions plotted in the figure herein.
For small γ, i.e., low wind stress, the steady-state, double-gyre solution is stable. A supercritical Hopf bifurcation to a stable limit cycle occurs at γ = 0.348 but the amplitude of the latter is rather small up to γ ∼ = 0.9, after which it increases quite rapidly; the filled circle in the figure indicates the original Hopf bifurcation. The critical value γ = γ c = 1 corresponds to a TP that marks the abrupt transition from small-amplitude limit cycles to large-amplitude self-sustained ROs. Besides these limit cycles, ESs show the existence of stable fixed points emanating from very small basins of attraction. Moreover, Pierini et al.
[66] showed that the system enters the chaotic regime at γ = γ 0 = 1.3475. [66] clarifies the character of the critical transition at γ = 1. For γ = 0.99, shown in , the Ψ 3 (t) time series of two trajectories (red and blue lines) that start out near each other exhibit small-amplitude oscillations in both, with no visible divergence. For γ = 1.01 in panel (c), the two trajectories still coincide, although larger amplitude ROs appear at regular intervals in the periodic signal. Note that, for γ = 1 in panel (b), the two trajectories differ, yielding small-and large-amplitude oscillations that alternate at irregular intervals; in fact, it was shown [66] that chaotic dynamics occurs in an extremely restricted range that separates the two different types of limit cycles. This critical transition may be due to a canard explosion . provides an example of ROs that are excited by noise when the system is in the excitable regime γ s ≤ γ ≤ γ c , where γ s denotes the expectation of the random variable γ s . The forcing is the same as in , with γ = 0.9 (straight purple line), but now a normalized Ornstein-Uhlenbeck noise εζ (t) is added to G(t) of Eq. (2), in which ε = 0.2 and ζ has a decorrelation time T d = 2 yr. A realization of G(t) in the time interval 150-300 yr is shown in (squiggly purple line), while in the system's response is shown by the heavy orange line.
For the sake of comparison, the light orange and black lines show the autonomous response for γ = 0.9 and γ = 1.2, respectively, as already reported in . Thus, the heavy orange line will tend to the light one as the noise vanishes, ε → 0. The excited ROs of are quite similar to the self-sustained ROs of the autonomous case, despite the large difference in the climatological value of the forcing, i.e. γ = 0.9 vs. 1.2. The only difference is in the greater intermittency of the large minima in Ψ 3 (t) in the presence of the noise, but the latter has little effect on the local smoothness of the solution [39]. Naturally, the two-dimensional evolutions of the streamfunction fields ψ(x,t) of the excited and self-sustained ROs are very similar as well; the latter are not shown here, but see [62] and therein.
Finally, for the sake of clarity shows, as an example, the time dependence of the forcing G(t) for the smallest and largest τ-value used in Exp1.
## Supplementary information on: "results: the basic numerical experiment"
In Figs. S5-S8 we investigate in further detail the anomalous behavior noted in . shows the 80 time series of Ψ 3 (t) for the three ESs that correspond to the points P 1 − P 3 of .
At P 1 , τ = 877.5 yr and virtually all the ensemble members yield the first RO at t tp ∼ = 750 yr . At P 2 , τ = 910 yr and t tp changes slightly to t tp ∼ = 770 yr, but only a few ensemble members exhibit their first RO at that TP, cf. : for all the remaining members, the first RO appears much later, at t ∼ = 850 yr. It is this group of members that determines the subsequent evolution of t tp : at P 3 , with τ = 942.5 yr, all the members yield, in fact, the first RO at t tp ∼ = 870 yr, cf. . After P 3 , the behavior is the same as the one before P 1 , until a further jump at τ ∼ = 1 140 yr occurs due to a similar phenomenon.
It follows that this anomalous dependence of t tp and G tp upon τ is associated with the sudden appearance of two clusters in the ES and the equally sudden disappearance of the first cluster, which leads to an abrupt forward shift of the TP, as defined in this study. The coexistence of such clusters suggests the possibility of two distinct local PBAs, as discussed in . The authors showed there that the dissipative model of Eq. (1) possesses a unique global PBA but suggested that the numerically found coexistence of two local PBAs with distinct stability properties was consistent with the well-studied simpler case of a Van der Pol-Duffing oscillator.
## 8/9
Another interesting aspect that is worth considering is the sudden lack of TPs for τ ∼ 1 300 yr. In , P 4 denotes the ES (orange lines of with the highest τ-value, τ = 1 267.5 yr, that still yields ROs. For greater values, all trajectories collapse onto an extremely small-amplitude limit cycle, as illustrated by the cyan lines of , which corresponds to τ = 1 300 yr. This filamentary PBA is, however, nonlinearly unstable, as seen in the perturbed ES shown in . Here, a very small-amplitude periodic perturbation acting over a short time interval on the forcing G(t), cf. , makes the model transition from the cyan to the orange trajectories in .
The cyan and orange ensembles of trajectories in appear therewith as estimates of two coexisting PBAs with very different stability properties. Additional numerical experiments (not shown) confirm that not even much stronger perturbations cause the system to shift from the RO-PBA (orange) to the small-size, sinusoidal PBA (cyan).
The four red bars of refer to the ESs reported in . For τ = 97.5 yr, the very small value ofC 0.01 indicates a notable phase spread of the ROs , while the higher value for τ = 520 yr indicates a more limited spread . For τ = 552.5 yr, the valueC = 0.86 indicates an appreciable clustering , which disappears for a small increment of τ, up to τ = 568.75 yr, to yield a very smallC = 0.063 in .
Finally, shows the dependence of the numberC of trajectory clusters on τ for Exp2 (see the main text for a discussion).
## Supplementary information on: "sensitivity to periodic perturbations (exp3-exp6)"
Figure S10 provides interesting results concerning the dependence of the phase distribution of ROs on the presence and amplitude of periodic forcing. for Exp3 displays a more uniform distribution ofC-values than in for Exp1, with the high values being reduced and the very small ones being increased. Note that the disappearance of values close to unity at τ 1 300 yr is obvious in view of the instability of the corresponding filamentary PBAs, as seen in . However, for a higher perturbation amplitude, several cases with a fairly high value ofC appear, as found in for Exp4. Hence, although t tp and G * tp are almost equal in Exp3 and Exp4, cf. , the RO phases in Exp4 are more prone to cluster into STs than in Exp3.
Another intriguing result emerges when comparingC of Exp5 and Exp6 in Figs. S10(c,d) with that of Exp2 in . The TIID noticed in Exp2 is generally preserved in the presence of periodically perturbed forcing up to τ 800 yr; on the other hand,C undergoes a notable reduction for higher values of τ. Again, the perturbation amplitude does not appear to play a major role inasfar as this effect is concerned: compare panels (c) and (d) of . We thus conclude that TIID is a robust feature for high-to-moderate forcing drift rates δ but tends to disappear for lower α (Exp3 and Exp4, with α = 0.3), and hence δ .
Finally, we complement the analysis with the study of four particularly interesting ESs for each one of Exp2, Exp5 and Exp6; these ESs are carried out in steps of ∆τ = 32.5 yr and plotted in in three columns of four panels each. For Exp2, with no periodic forcing, in the first column of , we notice four interesting features:
- For panels (a,b,d), the TP is reached after the end of the ramp, as can also be seen in , since the red line that corresponds to Exp2 there lies in the gray area. A TP being reached after the forcing strength has increased and achieved its maximum level appears to be novel, and it deserves further investigation elsewhere.
- In panel (b), two single trajectories emerge (see the corresponding definition after Eq. (5)). This represents the coexistence of two disjoint PBAs with distinct properties: nearly constant vs periodic.
- For τ = 97.5, 130 yr in panels (c) and (d), TIID occurs; this is also revealed by the corresponding valuesC = 1 in .
- For τ = 97.5 yr in panel (c), in particular, the filamentary nature of the PBA implies the absence of a TP; this is also revealed by the absence of a filled circle corresponding to that value of τ on the red line of .
The features listed above, though, are not robust, as shown in the second column of . The only striking feature that survives a perturbation by the small periodic component present in Exp5 is the occurrence of TIID, as confirmed by the values ofC ∼ = 1 for the corresponding τ-values in .
Finally, if the periodic perturbation's amplitude doubles, as shown for Exp6 in the third column of , no substantial changes in the TP timing t tp occur, but a novel feature does emerge: namely, the ROs undergo irregular oscillations, while still maintaining TIID.
Moreover, 160-180 yr after t tp , phase coherence is rapidly lost. Remarkably, this coherence loss occurs while the forcing is constant in time, having passed the ramp in R τ (t) already; once again, this rather unexpected feature deserves further analysis in future work.
[fig] Figure S 1, Figure S 3, Figure S 5: Bifurcation diagram of the autonomous system (α = β = 0) showing the range of variability of Ψ 1 vs the normalized forcing amplitude γ; the filled circle indicates the Hopf bifurcation at γ = 0.348, while the black dashed line indicates the unstable steady state. The light dashed vertical line shows the sharp transition from small-amplitude oscillations to ROs that occurs at γ = γ c = 1. Adapted from [62] ( c American Meteorological Society; used with permission). Supplementary Figure S 2. Critical transition in the autonomous system at γ = γ c = 1. Evolution of Ψ 3 (t) for two trajectories (blue and red lines) started at nearby points for (a) γ = 0.99, (b) γ = 1, and (c) γ = 1.01. Adapted from [66]. . Relaxation oscillation (RO) excitation by noise. (a) Time dependence of the factor G(t) in the external forcing with γ = 0.9 (solid purple line) plus a red noise εζ (t) with ε = 0.2 and T d = 2 yr. (b) Heavy orange line: model response in terms of Ψ 3 . The light orange and black lines show the autonomous response for γ = 0.9 and γ = 1.2, respectively, as already reported in Figs. 2(b,d). t=32.5 yr t=1316.25 yr Supplementary Figure S 4. Time dependence of the factor G(t) in the forcing for the smallest and largest τ-value used in Exp1. Nonmonotonic TP-dependence on ramp duration τ. This dependence is illustrated by the Ψ 3 (t) time series for the ESs of Exp1, with three distinct τ-values: (a) τ = 877.5 yr; (b) τ = 910 yr; and (c) τ = 942.5 yr.These three cases correspond to the filled circles for the points P 1 − P 3 inFig. 5(b). [/fig]
[fig] t=1267. 5, Figure S 7: yr (orange) t=1300 yr (cyan) Supplementary Figure S 6. Same as Figs. S5(a-c), but for τ = 1 267.5 yr (orange lines) and τ = 1 300 yr (cyan lines).The orange ES corresponds to the filled circle P 4 inFig. 5(b); the cyan ES is unstable, as shown inFig. S7. Nonlinear instability of an ES with the parameter values of Exp1, cf. [/fig]
[fig] Figure S 8, Figure S 9: Same as Figs. S5 and S6 but for the ESs of Exp1 corresponding to the red bars in Fig. 7: (a) τ = 97.5 yr; (b) τ = 520 yr; (c) τ = 552.5 yr; and (d) τ = 568.75 yr. Same as Fig. 7 but for Exp2. [/fig]
[fig] Supplementary: Figure S 10. Same asFig. 7but for Exp3-Exp6. [/fig]
|
MRI findings guiding selection of active surveillance for prostate cancer: a review of emerging evidence
Active surveillance (AS) for prostate cancer (PCa) is generally considered to be a safe strategy for men with low-risk, localized disease. However, as many as 1 in 4 patients may be incorrectly classified as AS-eligible using traditional inclusion criteria. The use of multiparametric magnetic resonance imaging (mpMRI) may offer improved risk stratification in both the initial diagnostic and disease monitoring setting.We performed a review of recently published studies to evaluate the utility of this imaging modality for this clinical setting. An English literature search was conducted on PubMed for original investigations on localized PCa, AS, and magnetic resonance imaging. Our Boolean criteria included the following terms: PCa, AS, imaging, MRI, mpMRI, prospective, retrospective, and comparative. Our search excluded publication types such as comments, editorials, guidelines, reviews, or interviews. Our literature review identified 71 original investigations. Among these, 52 met our inclusion criteria. Evidence suggests mpMRI improves characterization of clinically significant prostate cancer (csPCa) foci, and the enhanced detection and riskstratification afforded by this modality may keep men from being inappropriately placed on AS. Use of serial mpMRI may also permit longer intervals between confirmatory biopsies. Multiple studies demonstrate the benefit of MRI-targeted biopsies. The use of mpMRI of the prostate offers improved confidence in riskstratification for men with clinically low-risk PCa considering AS. While on AS, serial mpMRI and MRItargeted biopsy aid in the detection of aggressive disease transformation or foci of clinically-significant cancer undetected on prior biopsy sessions.
# Introduction
In 2017 there will be an estimated 161,000 new cases of prostate cancer (PCa) in the United States. This represents nearly 20% of new cancer diagnoses, the most common among non-cutaneous neoplasms in men [bib_ref] Cancer Statistics, Siegel [/bib_ref]. Following the introduction of prostate specific antigen (PSA) screening in the 1990s, the incidence of PCa has steadily risen while cancer-specific mortality has declined considerably [bib_ref] Limitations of basing screening policies on screening trials: The US Preventive Services..., Etzioni [/bib_ref].
The former has been attributed to increased detection of low-risk, localized disease which may not pose a significant threat to a patient's longevity. Consequently, the appropriateness of aggressive intervention in the setting of increased detection has been brought under question [bib_ref] Gleason 6 Prostate Cancer: Translating Biology into Population Health, Eggener [/bib_ref]. For those individuals with low-risk, localized disease, a less invasive management approach may be more appropriate to avoid the potential negative impact the effects of medications, radiation therapy, and surgery may have on a Review Article MRI findings guiding selection of active surveillance for prostate cancer: a review of emerging evidence patient's quality of life [bib_ref] 20-year outcomes following conservative management of clinically localized prostate cancer, Albertsen [/bib_ref].
Close monitoring of low-risk patients by active surveillance (AS) includes serial serum PSA level assessments, digital rectal examinations, and transrectal ultrasound (TRUS) biopsies, and more recently, multiparametric magnetic resonance imaging (mpMRI). The decision to place a patient on AS, and ultimately determining when to depart from AS, is challenging due to limitations in both disease monitoring and reliable risk stratification criteria [bib_ref] Magnetic Resonance Imaging-Transrectal Ultrasound Guided Fusion Biopsy to Detect Progression in Patients..., Frye [/bib_ref] [bib_ref] Prostate Cancer Imaging and Biomarkers Guiding Safe Selection of Active Surveillance, Glaser [/bib_ref]. It is estimated that as many as 1 in 4 patients may be improperly placed on AS based on data supporting the undergrading and underassessment of tumor volume on systematic prostate biopsy for diagnosis and risk-stratification [bib_ref] Upgrading and downgrading of prostate needle biopsy specimens: risk factors and clinical..., Freedland [/bib_ref]. Indeed, nearly half of men initially placed on AS will at some point have pathologic progression and require oncologic intervention [bib_ref] Intermediate and Longer-Term Outcomes From a Prospective Active-Surveillance Program for Favorable-Risk Prostate..., Tosoian [/bib_ref]. Therefore, it is incumbent upon clinicians to improve risk-stratification and cancer surveillance protocols to minimize patient oncologic risk.
Over the past decade, mpMRI has emerged as a reliable diagnostic and monitoring adjunct for men on AS [bib_ref] Dramatic increase in the utilization of multiparametric magnetic resonance imaging for detection..., Oberlin [/bib_ref] [bib_ref] Role of Multi-Parametric Magnetic Resonance Image and PIRADS Score in Patients with..., Almeida [/bib_ref] [bib_ref] In-parallel comparative evaluation between multiparametric magnetic resonance imaging, prostate cancer antigen 3..., Porpiglia [/bib_ref]. This approach affords better visualization of the prostate and surrounding structures than traditional TRUS imaging used primarily for systematic tissue sampling at the time of biopsy [bib_ref] Multiparametric magnetic resonance imaging for prostate cancer: A review and update for..., Yoo [/bib_ref]. Specifically the improvements in magnetic resonance imaging technology and optimization of functional imaging sequence techniques such as diffusion weighted imaging (DWI) and volumetric estimation algorithms have permitted superior tumor characterization [bib_ref] Risk stratification of prostate cancer utilizing apparent diffusion coefficient value and lesion..., Salami [/bib_ref] [bib_ref] Nine-year Follow-up for a Study of Diffusion-weighted Magnetic Resonance Imaging in a..., Henderson [/bib_ref] [bib_ref] Diffusion-weighted magnetic resonance imaging for prediction of insignificant prostate cancer in potential..., Kim [/bib_ref].
While variable, mpMRI sequences typically consist of high-resolution T2-weighted imaging, diffusion-weighted imaging (DWI) with apparent diffusion coefficient (ADC) mapping, and dynamic contrast-enhanced imaging (DCEI) [bib_ref] Multiparametric magnetic resonance imaging for prostate cancer: A review and update for..., Yoo [/bib_ref] [bib_ref] Nine-year Follow-up for a Study of Diffusion-weighted Magnetic Resonance Imaging in a..., Henderson [/bib_ref]. T2-weighted imaging reflects the generalized water content of tissues which is applicable since normal benign prostatic tissue is water-rich, cancerous tissue is water-poor, and the prostatic capsule is commonly well-defined [bib_ref] Multiparametric magnetic resonance imaging for prostate cancer: A review and update for..., Yoo [/bib_ref]. DWI quantifies the degree of water diffusing through tissues, and similarly aids in differentiating benign tissue from more densely packed malignant prostate tissue [bib_ref] Multiparametric magnetic resonance imaging for prostate cancer: A review and update for..., Yoo [/bib_ref]. The ADC, a measurement of the impedance of water molecule diffusion based on DWI sequences, further distinguishes benign from malignant prostatic tissue with a quantified value that can be mapped as a surrogate image [bib_ref] Multiparametric magnetic resonance imaging for prostate cancer: A review and update for..., Yoo [/bib_ref]. DCEI evaluates tissue vasculature and differentiates benign from malignant tissue based on the altered patterns of angiogenesis observed in cancerous lesions [bib_ref] Prostate cancer: evaluation of vascular characteristics with dynamic contrast-enhanced T1-weighted MR imaging--initial..., Buckley [/bib_ref]. We therefore performed a review of recently published literature to characterize the emerging evidence in support of using mpMRI to properly select patients for AS.
# Methods
An English literature search was conducted on PubMed for original investigations on localized PCa, AS, and MR imaging. All articles published within the past few years (January 1 st , 2014 through November 28 th , 2017) were considered. Our Boolean criteria included the following terms: "PCa", "AS", "imaging", "MRI", "mpMRI", "prospective", "retrospective", and "comparative". Our search excluded publication types including commentaries, editorials, guideline statements, review articles, or interviews. We identified 71 original studies. Among these, 52 publications met our final inclusion criteria. A total of 28 studies evaluated the usefulness of mpMRI for detection of clinically significant prostate cancer (csPCa) in the setting of AS. We identified 18 studies examining the role of mpMRI specifically in guiding prostate biopsy in the context of AS management protocols. Six studies considered using serial mpMRIs in place of routine biopsy to survey men on AS meeting certain risk criteria.
## Review
## Improved detection of significant pca
We identified multiple studies that demonstrated the value of using mpMRI during the initial diagnostic workup to classify patients as AS-eligible better than traditional criteria alone without advanced imaging [bib_ref] In-parallel comparative evaluation between multiparametric magnetic resonance imaging, prostate cancer antigen 3..., Porpiglia [/bib_ref] [bib_ref] Magnetic Resonance Imaging Targeted Biopsy Improves Selection of Patients Considered for Active..., Ouzzane [/bib_ref] [bib_ref] Further reduction of disqualification rates by additional MRItargeted biopsy with transperineal saturation..., Radtke [/bib_ref]. Ouzzane et al. reviewed 281 patients who were initially deemed appropriate for AS based on clinical and biopsy results. They found 10% of the cohort was later reclassified as ineligible for AS following mpMRI and subsequent biopsy of clinically occult lesions found and sampled based on mpMRI results [bib_ref] Magnetic Resonance Imaging Targeted Biopsy Improves Selection of Patients Considered for Active..., Ouzzane [/bib_ref]. Porpiglia et al. went one step further to suggest the use of mpMRI alone may reliably predict pathologically significant disease without confirmatory biopsy [bib_ref] In-parallel comparative evaluation between multiparametric magnetic resonance imaging, prostate cancer antigen 3..., Porpiglia [/bib_ref].
DWI sequencing, which affords calculation of the ADC values for different imaging voxels, gives providers an additional tool to evaluate suspicious prostatic lesions. In a retrospective study of 86 AS-eligible patients who eventually underwent radical prostatectomy, Henderson et al. demonstrated a low ADC, defined as lower than their single-institution median value, may independently predict time to undergoing curative intervention and/or detection of adverse histology [bib_ref] Nine-year Follow-up for a Study of Diffusion-weighted Magnetic Resonance Imaging in a..., Henderson [/bib_ref]. Morgan et al. compared interval suspicious lesion growth and ADC change in a cohort of 151 men on AS who underwent serial mpMRI over a median two-year interval. They found tumor growth was inversely correlated with change in the ADC, and therefore a significant decrease in ADC may be a sign of impending AS failure based on PCa progression [bib_ref] Monitoring Tumor Volume in Patients With Prostate Cancer Undergoing Active Surveillance: Is..., Morgan [/bib_ref].
As processing software continues to improve, there is emerging evidence to suggest even greater PCa detection sensitivity may be achieved with mpMRI. Sharif-Afshar et al. conducted a pilot trial comparing standard versus a novel high resolution DWI (HR-DWI) sequencing software in the evaluation of biopsy-confirmed PCa lesions. This technique uses smaller voxel size and also achieves a greater signal-to-noise ratio for greater spatial resolution. They found a 5-fold improvement in spatial resolution with a nearly 35% greater sensitivity in detecting biopsy-proven csPCa [bib_ref] Prospective Pilot Trial to Evaluate a High Resolution Diffusion-Weighted MRI in Prostate..., Sharif-Afshar [/bib_ref]. Of note, use of 5-alpha-reductase inhibitors does not appear to alter the ability to detect cancerous tissue on prostate mpMRI [bib_ref] Prostate cancer detection using quantitative T2 and T2 -weighted imaging: The effects..., Giganti [/bib_ref].
It has been shown that automated calculations of lesion volume on mpMRI may correspond with PCa presence [bib_ref] Nine-year Follow-up for a Study of Diffusion-weighted Magnetic Resonance Imaging in a..., Henderson [/bib_ref] [bib_ref] Comparison of semi-automated and manual methods to measure the volume of prostate..., Marin [/bib_ref]. Marin et al. found that using semi-automated sizing algorithms to measure tumor dimensions reliably correlates with actual tumor diameter on final pathology [bib_ref] Comparison of semi-automated and manual methods to measure the volume of prostate..., Marin [/bib_ref]. Stensland et al. retrospectively evaluated 1,633 patients with available mpMRIs who underwent radical prostatectomy, and concluded tumor lesions <5 mm on mpMRI most likely represent clinically-insignificant disease on final pathology [bib_ref] Are magnetic resonance imaging undetectable prostate tumours clinically significant? Results of histopathological..., Stensland [/bib_ref]. However in a retrospective study of 118 patients, Dianat et al. observed 8.3% of men with mpMRI invisible tumors harbored csPCa [bib_ref] Magnetic resonance-invisible versus magnetic resonance-visible prostate cancer in active surveillance: a preliminary..., Dianat [/bib_ref]. In a separate study of 298 patients by Park et al., 14% of AS-eligible patients without an identifiable lesion had their PCa upgraded on final pathology following radical prostatectomy, but just one was found to have a positive surgical margin and no patients had greater than either form of Gleason 7 disease (28). Sahibzada et al. reached a similar conclusion in their retrospective cross-sectional validation study of 100 patients, and suggest mpMRI may have greater reliability in the post-TRUS biopsy surveillance setting [bib_ref] Validating multiparametric MRI for diagnosis and monitoring of prostate cancer in patients..., Sahibzada [/bib_ref].
## Incorporation of pirads into as protocols
Over the past ten years, the Prostate Imaging Reporting and Data System (PIRADS) has become an increasingly useful tool for evaluating suspicious prostatic neoplasms [bib_ref] Diagnostic accuracy of magnetic resonance imaging (MRI) prostate imaging reporting and data..., Grey [/bib_ref] [bib_ref] Results of Targeted Biopsy in Men with Magnetic Resonance Imaging Lesions Classified..., Venderink [/bib_ref] [bib_ref] The performance of PI-RADSv2 and quantitative apparent diffusion coefficient for predicting confirmatory..., Nougaret [/bib_ref].
Suspicious lesions are rated on a five-point Likert scale with a score of 5 being the most concerning for a malignant tumor [bib_ref] ESUR prostate MR guidelines 2012, Barentsz [/bib_ref] [bib_ref] PI-RADS Prostate Imaging -Reporting and Data System: 2015, Version 2, Weinreb [/bib_ref]. Venderink et al. retrospectively evaluated 1,000 patients on AS, and when compared to PSA density, they found a PIRADS score of ≥3 better predicts significant PCa on repeat biopsy [bib_ref] Results of Targeted Biopsy in Men with Magnetic Resonance Imaging Lesions Classified..., Venderink [/bib_ref]. In a study by Grey et al. that retrospectively reviewed 201 men on AS who underwent both mpMRI and prostate biopsy, they similarly concluded that those with a PIRADS score of <3 could safely forgo a subsequent repeat biopsy. However, 2.3% of the men with PIRADS <3 lesion(s) still harbored csPCa (Gleason pattern 4 or ≥6 mm cancer core length), bringing into question whether it is acceptable to potentially 'miss' a small number of presumably indolent cancers in the population and rely on other parameters in the surveillance protocol to pick these up at a later time point [bib_ref] Diagnostic accuracy of magnetic resonance imaging (MRI) prostate imaging reporting and data..., Grey [/bib_ref]. A different study by Porpiglia et al. retrospectively analyzed 126 patients who underwent radical prostatectomy, and found incorporating PIRADS into the existing Epstein and/or Prostate Cancer Research International Active Surveillance (PRIAS) criteria would have increased csPCa detection by 5% and 7%, respectively [bib_ref] Multiparametric magnetic resonance imaging and active surveillance: How to better select insignificant..., Porpiglia [/bib_ref]. We identified three additional original investigations that compared a widely-accepted AS protocol with a predictive nomogram incorporating PIRADS, and they also demonstrated a significant improvement in riskstratification [bib_ref] Value of 3-T multiparametric magnetic resonance imaging and magnetic resonance-guided biopsy for..., Hoeks [/bib_ref] [bib_ref] Accuracy of multiparametric magnetic resonance imaging in confirming eligibility for active surveillance..., Stamatakis [/bib_ref] [bib_ref] Factors predicting prostate cancer upgrading on magnetic resonance imaging-targeted biopsy in an..., Lai [/bib_ref].
More recently, a proposed PIRADS version 2 (PIRADSv2) was developed to capture the increasingly complex MRI characterization of a single lesion with the application of multiple sequences such as DWI and DCEI for interpretation [bib_ref] The performance of PI-RADSv2 and quantitative apparent diffusion coefficient for predicting confirmatory..., Nougaret [/bib_ref] [bib_ref] PI-RADS Prostate Imaging -Reporting and Data System: 2015, Version 2, Weinreb [/bib_ref] [bib_ref] Updated prostate imaging reporting and data system (PIRADS v2) recommendations for the..., Vargas [/bib_ref]. Studies comparing it to the original PIRADS algorithm are underway in the setting of AS. Yim et al. recently found that using the PIRADSv2 scoring system may reliably classify suspicious lesions as clinically-insignificant PCa, therefore permitting safe selection for AS [bib_ref] Clinically insignificant prostate cancer suitable for active surveillance according to Prostate Cancer..., Yim [/bib_ref]. Lim et al. found patients with PIRADSv2 scores ≥3 on mpMRI with a prior TRUS biopsy of 3+4=7 PCa have a higher chance of pathological upgrading at the time of radical prostatectomy. Therefore, the PIRADSv2 system could predict AS failure in this select patient population [bib_ref] Prostate Imaging Reporting and Data System, Version 2, Assessment Categories and Pathologic..., Lim [/bib_ref].
Alternatively, Nougaret et al. suggest PCa may be overlooked as often as 5% of the time when using PIRADSv2 scores of ≥3 as a threshold cutoff [bib_ref] The performance of PI-RADSv2 and quantitative apparent diffusion coefficient for predicting confirmatory..., Nougaret [/bib_ref]. In addition, there is concern that central zone (CZ) lesions may not be accurately characterized when using the PIRADS algorithm. In a review of 73 patients who underwent MRIfusion biopsy, only two (7.7%) of 26 CZ lesions that were designated PIRADS ≥3 actually contained clinically-significant disease [bib_ref] Central zone lesions on magnetic resonance imaging: Should we be concerned?, Tan [/bib_ref]. Since there is evidence to suggest PCa originating from the CZ can be more aggressive, relying on the PIRADS score alone may overcall lesions and lead to unnecessary confirmatory biopsy sessions [bib_ref] Central zone carcinoma of the prostate gland: a distinct tumor type with..., Cohen [/bib_ref] [bib_ref] Normal central zone of the prostate and central zone involvement by prostate..., Vargas [/bib_ref].
## Prostate biopsy in the era of mri-us fusion
MRI-US fusion technology utilizes the improved visualization of anatomy afforded by MRI to perform targeted biopsy of suspicious prostatic lesions [bib_ref] Utility of multiparametric magnetic resonance imaging suspicion levels for detecting prostate cancer, Rais-Bahrami [/bib_ref] [bib_ref] Current status of magnetic resonance imaging (MRI) and ultrasonography fusion software platforms..., Logan [/bib_ref]. Multiple investigations suggest performing MRI-US fusion targeted biopsy may be superior to standard systematic template TRUS-guided tissue sampling to detect new csPCa for men on AS (47-52). Siddiqui et al. suggest performing MRI-US fusion targeted biopsy may reduce the number of insignificant PCa diagnoses, thus sparing patients from unnecessary biopsies [bib_ref] Comparison of MR/ultrasound fusion-guided biopsy with ultrasound-guided biopsy for the diagnosis of..., Siddiqui [/bib_ref]. We identified two studies showing improved csPCa detection when utilizing mpMRI-US fusion technology for transperineal prostate biopsy [bib_ref] Magnetic Resonance and Ultrasound Image Fusion Supported Transperineal Prostate Biopsy Using the..., Hansen [/bib_ref] [bib_ref] Transperineal in-bore 3-T MR imaging-guided prostate biopsy: a prospective clinical observational study, Penzkofer [/bib_ref]. Penzkofer et al. found this approach to be of particular benefit in the setting of anteriorly located tumors when utilizing an in-bore MRI-guided approach (55). Felker et al. also achieved satisfactory cancer detection rates when performing in-bore MRI-guided biopsy via transrectal approach [bib_ref] In-bore magnetic resonance-guided transrectal biopsy for the detection of clinically significant prostate..., Felker [/bib_ref].
However, there is conflicting data over whether performing MRI targeted biopsy in isolation, abandoning the standard twelve core template sampling approach, is a safe monitoring strategy for men on AS [bib_ref] Multiparametric magnetic resonance imaging vs. standard care in men being evaluated for..., Panebianco [/bib_ref] [bib_ref] A prospective comparison of MRI-US fused targeted biopsy versus systematic ultrasound-guided biopsy..., Da Rosa [/bib_ref] [bib_ref] Targeted Biopsy to Detect Gleason Score Upgrading during Active Surveillance for Men..., Nassiri [/bib_ref] [bib_ref] The role of MRItargeted and confirmatory biopsies for cancer upstaging at selection..., Marliere [/bib_ref]. Nassiri et al. retrospectively evaluated 250 patients undergoing MRI-fusion biopsy and observed that 32 of 33 cases with pathological upgrading were a result of positive MRItargeted cores [bib_ref] Targeted Biopsy to Detect Gleason Score Upgrading during Active Surveillance for Men..., Nassiri [/bib_ref]. Da Rosa et al. reported a 100% negative predictive value for detecting a Gleason score 6 to 7 upgrade when using MRI-fusion technology in their cohort of 72 men on AS [bib_ref] A prospective comparison of MRI-US fused targeted biopsy versus systematic ultrasound-guided biopsy..., Da Rosa [/bib_ref]. Conversely, Marliere et al. observed that standard template biopsy at the time of MRI-targeted sampling still has utility for the detection of new and significant cancer foci. In their cohort of 41 men on AS undergoing combined standard template and MRI-targeted confirmatory biopsy, pathological upstaging was attributable to standard template core tissue more than half of the time [bib_ref] The role of MRItargeted and confirmatory biopsies for cancer upstaging at selection..., Marliere [/bib_ref].
Of all of the prostate biopsy modalities, there is strong evidence in support of saturation biopsy (24 or 30 cores templated sampling) as the technique with greatest sensitivity for detecting significant PCa in the initial AS period [bib_ref] Potential benefit of transrectal saturation prostate biopsy as an initial biopsy strategy:..., Li [/bib_ref] [bib_ref] Detection rate for significant cancer at confirmatory biopsy in men enrolled in..., Pepe [/bib_ref] [bib_ref] Cognitive zonal fusion biopsy of the prostate: Original technique between target and..., Galosi [/bib_ref]. However, it subjects patients to the burden of acquiring a significant amount of tissue, and may not reliably predict the location or extent of disease on pathology following radical prostatectomy [bib_ref] Pathological Findings in Multiparametric Magnetic Resonance Imaging/Ultrasound Fusion-guided Biopsy: Relation to Prostate..., Gordetsky [/bib_ref]. Alternatively, Galosi et al. proposed a hybrid approach to saturation biopsy called 'cognitive zonal fusion biopsy' [bib_ref] Cognitive zonal fusion biopsy of the prostate: Original technique between target and..., Galosi [/bib_ref]. For this, patients undergo mpMRI prior to fusion biopsy. If during the biopsy there is a discrepancy between what was found on MRI and what is seen on US, several cores are obtained from the MRI region of interest. This approach reliably detected csPCa in their prospective study of 58 men who were either biopsy naïve, had a prior negative biopsy, or were on AS [bib_ref] Cognitive zonal fusion biopsy of the prostate: Original technique between target and..., Galosi [/bib_ref]. In a study of 48 men on AS with prior negative TRUS biopsy, Lai et al. also achieved satisfactory PCa detection using this technique [bib_ref] Cognitive MRI-TRUS fusion-targeted prostate biopsy according to PI-RADS classification in patients with..., Lai [/bib_ref].
## Could mpmri allow safe surveillance without repeat tissue biopsy?
There is emerging evidence to suggest that serial mpMRIs alone may be sufficient to monitor men on AS, and the time interval for repeat confirmatory biopsy could be prolonged [bib_ref] Use of serial multiparametric magnetic resonance imaging in the management of patients..., Diaz [/bib_ref] [bib_ref] Clinical implications of a multiparametric magnetic resonance imaging based nomogram applied to..., Siddiqui [/bib_ref] [bib_ref] Serial Magnetic Resonance Imaging in Active Surveillance of Prostate Cancer: Incremental Value, Felker [/bib_ref] [bib_ref] Natural history of small index lesions suspicious for prostate cancer on multiparametric..., Rais-Bahrami [/bib_ref] [bib_ref] Reporting Magnetic Resonance Imaging in Men on Active Surveillance for Prostate Cancer:..., Moore [/bib_ref]. Both Walton reported that stable mpMRI findings reliably correlated with Gleason score stability in their cohorts of men on AS meeting standard inclusion criteria [bib_ref] Use of serial multiparametric magnetic resonance imaging in the management of patients..., Diaz [/bib_ref] [bib_ref] Serial Magnetic Resonance Imaging in Active Surveillance of Prostate Cancer: Incremental Value, Felker [/bib_ref]. Frye et al. also appreciated reliable detection of cancer progression using mpMRI alone in a retrospective review of 162 men [bib_ref] Magnetic Resonance Imaging-Transrectal Ultrasound Guided Fusion Biopsy to Detect Progression in Patients..., Frye [/bib_ref].
Several recent investigations evaluated the usefulness of predicting pathologic progression of an index lesion based on size criteria. In a review of more than 150 men on AS, those with index lesions 7mm or less were found to have no change in either size or pathologic characteristics during a two-year follow-up period [bib_ref] Natural history of small index lesions suspicious for prostate cancer on multiparametric..., Rais-Bahrami [/bib_ref]. Thus, men otherwise meeting AS criteria could potentially defer PCa surveillance for up to a two-year interval of time without compromising care. Based on serial mpMRIs on a cohort of men who underwent regularly scheduled biopsies, Siddiqui et al. developed a predictive nomogram for pathological progression. Their nomogram would have theoretically avoided repeat biopsy in 68% of men in their study [bib_ref] Clinical implications of a multiparametric magnetic resonance imaging based nomogram applied to..., Siddiqui [/bib_ref]. Lai et al. developed a similar predictive nomogram for low-risk men on AS with certain mpMRI criteria but also incorporating clinical parameters for each case [bib_ref] Factors predicting prostate cancer upgrading on magnetic resonance imaging-targeted biopsy in an..., Lai [/bib_ref].
# Discussion
AS for low-risk, localized PCa has become a widely adopted strategy, and offers adequate disease control while optimizing quality of life. Nonetheless, this strategy remains a challenge due to disease variability, inconsistencies on optimal surveillance regimens and a wide variety of available diagnostic tests. A majority of AS protocols use serial digital rectal examinations, serum PSA levels, and TRUS-guided systematic biopsies to monitor patients [bib_ref] Increasing use of observation among men at low risk for prostate cancer..., Ritch [/bib_ref] [bib_ref] 20-year outcomes following conservative management of clinically localized prostate cancer, Albertsen [/bib_ref]. However, since nearly half of all patients on AS have been shown to ultimately require some form of intervention, improved surveillance strategies are needed to monitor for disease progression in an effective and efficient manner [bib_ref] Magnetic Resonance Imaging-Transrectal Ultrasound Guided Fusion Biopsy to Detect Progression in Patients..., Frye [/bib_ref] [bib_ref] Upgrading and downgrading of prostate needle biopsy specimens: risk factors and clinical..., Freedland [/bib_ref] [bib_ref] Intermediate and Longer-Term Outcomes From a Prospective Active-Surveillance Program for Favorable-Risk Prostate..., Tosoian [/bib_ref] [bib_ref] Treatment outcomes of radical prostatectomy in potential candidates for 3 published active..., Thaxton [/bib_ref].
Novel MRI sequencing techniques and improved technology has allowed for meticulous characterization of suspicious intraprostatic lesions [fig_ref] Figure 1: A 65-year-old male with a history of TRUS biopsy performed at another... [/fig_ref]. Recent evidence suggests that using mpMRI to characterize suspicious foci within the prostate allows for better detection than prior imaging techniques and systematic sampling alone, and may allow for safe AS while potentially decreasing the frequency of invasive biopsy sessions [bib_ref] In-parallel comparative evaluation between multiparametric magnetic resonance imaging, prostate cancer antigen 3..., Porpiglia [/bib_ref] [bib_ref] Risk stratification of prostate cancer utilizing apparent diffusion coefficient value and lesion..., Salami [/bib_ref] [bib_ref] Nine-year Follow-up for a Study of Diffusion-weighted Magnetic Resonance Imaging in a..., Henderson [/bib_ref] [bib_ref] Diffusion-weighted magnetic resonance imaging for prediction of insignificant prostate cancer in potential..., Kim [/bib_ref] [bib_ref] Magnetic Resonance Imaging Targeted Biopsy Improves Selection of Patients Considered for Active..., Ouzzane [/bib_ref] [bib_ref] Further reduction of disqualification rates by additional MRItargeted biopsy with transperineal saturation..., Radtke [/bib_ref]. Furthermore, mpMRI may even detect more aggressive disease not found on the initial standard template biopsy, and, therefore, may keep the patient from being inappropriately placed on AS [bib_ref] Magnetic Resonance Imaging Targeted Biopsy Improves Selection of Patients Considered for Active..., Ouzzane [/bib_ref]. For these reasons, there has been a push towards formally incorporating mpMRI findings into existing AS criteria such as the Epstein criteria and PRIAS [bib_ref] Multiparametric magnetic resonance imaging and active surveillance: How to better select insignificant..., Porpiglia [/bib_ref] [bib_ref] Value of 3-T multiparametric magnetic resonance imaging and magnetic resonance-guided biopsy for..., Hoeks [/bib_ref] [bib_ref] Accuracy of multiparametric magnetic resonance imaging in confirming eligibility for active surveillance..., Stamatakis [/bib_ref] [bib_ref] Factors predicting prostate cancer upgrading on magnetic resonance imaging-targeted biopsy in an..., Lai [/bib_ref].
When performing a prostate biopsy is warranted, novel MRI fusion technology permits targeted tissue sampling with unparalleled accuracy [fig_ref] Figure 2: Tissue core at 20× magnification obtained from MRI-US fusion biopsy of 65-year-old... [/fig_ref] [bib_ref] Multiparametric magnetic resonance imaging vs. standard care in men being evaluated for..., Panebianco [/bib_ref] [bib_ref] A prospective comparison of MRI-US fused targeted biopsy versus systematic ultrasound-guided biopsy..., Da Rosa [/bib_ref] [bib_ref] Targeted Biopsy to Detect Gleason Score Upgrading during Active Surveillance for Men..., Nassiri [/bib_ref] [bib_ref] Multiparametric magnetic resonance imaging enhances detection of significant tumor in patients on..., Abdi [/bib_ref]. Compared to saturation biopsies, recent evidence suggests that MRItargeted biopsies may be just as reliable for csPCa detection with improved efficiency [bib_ref] Potential benefit of transrectal saturation prostate biopsy as an initial biopsy strategy:..., Li [/bib_ref] [bib_ref] Detection rate for significant cancer at confirmatory biopsy in men enrolled in..., Pepe [/bib_ref] [bib_ref] Cognitive zonal fusion biopsy of the prostate: Original technique between target and..., Galosi [/bib_ref] [bib_ref] Efficiency of Prostate Cancer Diagnosis by MR/Ultrasound Fusion-Guided Biopsy vs Standard Extended-Sextant..., Siddiqui [/bib_ref]. This would spare patients, urologists and pathologists the task of acquiring and interpreting numerous cores obtained from a saturation biopsy approach and may decrease the frequency of finding [bib_ref] Detection rate for significant cancer at confirmatory biopsy in men enrolled in..., Pepe [/bib_ref]. Furthermore, we identified two studies suggesting that in-bore MRI-guided biopsy may offer an even greater detection rate [bib_ref] Transperineal in-bore 3-T MR imaging-guided prostate biopsy: a prospective clinical observational study, Penzkofer [/bib_ref] [bib_ref] In-bore magnetic resonance-guided transrectal biopsy for the detection of clinically significant prostate..., Felker [/bib_ref]. Conversely, other evidence suggests MRI-US fusion alone may not be adequate in the initial diagnostic setting, and standard template biopsy may be equally diagnostic in biopsy naïve men [bib_ref] The role of MRItargeted and confirmatory biopsies for cancer upstaging at selection..., Marliere [/bib_ref]. Several other studies suggest that men with small enough index lesions could avoid repeat biopsy altogether, and instead be followed by serial mpMRIs with equivocal detection and omission rates [bib_ref] Clinical implications of a multiparametric magnetic resonance imaging based nomogram applied to..., Siddiqui [/bib_ref] [bib_ref] Natural history of small index lesions suspicious for prostate cancer on multiparametric..., Rais-Bahrami [/bib_ref].
[formula] A B C D E F G H insignificant disease [/formula]
# Conclusions
In the era of ever increasing use of AS for men with lowrisk PCa, improved strategies for proper stratification are needed to balance overtreatment with underassessment of true risk. mpMRI has dramatically enhanced the detection of clinically-significant PCa, and may permit less-invasive surveillance strategies compared to currently accepted protocols. Further investigation is warranted to determine the most appropriate utilization of mpMRI in the setting of serial imaging and to also identify to what extent targeted versus templated systematic prostate biopsy should be performed. A B
[fig] Figure 1: A 65-year-old male with a history of TRUS biopsy performed at another institution presents for repeat biopsy. Pathology from the TRUS biopsy showed atypical small acinar proliferation and the patient's PSA was 5.35ng/mL. Prior to the repeat biopsy, the patient underwent prostate mpMRI (A-D). A circumscribed, focal lesion in the right mid lateral peripheral zone (A) was identified with hypointensity on ADC (B) with associated hyperintensity on high b-value DWI (C). This lesion demonstrated associated abnormal perfusion (D) and was suspicious for csPCa. The patient underwent MR-US fusion biopsy with Gleason 3+3=6 disease on pathology and elected for AS. Subsequent mpMRI in one year demonstrated stability of this focal lesion on T2-weighted images (E) and ADC (F). The lesion remains stable at two-year follow up on T2-weighted images (G) and ADC (H). [/fig]
[fig] Figure 2: Tissue core at 20× magnification obtained from MRI-US fusion biopsy of 65-year-old male in Figure 1. (A) Hematoxylin and eosin stained slide showing infiltrating small, well-formed glands with cytologic atypia, consistent with prostatic adenocarcinoma, Gleason score 3+3=6 (grade group 1); (B) immunohistochemical-stained slide showing positivity in the atypical glands for racemase (pink) and lack of staining for p63 (nuclear staining, brown, and high molecular weight cytokeratin (cytoplasmic staining, brown) confirming prostatic adenocarcinoma. [/fig]
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How do tenascins influence the birth and life of a malignant cell?
Tenascins are large glycoproteins found in embryonic and adult extracellular matrices. Of the four family members, two have been shown to be overexpressed in the microenvironment of solid tumours: tenascin-C and tenascin-W. The regular presence of these proteins in tumours suggests a role in tumourigenesis, which has been investigated intensively for tenascin-C and recently for tenascin-W as well. In this review, we follow a malignant cell starting from its birth through its potential metastatic journey and describe how tenascin-C and tenascin-W contribute to these successive steps of tumourigenesis. We consider the importance of the mechanical aspect in tenascin signalling. Furthermore, we discuss studies describing tenascin-C as an important component of stem cell niches and present examples reporting its role in cancer therapy resistance.
# Introduction
Tumourigenesis has traditionally been described as a cellautonomous process when a cell, after accumulation of genetic alterations, loses its control over growth, undergoes aberrant proliferation and thus gives rise to a tumour. It is now accepted that the microenvironment surrounding a potential tumour cell plays a crucial role influencing its fate [bib_ref] Jekyll and Hyde: the role of the microenvironment on the progression of..., Allen [/bib_ref]. For instance, experiments exposing pre-neoplastic or tumour cells to various mesenchymal environments have underlined the critical role of the stroma in this context [bib_ref] Effect of radiation-induced injury of tumour bed stroma on metastatic spread of..., Milas [/bib_ref] [bib_ref] Carcinoma-associated fibroblasts direct tumour progression of initiated human prostatic epithelium, Olumi [/bib_ref] [bib_ref] Breast stroma plays a dominant regulatory role in breast epithelial growth and..., Shekhar [/bib_ref]. Among the proteins known to be overexpressed in tumour-associated stroma are tenascin-C and tenascin-W. Tenascins are large glycoproteins found in the extracellular compartment of various tissues. The tenascin family has four members: tenascin-C, tenascin-R, tenascin-X and tenascin-W. They all share a characteristic modular structure with an oligomerization domain followed by EGF-like repeats, fibronectin (FN) type III repeats and a fibrinogen globe (see Ref.and . In the case of tenascin-C and tenascin-R, alternative splicing can lead to the generation of multiple isoforms that contain additional FN type III repeats. Only two of the tenascin members, the original tenascin-C [bib_ref] Human glioma-mesenchymal extracellular matrix antigen defined by monoclonal antibody, Bourdon [/bib_ref] and the more recently discovered tenascin-W , have been shown to be overexpressed in tumours compared to healthy tissues (for review, see Ref. . In most cases, healthy tissues are not completely deprived of tenascin-C . However, corresponding tumour tissues express much higher levels of tenascin-C or harbour different, larger isoforms . Tenascin-W expression is much more restricted in adult healthy tissues , which makes it an excellent tumour marker: it is absent in healthy tissue but present in most breast , colon Tumor Microenvironment Review Series
## - introduction - birth of a malignant cell - niches for self-renewable cells - expression patterns in tumours - promotion of tumour cell proliferation - promotion of tumour cell migration - contribution to epithelial-mesenchymal transition (emt) - promotion of angiogenesis - promotion of metastasis - how do tenascins signal to cells? - importance of the mechanical aspect - evasion of tumour cells from conventional therapy - conclusions
[13] and brain tumours. In contrast, tenascin-R and tenascin-X expression is rather constant and is not modulated by tissues malignancy (for review, see Ref. . In this review, we will follow the major steps in the life of a tumour cell and its descendants and describe how tenascin-C and tenascin-W influence their fate.
## Birth of a malignant cell
The birth of a malignant cell is a multi-step process, which starts with the acquisition of alterations in genes encoding proteins implicated in cellular growth control. For this reason, the first defense of an organism against cancer relies on the prevention of genomic alterations. At the cellular level, many responses to mutagenic challenges have been developed. For instance, cell cycle arrest is triggered to enable proofreading of DNA and ensure genomic stability. Interestingly, the state of cell adhesion is suspected to alter pathways controlling genomic stability (for review, see Ref. . Because tenascins modulate the adhesion status of cells , it is tempting to speculate that they may also influence genomic stability . Consistent with this hypothesis, BRCA1associated RING domain 1 (Brad1), H2A histone family member X (H2AX), as well as other molecules with assigned functions in genome stability were found to be down-regulated in the presence of tenascin-C in glioblastoma cells . The hypothesis that tenascin-C could favour the birth of malignant cells implies its expression before the development of the tumour. Remarkably, transgenic mice expressing an autoactivating mutant of the matrix metalloproteinase stromelysin-1 in mammary epithelia show inappropriate expression of tenascin-C, starting by day 6 of pregnancy and followed by the development of mammary pre-neoplastic and neoplastic lesions . Furthermore, fibroblasts expanded from Gorlin syndrome patients, which are prone to develop basal cell carcinomas, show phenotypic traits reminiscent of cancer-associated fibroblasts. In particular, tenascin-C is highly expressed by these cells . It is believed that the dermis contributes to the predisposition of these patients to develop basal cell carcinomas, which is compatible with the hypothesis that a tenascin-C rich environment favours the birth of malignant cells.
## Niches for self-renewable cells
The capacity to proliferate indefinitely and to self-renew is a shared hallmark of stem cells and tumour cells. Hence, major signalling pathways initially aimed at regulating normal stem cells, such as Wnt, Shh and Notch signalling are re-utilized by the tumour cell machinery (for review, see Ref. . It is interesting to note that tenascin-C was shown to be associated with stem cell niches in the neural, haematopoietic and epidermal system (for review, see Ref. . Tenascin-C in brain is highly expressed in specialized germinal zones such as the subventricular zone where it may play an important role in the regulation of stem cells because transgenic mice lacking tenascin-C show altered numbers of central nervous system stem cells . Specific carbohydrate side chains of tenascin-C could play a key role in the regulation of embryonic neural stem cell proliferation. For instance, human natural killer 1 (HNK1) carbohydrate epitopes carried by large splice variants of tenascin-C are involved in the proliferation of neural stem cells via modulation of the expression level of epidermal growth factor (EGF) receptor . In parallel, tenascin-C was identified as a cell surface glycoprotein marker for glioblastoma-derived stem-like cells . Tenascin-C is also present in . In addition, tenascin-C could be part of the specialized extracellular matrix characterizing the niche of epidermal stem cells because it is strongly up-regulated in the hair follicle bulge, which is known to be a stem cell reservoir .
## Expression patterns in tumours
In tumours arising from epithelial organs (i.e. in most carcinomas), the source of tenascins is restricted to the structural, mesenchymal compartment: very strong tenascin immunostaining is found in the tumour stroma surrounding tenascin-free tumour nests (see
## , for a typical staining pattern). the stromal tenascin-c expression can be influenced by cancer cells because co-culture of fibroblasts with tumour cells has been shown to stimulate tenascin-c expression in the fibroblasts [29, 30]. in other tumours such as melanoma and brain tumours, the tumour cells themselves are the source of tenascin-c [31, 32]. interestingly, brain tumours arise from structural cells (oligodendrocytes for oligodendrogliomas, astrocytes for astrocytomas and glioblastomas), which may explain that these cells have common features with mesenchymal cells rather than with carcinoma cells. melanomas develop from neural crest-derived melanocytes. neural crest cells express high levels of tenascin-c [33]
, which is required for their migration in embryogenesis . In contrast to tenascin-C, tenascin-W expression has yet to be described in tumour cells themselves. However, tenascin-W is also expressed in the stroma of breast and colon carcinomas. In brain tumours, both tenascin-C and tenascin-W can be observed around blood vessels. However, their pattern of expression is distinct because tenascin-C surrounds both endothelial and pericyte cell layers, whereas tenascin-W is restricted to the endothelial cell layer .
## Promotion of tumour cell proliferation
An important feature of tumour cells is their ability to overproliferate. Interestingly, tenascin-C has been shown to promote tumour cell proliferation in standard cell culture conditions . More specifically, culturing glioblastoma and breast carcinoma cells on mixed fibronectin/tenascin-C substrata not only attenuated their adhesion but also increased their proliferation rate compared to a pure fibronectin substratum .
## Promotion of tumour cell migration
Tenascin-C was initially described as an anti-adhesive molecule that antagonizes the capacity of tumour cells to adhere and spread on fibronectin . Experiments performed in three-dimensional fibrin matrices containing fibronectin have revealed that tenascin-C suppresses actin stress fibre formation via inhibition of RhoA activation, and instead induces actin-rich filopodia [bib_ref] Cyclic mechanical stretch reduces myofibroblast differentiation of primary lung fibroblasts, Blaauboer [/bib_ref]
## Contribution to epithelial-mesenchymal transition (emt)
## Epithelial-mesenchymal transition is a developmental process re-utilized by cancer cells and characterized by loss of cell adhesion, repression of e-cadherin expression as well as increased cell mobility. tenascin-c has been shown to be associated with emt because tumour cell lines undergoing transforming growth factor- (tgf-) induced emt secrete tenascin-c [49]. furthermore, vimentin (an emt marker) and tenascin-c expressions are associated in cancer cells [50], and cancer cell lines with clear epithelial morphology secrete far less tenascin-c than cancer cell lines characterized by a fibroblastic morphology (our observations). finally, a recent study reports that tenascin-c is required for injury-induced emt of lens epithelium [51]. however, except for the last example, it remains to be determined whether tenascin-c expression is only a consequence of emt or may also be required for this process.
## Promotion of angiogenesis
When a tumour reaches a critical size, it has to attract the formation of new vessels to supply nutrients and oxygen to the proliferating cells. Both tenascin-C and tenascin-W can been classified as pro-angiogenic factors because they trigger endothelial cells to acquire a sprouting phenotype and increase their migration capacities . Tenascin-C is known to play a crucial role in postnatal cardiac angiogenesis because tenascin-C-deficient mice fail to vascularize cardiac allografts in an established cardiac transplantation model . Furthermore, in a xenograft tumour model it was shown that tumours were much more vascularized when they were grown in wild-type mice than in tenascin-C-deficient mice, suggesting that the presence of tenascin-C in the microenvironment is very important for tumour angiogenesis .
## Tenascin-c was also associated with lymphangiogenesis [56], particularly in the context of podoplanin induction [57]. the proximity of tenascin-c and tenascin-w around blood vessels prompts the question whether or not these proteins can extravasate from the tumour site and circulate in the bloodstream. indeed, tenascin-c was found in the serum of cancer patients, but because its presence in serum was also associated with conditions other than cancer (e.g. as a consequence of infection and inflammation, see ref. [10]), tenascin-c turned out to be a questionable serum tumour marker [58]. in contrast, tenascin-w function in adults seems to be restricted to tumourigenesis and osteogenesis (for review, see
Ref.
[59]), so it may become a better tumour biomarker. Indeed, tenascin-W levels in serum from breast and colorectal cancer patients were found to be elevated compared to serum collected from healthy, control individuals . However, individual tenascin-W levels were heterogeneous, with some patients exhibiting highly elevated values and others with values within the range of controls. Interestingly, most patients that developed early tumour recurrence were initially characterized by high serum tenascin-W levels. A follow-up study including a large cohort of patients should therefore be performed to confirm this tendency. Together, these results suggest that tenascin-W is a promising tumour biomarker, but should be used in combination with other markers to avoid generation of false negative distortions.
## Promotion of metastasis
After escape from the primary tumour and circulation in the blood or lymphatic vessels cancer cells will, as an ultimate step, colonize other organs of the body. The nature of the 'soil' influences greatly the choice of host tissues in which the tumour cells will 'seed' . Interestingly, extracellular matrix proteins produced by tumour cells themselves also determine their metastatic capacity. For instance, knocking-down tenascin-C in melanoma cells significantly decreased their capacity to form pulmonary metastases . This holds true as well for certain breast tumour xenograft models . In support for a human relevance of these observations, high tenascin-C expression was associated with an 8-fold increased risk of metastasis in classic giant cell tumours of bone . A recent study suggests that the fibrillar organization of tenascin-C, requiring stromal fibroblasts and active MMP2, is associated with metastatic pancreatic cancers . However, activation of tenascin-C-dependent metastatic pathways may depend on the initial oncogenic alterations because tenascin-C is not found in all metastatic transcript profiles . To our knowledge, tenascin-W expression has not been associated with metastasis in human beings. However, this possibility is difficult to investigate in experimental models because cancer cell lines expanded from any type of tumours were negative for tenascin-W expression (our own observation).
## How do tenascins signal to cells?
We described above the effects triggered by tenascins on tumour and endothelial cells, but a main question remains: how do the signals activated by tenascins reach the nucleus where regulation of gene expression takes place? There are many possibilities how this can occur. For example, the signal can be transmitted by an indirect, 'classical' pathway, that is through activation of transmembrane receptor proteins, cascades of kinases, and activation of transcription factors, which finally act in the nucleus to modulate gene expression. Alternatively, the signal could also be transduced mechanically in a more direct manner through the cell cytoskeleton, which physically connects the outside of a cell with the chromatin . Concerning indirect ways of activation, there is no consensus for a given signalling pathway, suggesting that tenascin-C can activate many parallel context-and tissue-specific signalling cascades. On the level of cell surface receptors, a large set of integrins have been identified as receptors for tenascin-C (␣21, ␣v3, ␣71, ␣81, ␣91, ␣53, ␣56 [bib_ref] Stromal fibroblasts influence oral squamous-cell carcinoma cell interactions with tenascin-C, Ramos [/bib_ref] [bib_ref] Endothelial cell attachment and spreading on human tenascin is mediated by alpha..., Sriramarao [/bib_ref] [bib_ref] The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD..., Yokosaki [/bib_ref] [bib_ref] The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick..., Varnum-Finney [/bib_ref] [bib_ref] Differential effects of the integrins alpha9beta1, alphavbeta3, and alphav-beta6 on cell proliferative..., Yokosaki [/bib_ref] and for tenascin-W [fig_ref] Figure 2: Summary of the actions of tenascins in cancer [/fig_ref]. The fact that tenascins contain epidermal growth factor motifs suggests that they could bind to EGF receptors. Tenascin-C was classified as an 'insoluble growth factor ligand' [bib_ref] Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF..., Swindle [/bib_ref] because it can bind to EGFR with a low affinity insufficient to trigger ligand internalization. This binding favours activation of motility rather than proliferation responses [bib_ref] Cell surface restriction of EGFR by a tenascin cytotactin-encoded EGF-like repeat is..., Iyer [/bib_ref]. Interestingly, tenascin-C-enriched medium can stimulate invasion of colon cancer cells only in presence of a functional EGFR . The receptor of hepatocyte growth factor, c-Met, was shown to work in close association with tenascin-C both in colon and mammary culture models . Tenascin-C triggers in vitro the formation of filled (instead of hollow) acini surrounded by an abnormal basement membrane. This effect is abrogated in the absence of functional c-Met receptors . Another transmembrane protein, TLR4, a member of the toll-like receptor family which is known to contribute to infection and inflammation responses, is activated by tenascin-C [bib_ref] Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential..., Midwood [/bib_ref]. Whether tenascin-C binds as a ligand to TLR4 remains to be investigated. In addition to EGFR, HGF/c-Met and TLR4, two other welldescribed pathways are suspected to be modulated by tenascin-C: these are TGF- and Wnt signalling pathways. Gene expression profile analysis of T98G glioblastoma cells cultured in presence of tenascin-C revealed, for instance, down-regulation of follistatin, a known inhibitor of activins . This study also led to the identification of Dkk1, a known inhibitor of the Wnt pathway, as a gene down-regulated by tenascin-C. Interestingly, a potential mechanism of cell cycle regulation by tenascin-C could also be through induction of 14-3-3 [bib_ref] Tenascin-C signaling through induction of 14-3-3 tau, Martin [/bib_ref] followed by p21 down-regulation [bib_ref] 14-3-3 Tau regulates ubiquitin-independent proteasomal degradation of p21, a novel mechanism of..., Wang [/bib_ref].
Another more direct way for tenascin-C-triggered signals to reach the nucleus has been recently investigated in our laboratory [bib_ref] Interfering with the connection between the nucleus and the cytoskeleton affects nuclear..., Brosig [/bib_ref]. These studies show that disruption of the physical link connecting the cell membrane to the nucleus perturbs the mechanical control of cell differentiation in the context of myogenesis. This suggests that tenascin-C-induced cytoskeletal rearrangements could also affect mechanotransduction in the frame of tumourigenesis.
It is important to note that transduction of tenascin signals to cells could also be influenced by other extracellular matrix molecules. For instance, through their binding to tenascin-C, fibronectin [bib_ref] Binding of tenascin-C to soluble fibronectin and matrix fibrils, Chung [/bib_ref] , perlecan, lectican [bib_ref] Alternative splicing in the aggrecan G3 domain influences binding interactions with tenascin-C..., Day [/bib_ref] , heparin, contactin [bib_ref] Functional interactions of the immunoglobulin superfamily member F11 are differentially regulated by..., Zacharias [/bib_ref] [bib_ref] Regulatory mechanisms that mediate tenascin C-dependent inhibition of oligodendrocyte precursor differentiation, Czopka [/bib_ref] and SMOC1 [bib_ref] SMOC1 is a tenascin-C interacting protein overexpressed in brain tumours, Brellier [/bib_ref] have been shown to modify its effects.
## Importance of the mechanical aspect
An important parameter for the progression of a tumour is the stiffness of the tissue in which it develops. Reconstruction of mammary epithelial acini in vitro has shown that the rigidity of the matrix strongly influences epithelial morphogenesis [bib_ref] Tensional homeostasis and the malignant phenotype, Paszek [/bib_ref]. Consistently, in vivo reduction of lysyl oxidase-mediated collagen crosslinking to weaken the collagen fibres and to reduce their tensile strength decreases mammary tumour incidence [bib_ref] Matrix crosslinking forces tumour progression by enhancing integrin signaling, Levental [/bib_ref]. Interestingly, mechanical strain not only influences features of the epithelial tumour cells themselves but may also stimulate neovascularization through activation of angiogenic genes [bib_ref] Mechanical strain activates a program of genes functionally involved in paracrine signaling..., Yang [/bib_ref]. It is therefore interesting to note that tenascin-C is induced by mechanical strain, requiring integrin-linked kinase [bib_ref] Tenascin-C induction by cyclic strain requires integrin-linked kinase, Maier [/bib_ref] followed by RhoA/ROCK-dependent actin contractility [bib_ref] Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic..., Sarasa-Renedo [/bib_ref]. Interestingly, fibroblasts cultured on attached, restrained collagen gels express much more tenascin-C than those cultured on floating, unrestrained collagen gels [bib_ref] Tenascin-C expression by fibroblasts is elevated in stressed collagen gels, Chiquet-Ehrismann [/bib_ref]. This may be an explanation why cancer-associated fibroblasts, which are exposed to high mechanical strain, overexpress tenascin-C compared to 'normal' fibroblasts populating healthy tissues. Stiffness of the tumour environment may also favour transdifferentiation of cancer-associated fibroblasts into myofibroblasts as myodifferentiation of primary lung fibroblasts has been recently reported to be influenced by cyclic mechanical stress .
## Evasion of tumour cells from conventional therapy
Interestingly, tenascin-C has been shown to mediate chemotherapy-resistance in various contexts. Knocking-down tenascin-C sensitizes melanoma cells to doxorubicin . This happens most likely via down-regulation of ATP-binding cassette B5, a transporter known to provide the cells high efflux capacity for antimitotic drugs. In the frame of breast cancer, tenascin-C could be associated with resistance to tamoxifen therapy [bib_ref] Association of an extracellular matrix gene cluster with breast cancer prognosis and..., Helleman [/bib_ref] and doxorubicin therapy [bib_ref] 14-3-3 Tau regulates ubiquitin-independent proteasomal degradation of p21, a novel mechanism of..., Wang [/bib_ref]. Interestingly, the doxorubicin-induced G1/S arrest could be abrogated through p21 regulation, which in turn is known to be affected by the presence of tenascin-C. It was also shown that large isoforms of tenascin-C and cell surface protein annexin A2 can interact and together decrease gemcitabineinduced cytotoxicity in pancreatic cancer cells [bib_ref] Gemcitabine resistance induced by interaction between alternatively spliced segment of tenascin-C and..., Gong [/bib_ref]. Tenascin-C might also be linked to resistance to anti-angiogenesis treatment because hypoxic tumour cells adapt by inducing new sets of genes, among which tenascin-C is found [bib_ref] Transcriptional response to hypoxia in human tumours, Lal [/bib_ref].
# Conclusions
We have summarized how tenascins have been shown to influence the successive steps of tumourigenesis (summarized in [fig_ref] Figure 2: Summary of the actions of tenascins in cancer [/fig_ref]. Based on our knowledge of the properties of tenascin-C, two types of anti-cancer therapies have been established. The first type aims at the neutralization of its pro-tumourigenic effects by down-regulating its expression using anti-sense oligonucleotides or aptamers (for review, see Refs. [bib_ref] Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas, Caruso [/bib_ref] [bib_ref] Discovery and development of anticancer aptamers, Ireson [/bib_ref] , respectively). The second type takes advantage of the specific overexpression of tenascin-C isoforms at tumour sites to selectively target tumour cells, using anti-tenascin-C antibody fragments coupled to a variety of anti-cancer molecules (for review, see Ref. [bib_ref] Antibody-based vascular tumour targeting, Schliemann [/bib_ref]. Further studies will be necessary to extend our knowledge to tenascin-W, but studies reported so far suggest a promising future for tenascin-W as a tumour marker and potential drug target.
[fig] Fig: 1. Tenascin structure and tumour stroma staining. A schematic model representing one subunit of the hexameric tenascin-C and tenascin-W proteins is shown above a breast carcinoma section stained with antibodies against the two proteins. Both tenascins are built from the following domains: a central, N-terminal oligomerization domain (black line), EGFlike repeats (EGF, yellow), FN type III repeats (FN3, green; FN3 repeats subject to alternative splicing in light green) and a C-terminal fibrinogen related domain (FReD, violet). Antibodies against tenascin-C and tenascin-W strongly stain the stroma of the breast carcinoma, although the epithelial tumour nests (nuclei in blue) are negative. [/fig]
[table] 36 © 2011: The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd [/table]
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IgA Vasculitis With Adalimumab Therapy for Hidradenitis Suppurativa: A Case Report and Review of Literature
# Introduction
Immunoglobulin A (IgA) vasculitis, formerly Henoch-Schönlein purpura, is a systemic immune complex vasculitis primarily affecting the small vessels. IgA vasculitis is the most common systemic vasculitis in the pediatric population. Its annual incidence is three to 26 cases per 100,000 children [bib_ref] Incidence of Henoch-Schonlein purpura, Kawasaki disease, and rare vasculitides in children of..., Gardner-Medwin [/bib_ref]. It is more common in winter and fall and in pediatric patients between the ages of four and seven years. The condition is less common in adults, with an annual incidence of 0.1 to 1.8 per 100,000 adults, and is noted to occur more frequently in summer and winter [bib_ref] Henoch-Schönlein purpura in northern Spain: clinical spectrum of the disease in 417..., Calvo-Río [/bib_ref] [bib_ref] Henoch-Schönlein purpura in adults: outcome and prognostic factors, Pillebout [/bib_ref].
Until now, the etiology of IgA vasculitis remains unknown. However, the deposition of IgA1-dominant immune complexes in target organs plays a pivotal role in the pathophysiology of the condition [bib_ref] Serum levels of galactose-deficient IgA in children with IgA nephropathy and Henoch-Schönlein..., Lau [/bib_ref].
The clinical scope of the disorder essentially includes cutaneous purpura, arthralgias and/or arthritis, glomerulonephritis, and acute enteritis [bib_ref] Henoch-Schönlein purpura in adults: outcome and prognostic factors, Pillebout [/bib_ref]. These clinical presentations may develop within days to weeks and differ in their sequence of manifestation. Palpable purpura has been reported in 96-100% of cases at presentation, arthralgia and/or arthritis are seen in 61%, and gastrointestinal features in 48-53% of the cases. Approximately one-third of the cases develop renal failure with a creatinine clearance of <50 ml/min/m² within four months of presentation [bib_ref] Henoch-Schönlein purpura in adults: outcome and prognostic factors, Pillebout [/bib_ref].
The American College of Rheumatology 1990 criteria for the classification of IgA vasculitis are utilized for diagnostic purposes. These criteria include the following: age at onset of 20 years or more, the presence of a slightly elevated palpable purpuric rash, acute abdominal pain, and biopsy showing granulocyte in the walls of small arteries. The presence of at least two criteria carries a sensitivity of 87.1% and a specificity of 87.7% for the diagnosis of IgA vasculitis [bib_ref] The American College of Rheumatology 1990 criteria for the classification of Henoch-Schönlein..., Mills [/bib_ref].
Hidradenitis suppurativa (HS) is a chronic inflammatory, recurrent, and debilitating skin disorder that usually presents after puberty with painful, deeply located, and inflamed lesions in the apocrine glandbearing tissues, usually in the axillaries, inguinal, and anogenital areas [bib_ref] What causes hidradenitis suppurativa, Paus [/bib_ref]. It affects 1% of the global population.
HS significantly decreases the quality of life. Patients report self-consciousness, embarrassment, and inability to participate in athletic and social activities [bib_ref] A cross-sectional epidemiological study of hidradenitis suppurativa in an Irish population (SHIP), Delany [/bib_ref].
This report presents a case of IgA vasculitis in a patient receiving adalimumab for HS. A 26-year-old man presented to the emergency department with moderate diffuse abdominal pain and painless purpuric erythematous rash over his lower extremities. It was associated with bilateral lower limb swelling and pain with swelling and stiffness of both ankles and left knee. His symptoms began two weeks ago and progressed gradually. He was clinically assessed in a private clinic and was treated with prednisone 30 mg daily for a short period and a quick taper. His symptoms responded marginally to prednisone for a few days. Then, his abdominal pain became severe with associated nausea, non-projectile and non-bloody vomiting, and a single episode of bloody diarrhea. His rash further extended to involve his upper thigh and buttock area. He denied any constitutional symptoms, headache, dizziness, chest pain, palpitation, shortness of breath, or change in his urine color.
## Case presentation
His past medical history is significant for hidradenitis suppurativa for three years, for which he follows up with dermatology and has been on adalimumab 80 mg every other week for two years. He is not on any other medications and is allergic to penicillin and sodium diclofenac. There was no history of recent travel, contact with sick patients, or infection. He has no family history of autoimmune diseases, malignancies, or similar conditions. Past surgical history showed gastric sleeve surgery three years ago with no complications.
On physical examination, he looked in pain but not in distress. His weight is 72 kg, with a height of 168 cm and a body mass index of 25.5. His vital signs were all normal. His abdomen was soft and lax, with only moderate epigastric tenderness and no organomegaly or guarding. Lower extremity examination revealed diffuse non-tender raised purpuric rash over both shins and thighs with no evidence of bleeding or discharge [fig_ref] FIGURE 1: Classic palpable purple-colored purpura involving the lower extremities of the patient [/fig_ref]. Chest, cardiovascular and neurological examination was unremarkable. The results of the laboratory investigation are listed in [fig_ref] TABLE 1: Laboratory investigations for the patient were done upon his presentation to the... [/fig_ref]. Urinalysis did not show any evidence of hematuria. Abdominal ultrasound showed normal liver, gall bladder, spleen, and kidneys. Based on that, he was admitted to the medical ward as a case of IgA vasculitis associated with hidradenitis suppurativa under adalimumab therapy. Adalimumab was stopped, and the patient was treated with intravenous pulse methylprednisolone at a dose of 500 mg once daily for three days and supportive therapy. After three days of admission, he responded dramatically to this treatment regimen with significant improvement in his abdominal pain, arthritis, and skin rash. For that, he was discharged home after three days of inpatient management to continue on oral prednisone 60 mg once daily for two weeks with a quick taper to nil over one month with rheumatology follow-up since September 2021 until now.
# Discussion
Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine that is known to play a role in host defense mechanisms and initiates the response to local injury [bib_ref] Tumor necrosis factor antagonist mechanisms of action: a comprehensive review, Tracey [/bib_ref]. Currently, there are multiple anti-TNFα, which are monoclonal antibodies, one of which is adalimumab [bib_ref] Pharmacology of TNF blockade in rheumatoid arthritis and other chronic inflammatory diseases, Taylor [/bib_ref]. Many side effects are associated with anti-TNFα therapy, and they include increased risk of infections such as tuberculosis, malignancies, infusion-or injection-related skin reactions, cardiovascular disease, and autoimmunity [bib_ref] Investigating the potential side effects of anti-TNF therapy for rheumatoid arthritis: cause..., Atzeni [/bib_ref].
It is uncommon for adalimumab to be involved in association with IgA vasculitis. However, a review of the literature revealed two cases of IgA vasculitis in patients under adalimumab therapy.
The first case is a 19-year-old male, known case of Crohn's disease on adalimumab, who presented with a 10-day history of diffuse arthritis and a non-blanchable purpuric rash on both legs that were preceded by a short-term coryzal illness. Initially, the patient was managed conservatively, and a multi-disciplinary decision was made to commence a trial of adalimumab. However, the patient returned three days later with recurrence and an inability to bear weight in the lower extremity joints. The patient was then treated with right ankle intra-articular steroids along with systemic steroids as adalimumab was discontinued. His IgA vasculitis-related symptoms resolved entirely after a few weeks [bib_ref] Henoch-Schönlein purpura complicating adalimumab therapy for Crohn's disease, Rahman [/bib_ref].
The second case is a 33-year-old Caucasian man known to have ulcerative colitis for three years on adalimumab who was admitted to the hospital for a recurrent erythematous, palpable, non-blanching purpuric rash in the lower extremities bilaterally with arthritis in the ankles, knees, and elbows. The rash recurred with more severity and ascended to the buttocks, lower back, and abdomen. Adalimumab was stopped, and the patient was treated with methylprednisolone 20 mg intravenously every eight hours. As a result, the rash and arthritis improved significantly [bib_ref] Henoch-Schönlein purpura with adalimumab therapy for ulcerative colitis: a case report and..., Laconti [/bib_ref]. HS has been reported to be associated with multiple types of vasculitides. A case series and literature review revealed a rare association between HS and vasculitis seen in five reported cases. Three out of these showed an association between granulomatosis with polyangiitis and HS. Additionally, one reported case demonstrated an association between Behcet's disease and HS. Finally, one reported case showed an association between Takayasu's arteritis and HS [bib_ref] Hidradenitis suppurativa and vasculitis: a case series and literature review of a..., Alavi [/bib_ref].
According to our knowledge, our case is the first that may demonstrate an association between IgA vasculitis and adalimumab as a therapy for a co-associated HS. Additionally, our case adds to the observation of an increasing number of IgA vasculitis cases in association with adalimumab therapy. Based on the literature earlier, HS and/or adalimumab may be both contributing risk factors to the onset of IgA vasculitis through an unknown mechanism.
# Conclusions
There is a rare association between IgA vasculitis and anti-TNFα biological agents. This limited but seemingly growing evidence in the literature suggests that anti-TNFα may elicit the development of IgA vasculitis. We support this theory suggested by Rahman et al. and LaConti et al. Concurrently, literature also suggested that HS might promote vasculitis. We believe further studies are needed to determine the pathophysiological link and predisposition in the inhibition of TNFα and the presence of HS that lead to the deposition of IgA complexes systemically.
# Additional information disclosures
Human subjects: Consent was obtained or waived by all participants in this study. -issued approval -. This is a case report which does not require ethical approval. However, consent was taken from the patient himself with maintaining confidentiality. Our institution does not require ethical approval for this type of research. Thank you. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
[fig] FIGURE 1: Classic palpable purple-colored purpura involving the lower extremities of the patient. [/fig]
[table] TABLE 1: Laboratory investigations for the patient were done upon his presentation to the emergency department. [/table]
[table] Table 2: provides a summary of all cases of IgA vasculitis associated with adalimumab therapy that are reported in the literature. [/table]
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Dynamic Cerebral Autoregulation Is Acutely Impaired during Maximal Apnoea in Trained Divers
Aims: To examine whether dynamic cerebral autoregulation is acutely impaired during maximal voluntary apnoea in trained divers.Methods: Mean arterial pressure (MAP), cerebral blood flow-velocity (CBFV) and end-tidal partial pressures of O 2 and CO 2 (PETO 2 and PETCO 2 ) were measured in eleven trained, male apnoea divers (2862 yr; 18262 cm, 7667 kg) during maximal ''dry'' breath holding. Dynamic cerebral autoregulation was assessed by determining the strength of phase synchronisation between MAP and CBFV during maximal apnoea.Results: The strength of phase synchronisation between MAP and CBFV increased from rest until the end of maximal voluntary apnoea (P,0.05), suggesting that dynamic cerebral autoregulation had weakened by the apnoea breakpoint. The magnitude of impairment in dynamic cerebral autoregulation was strongly, and positively related to the rise in PETCO 2 observed during maximal breath holding (R 2 = 0.67, P,0.05). Interestingly, the impairment in dynamic cerebral autoregulation was not related to the fall in PETO 2 induced by apnoea (R 2 = 0.01, P = 0.75).Conclusions: This study is the first to report that dynamic cerebral autoregulation is acutely impaired in trained divers performing maximal voluntary apnoea. Furthermore, our data suggest that the impaired autoregulatory response is related to the change in PETCO 2 , but not PETO 2 , during maximal apnoea in trained divers.
# Introduction
Cerebral autoregulation is a compensatory mechanism which acts to maintain cerebral blood flow at a near constant value, despite fluctuations in arterial blood pressure or intracranial pressure [bib_ref] Cerebral autoregulation dynamics in humans, Aaslid [/bib_ref] [bib_ref] Transfer function analysis of dynamic cerebral autoregulation in humans, Zhang [/bib_ref]. It can be argued that autoregulation of cerebral blood flow and, by extension, cerebral O 2 delivery is nowhere more important for human survival than during prolonged asphyxia. And perhaps an extreme model of asphyxia can be found in the breath-hold diver performing a maximal apnoea [bib_ref] Breath-hold diving as a brain survival response, Dujić [/bib_ref] [bib_ref] Impact of breath holding on cardiovascular respiratory and cerebrovascular health, Dujic [/bib_ref].
In trained breath-hold divers, arterial O 2 tension may approach ,30 mmHg, and CO 2 tension may reach as high as 91 mmHg, by the breakpoint of maximal apnoea [bib_ref] Voluntary Breathholding. Iii. The Relation of the Maximum Time of Breathholding to..., Ferris [/bib_ref] [bib_ref] Breath holding after breathing of oxygen, Klocke [/bib_ref] [bib_ref] Observations on holding the breath, Schneider [/bib_ref] [bib_ref] Increased serum levels of the brain damage marker S100B after apnea in..., Andersson [/bib_ref] [bib_ref] Influence of lung volume, glossopharyngeal inhalation and P ET O2 and P..., Overgaard [/bib_ref]. In spite of this extreme asphyxia, cerebral tissue oxygenation is well-maintained during maximal apnoea due to a compensatory cerebral hyperaemia [bib_ref] Cerebral and peripheral hemodynamics and oxygenation during maximal dry breath-holds, Palada [/bib_ref]. The majority of the rise in cerebral blood flow during apnoea is achieved by vasodilation of cerebral resistance vessels, consequent to hypercapnia [bib_ref] Mechanisms of the cerebrovascular response to apnoea in humans, Przyby Owski [/bib_ref] [bib_ref] Facial immersion in cold water enhances cerebral blood velocity during breath-hold exercise..., Kjeld [/bib_ref]. Maximal apnoea also evokes a powerful vasoconstriction of the peripheral tissues, leading to a pronounced arterial hypertension [bib_ref] Cerebral and peripheral hemodynamics and oxygenation during maximal dry breath-holds, Palada [/bib_ref] [bib_ref] Cardiovascular Regulation During Apnea in Elite Divers, Heusser [/bib_ref] [bib_ref] Diving response and arterial oxygen saturation during apnea and exercise in breath-hold..., Andersson [/bib_ref]. Indeed, approxi-mately one-third of the cerebral blood flow response to brief apnoea (i.e., 20 s) is contributed by the increase in arterial blood pressure [bib_ref] Mechanisms of the cerebrovascular response to apnoea in humans, Przyby Owski [/bib_ref]. The observation that arterial hypertension partly explains the rise in cerebral blood flow suggests that cerebral autoregulation is functionally impaired during maximal voluntary apnoea. To date, no study has examined dynamic cerebral autoregulation (dCA) during prolonged breath holding.
The primary aim of this study was to quantify the effectiveness of dCA during maximal ''dry'' apnoea in trained breath-hold divers. Previous studies indicate that the relationship between arterial CO 2 on cerebrovascular pressure-reactivity is nonlinear, primarily affecting the phase more so than the amplitude dynamics of the cerebral autoregulatory response [bib_ref] Nonlinear modeling of the dynamic effects of arterial pressure and CO 2..., Mitsis [/bib_ref] [bib_ref] Effect of CO 2 on dynamic cerebral autoregulation measurement, Panerai [/bib_ref] [bib_ref] Assessment of Autoregulation by Means of Periodic Changes in Blood Pressure, Birch [/bib_ref] [bib_ref] Interaction among autoregulation, CO 2 reactivity, and intracranial pressure: a mathematical model, Ursino [/bib_ref]. Moreover, it is well-established that arterial O 2 tension exerts a modulatory effect on dCA, wherein arterial hypoxaemia seemingly impairs the autoregulatory response [bib_ref] The interaction of carbon dioxide and hypoxia in the control of cerebral..., Mardimae [/bib_ref] [bib_ref] Dynamic cerebral autoregulation during and following acute hypoxia: role of carbon dioxide, Querido [/bib_ref] [bib_ref] The effect of oxygen on dynamic cerebral autoregulation: critical role of hypocapnia, Ogoh [/bib_ref]. Consequently, dCA was assessed using phase synchronisation analysis [bib_ref] Phase dynamics in cerebral autoregulation, Latka [/bib_ref] [bib_ref] Increased phase synchronization and decreased cerebral autoregulation during fainting in the young, Ocon [/bib_ref] on spontaneous, beatto-beat values of mean arterial blood pressure (MAP) and middle cerebral artery blood flow-velocity (CBFV) during maximal apnoea. It was hypothesised that trained divers would display signs of impaired dCA by the breakpoint of apnoea, evidenced by a significant increase in the strength of phase synchronisation between arterial pressure and cerebral blood flow-velocity. Moreover, it was expected that those divers with the highest levels of arterial hypercapnia would demonstrate the largest increases in phase synchronisation by the apnoea breakpoint.
# Methods
# Ethics statement
The present study conformed to the principles outlined in the Declaration of Helsinki and was approved by the research ethics board at the School of Medicine, University of Split, Croatia.
## Subjects and experimental design
Eleven trained, male apnoea divers (2862 yr; 18262 cm, 7667 kg) volunteered to participate in the present study, and provided written informed consent. Within the 6 months prior to experimental testing, the divers were engaged in apnoea training at least 3 times a week, were each session lasted approximately 60 to 90 min. The subjects underwent a pre-participatory health screening to ensure they were physically active non-smokers, with no history of cardiac, pulmonary or metabolic disease.
Subjects first performed a number of pulmonary function tests in the supine position, after which they were instructed to remain supine for 10 min before performing breath-hold manoeuvres (i.e., rest period). Breath-hold manoeuvres commenced after full inflation to total lung capacity (TLC) while wearing a nose-clip. The subjects were instructed to keep their glottis closed during each breath-hold. Subjects were discouraged from performing preparatory hyperventilation, and were not allowed to perform glossopharyngeal insufflations prior to the manoeuvre. The subjects completed two practice trials, followed by 3 experimental breath holds, separated by at least 10 min of supine rest. A repeat apnoea trial did not begin until haemodynamic variables had returned to baseline. Subjects were encouraged to completely relax their respiratory musculature during the early period of the breath-hold (i.e., easy-going phase) and, should the urge arise, to allow respiratory contractions to develop ''naturally'' toward the end of breath holding. The subjects were also instructed to avoid activation of their peripheral musculature. No further explanation of the apnoeic effort was provided.
## Pulmonary function testing and end-tidal gases
Before each experiment, the subjects performed a forced vital capacity (FVC) manoeuvre while in the supine posture (Quark PFT, Cosmed, Rome, Italy). All pulmonary function testing was performed and reported in accordance with the American Thoracic Society guidelines [bib_ref] Standardisation of spirometry, Miller [/bib_ref]. Expired gases were continuously sampled at the mouth using a mass spectrometer (AMIS 2000, Innovision A/S, Odense, Denmark) and, from these data, the endtidal partial pressures of O 2 and CO 2 (PETO 2 and PETCO 2 ) were computed.
## Electrocardiogram, arterial oxygen saturation, blood pressure and cerebral blood flow-velocity
Arterial O 2 saturation of haemoglobin (SaO 2 ) was measured via finger pulse oximetry (Poet II, Criticare Systems, Waukesha, WI). Beat-by-beat arterial blood pressure was measured using a pneumatic cuff placed around the middle phalanx of the nondominant hand, and was connected to a photoplethysmograph (Finometer, Finapress Medical Systems, Arnhem, Netherlands). The hand bearing the finger cuff was positioned at the level of the heart in order to negate hydrostatic pressure artifact. The hand was kept in this position for the duration of the experimental protocol. Heart rate and rhythm were monitored continuously using a standard 3-lead electrocardiogram module that was interfaced with the Finometer unit. CBFV was obtained via transcranial Doppler ultrasound of the proximal middle cerebral artery, measured using a 2-MHz probe fixed at a constant angle over the right posterior temporal ''window'' (Transcranial Doppler, Multigon, Yonkers, NY, USA).
## Data collection and signal processing
All signals were sampled continuously at 1000 Hz (Powerlab 16 SP, ADInstruments Inc., Castle Hill, Australia) and stored on a personal computer for off-line analyses. Mean arterial pressure (MAP) and mean CBFV were computed as the arithmetic mean of the arterial pulse wave and transcranial Doppler flow-velocity signals over each cardiac beat interval, respectively [bib_ref] Spontaneous beat-by-beat fluctuations of total peripheral and cerebrovascular resistance in response to..., O'leary [/bib_ref]. Cerebrovascular resistance index (CVRi) was calculated as MAP/CBFV. The discontinuous, beat-by-beat time series were resampled to 2 Hz using cubic spline interpolation.
## Phase synchronisation and dynamic cerebral autoregulation
Phase synchronisation analysis describes the strength of interaction between the intrinsic phases (W) of two weakly-coupled, nonstationary, nonlinear chaotic oscillators [bib_ref] Synchronization in noisy systems and cardiorespiratory interaction, Rosenblum [/bib_ref] [bib_ref] Identification of coupling direction: application to cardiorespiratory interaction, Rosenblum [/bib_ref]. Two oscillators are said to be 'synchronised' or 'phase-locked' when the difference in their phases remain constant over a given length of time [bib_ref] Phase dynamics in cerebral autoregulation, Latka [/bib_ref] [bib_ref] Increased phase synchronization and decreased cerebral autoregulation during fainting in the young, Ocon [/bib_ref] [bib_ref] Comparison of Hilbert transform and wavelet methods for the analysis of neuronal..., Van Quyen [/bib_ref] [bib_ref] Performance of different synchronization measures in real data: a case study on..., Quiroga [/bib_ref]. In the present study, the strength of phase synchronisation between MAP and CBFV was used to assess the effectiveness of dCA during maximal apnoea. That is, MAP and CBFV were considered to act as two autonomous oscillators, weakly-coupled in their phases through the action of the effector, dCA. According to this paradigm, dCA is intact when the oscillators CBFV and MAP are not synchronised in their phase dynamics [bib_ref] Phase dynamics in cerebral autoregulation, Latka [/bib_ref] [bib_ref] Increased phase synchronization and decreased cerebral autoregulation during fainting in the young, Ocon [/bib_ref] ; i.e., the oscillator CBFV operates nearindependently of MAP. Conversely, when the dynamics of MAP and CBFV are tuned such that their phases become synchronised, fluctuations in arterial pressure are ''passively'' transmitted through the cerebral vasculature unabated, portending autoregulatory failure.
The strength of phase coupling between MAP and CBFV, and thus the effectiveness of dCA, was quantified using the phase synchronisation index (PhSI). The method used to calculate PhSI was modified from that described by others [bib_ref] Phase dynamics in cerebral autoregulation, Latka [/bib_ref] [bib_ref] Increased phase synchronization and decreased cerebral autoregulation during fainting in the young, Ocon [/bib_ref] [bib_ref] Respiration drives phase synchronization between blood pressure and RR interval following loss..., Ocon [/bib_ref]. In brief, the 2 Hz interpolated data for MAP and CBFV were band-pass filtered using a 5 th order, zero-phase Butterworth filter, with a low and high cut-off frequency of 0.01 and 0.15 Hz, respectively. These frequency cut offs were chosen based on previous observations that dCA is most active within the low to very-low frequency region [i.e., 0.01-0.15 Hz; 1,2]. This filtering approach also had the effect of removing variability in each time-series due to respiration and the cardiac cycle. The intrinsic phases of the filtered MAP and CBFV time series, W MAP (t) and W CBFV (t), were extracted using the Hilbert transform [bib_ref] Increased phase synchronization and decreased cerebral autoregulation during fainting in the young, Ocon [/bib_ref] [bib_ref] Respiration drives phase synchronization between blood pressure and RR interval following loss..., Ocon [/bib_ref]. The distribution of phase differences between MAP and CBFV was then determined as: DW(t)~W MAP (t){W CBFV (t). DW(t) was wrapped to the interval -180 to 180u, such that a negative value suggests that changes in MAP 'lead' those observed in CBFV. PhSI is a quantity that represents the time-dependent stability of DW(t), and was calculated as:
[formula] PhSI(t)~ffi ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi S cos DW(t)T 2 zS sin DW(t)T 2 qð1Þ [/formula]
where the brackets AENae denotes a 30-s moving average window. PhSI lies within the interval 0#PhSI #1. A value of PhSI = 1 indicates perfect phase synchronisation, while PhSI = 0 suggests complete absence of phase synchronisation between signals MAP and CBFV. Therefore, in the context of the present study, a decrease in PhSI during apnoea was taken to represent an increased effectiveness of dCA, whereas an increase in PhSI was interpreted as an impairment of autoregulatory processes while breath holding. Phase synchronisation analysis was performed using MATLABH (The MathWorks, Inc., Natick, USA).
## Statistical analyses
Data were averaged across repeated trials. The dependent variables were averaged into four time-epochs representing: the resting period, the first and last 30-s of maximal apnoea, and the final 30-s of the 2 min recovery period. PETO 2 and PETCO 2 could not be measured while subjects were breath holding. As such, PETO 2 and PETCO 2 data were organised into 4 bins representing: 1) the resting period; 2) the mean of values observed 30-s immediately prior to apnoea; 3) the minimum and maximum values of PETO 2 and PETCO 2 , respectively, within the first 3 breaths immediately following the apnoea breakpoint; and: 4) the final 30-s of the 2-min post-apnoea recovery period. One-way analyses of variance were used to evaluate the effect of these time epochs on cardiovascular and cerebrovascular variables, and on parameters derived the phase synchronisation analysis. Pair-wise comparisons were assessed using the Bonferroni post-hoc adjustment. Pearson's correlation coefficient was used to determine whether any change in dCA function, assessed via PhSI, could be related to the changes in either PETO 2 and PETCO 2 observed during maximal voluntary apnoea. Results are presented as mean 6 standard error of the mean (SEM) unless stated otherwise. All data were analysed using SPSS 20.0 (SPSS, Inc., Chicago, IL, USA). Statistical analyses were considered significant if P,0.05.
# Results
## Pulmonary function and breath holding performance
All subjects presented with normal pulmonary function, where FVC, forced expiratory volume in 1 s (FEV 1 ) and FEV 1 /FVC were 6.3060.41 L (11667% predicted), 5.1460.24 L (11365% predicted) and 0.8260.02 (10062% predicted). The average duration of maximal voluntary apnoea was 199611 s (range 154 to 261 s). The coefficient of variation computed for the subject's repeated apnoeic efforts was 762%.
## Expired gases, cardiovascular and cerebrovascular variables during maximal voluntary apnoea
During the resting period before apnoea, PETO 2 and PETCO 2 were 12163 mmHg and 4161 mmHg, respectively. In the 30-s period before apnoea, PETO 2 increased to 13163 mmHg (P,0.05) while PETCO 2 fell to 3861 mmHg (P,0.05). Thus, despite verbal instruction, the subjects hyperventilated (albeit mildly) before performing maximal apnoea. Immediately postapnoea, PETO 2 and PETCO 2 were 6164 mmHg and 5661 mmHg -both values were different from those observed at rest (P,0.05). The average changes in PETO 2 and PETCO 2 from rest until the apnoea breakpoint were -5767 mmHg and +1461 mmHg, respectively. By the end of the 2-min recovery period, PETO 2 and PETCO 2 remained different from rest (10465 mmHg and 4664 mmHg, respectively; both P-values were ,0.05). [fig_ref] Figure 1: also displays the changes in DW and PhSI during maximal apnoea for... [/fig_ref] displays the changes in MAP and CBFV during maximal apnoea for a representative trained diver. The time-course changes in cardiovascular and cerebrovascular variables during maximal apnoea are reported in [fig_ref] Table 1: Cardiovascular and cerebrovascular parameters before, during and after maximal voluntary apnoea [/fig_ref]. CVRi increased from rest to the beginning of maximal apnoea (P,0.05). Conversely, MAP and CBFV decreased over the same period of time (P,0.05). Thereafter, MAP and CBFV progressively increased (P,0.05), while CVRi and SaO 2 continuously decreased (P,0.05), from the beginning until the end of apnoea. After 2-min of recovery, the above parameters returned to baseline. Apart from an initial rise at the beginning of apnoea (P,0.05), HR was not different from rest during and after breath holding. The absolute rise in PETCO 2 from rest until the apnoea breakpoint was strongly, and positively correlated with the increase in PhSI over the same period of time (R 2 = 0.67, P,0.05, left-panel [fig_ref] Figure 3: The influence of CO 2 and O 2 on the dynamic cerebral... [/fig_ref]. That is, the larger the rise in PETCO 2 and, by extension, arterial tension of CO 2 , the greater the impairment in dCA during maximal voluntary apnoea.
## Dynamic cerebral autoregulation during maximal voluntary apnoea
Interestingly, the magnitude of rise in PETO 2 was not identified as significant correlate of the rise in PhSI throughout breath holding (R 2 = 0.01, P = 0.75, right-panel [fig_ref] Figure 3: The influence of CO 2 and O 2 on the dynamic cerebral... [/fig_ref].
# Discussion
The novel findings of the present study were that: (i) the effectiveness of dCA is reduced during maximal voluntary apnoea in trained divers; and that (ii) this reduction was strongly related to the rise in PETCO 2 during apnoea. Our data suggest that the ability of the cerebral vessels to autoregulate blood flow, and therefore to protect the brain against abrupt oscillations in driving pressure, is acutely impaired in divers performing maximal ''dry'' apnoea. These findings have important implications for the cerebrovascular health and safety of trained apnoea divers.
We report that dCA was acutely impaired during prolonged, voluntary apnoea. This inference is substantiated by two lines of evidence. First, PhSI had increased above resting values by the breakpoint of maximal apnoea. The rise in phase-coupling strength between MAP and CBFV indicates a progressive weakening of the autoregulatory response over time [bib_ref] Phase dynamics in cerebral autoregulation, Latka [/bib_ref] [bib_ref] Increased phase synchronization and decreased cerebral autoregulation during fainting in the young, Ocon [/bib_ref]. Second, not only had DW decreased during maximal apnoea, but its value approached zero by the breakpoint. It has been argued that when DW approximates zero, changes in MAP affect those in CBFV with no temporal delay, signifying autoregulatory failure [bib_ref] Phase Relationship Between Cerebral Blood Flow Velocity and Blood Pressure: A Clinical..., Diehl [/bib_ref] [bib_ref] Effect of Carotid Endarterectomy or Stenting on Impairment of Dynamic Cerebral Autoregulation, Reinhard [/bib_ref]. The present finding that maximal voluntary apnoea weakens dCA is consistent with observations made by others. For example, Przyby owski et al. [bib_ref] Mechanisms of the cerebrovascular response to apnoea in humans, Przyby Owski [/bib_ref] reported that approximately one-third of the cerebral blood flow response to brief apnoea (,20 s) is mediated by increases in arterial blood pressure, indicating a partial failure of cerebral autoregulatory mechanisms during voluntary breath holding. More recently, Dineen et al. [bib_ref] Continuous estimates of dynamic cerebral autoregulation during transient hypocapnia and hypercapnia, Dineen [/bib_ref] demonstrated that the effectiveness of dCA is transiently reduced during voluntary apnoeas lasting ,35 s. These findings, in conjunction with those of our own, supports the idea that voluntary breath holding acutely impairs dCA.
Many investigators have demonstrated that elevated arterial CO 2 acutely impairs dCA during spontaneous breathing [bib_ref] Cerebral autoregulation dynamics in humans, Aaslid [/bib_ref] [bib_ref] Nonlinear modeling of the dynamic effects of arterial pressure and CO 2..., Mitsis [/bib_ref] [bib_ref] Effect of CO 2 on dynamic cerebral autoregulation measurement, Panerai [/bib_ref] [bib_ref] Assessment of Autoregulation by Means of Periodic Changes in Blood Pressure, Birch [/bib_ref] [bib_ref] Interaction among autoregulation, CO 2 reactivity, and intracranial pressure: a mathematical model, Ursino [/bib_ref] [bib_ref] Does hypercapniainduced impairment of cerebral autoregulation affect neurovascular coupling? A functional TCD..., Maggio [/bib_ref] ] -the precise mechanism(s) by which hypercapnia weakens the autoregulatory response is unclear. Nevertheless, our findings are in agreement with the above reports, insofar as the trained divers who displayed the largest rise in PETCO 2 during maximal apnoea presented with the greatest impairment in dCA. It is wellestablished that isocapnic hypoxia impairs the cerebral autoregulatory response in awake, spontaneously breathing humans [bib_ref] The interaction of carbon dioxide and hypoxia in the control of cerebral..., Mardimae [/bib_ref] [bib_ref] Dynamic cerebral autoregulation during and following acute hypoxia: role of carbon dioxide, Querido [/bib_ref] [bib_ref] The effect of oxygen on dynamic cerebral autoregulation: critical role of hypocapnia, Ogoh [/bib_ref]. It is therefore surprising that we observed no direct relationship between the fall in PETO 2 and the rise in PhSI during maximal apnoea. This finding does not necessarily obviate the thesis that arterial hypoxaemia contributes to the weakened autoregulatory response observed during maximal apnoea. Rather, it is possible that arterial O 2 tension impacts on dCA in a manner not elucidated by the correlation analysis performed on our group of trained divers.
It must be appreciated that factors other than arterial blood gas tensions are known to modulate the effectiveness of dCA, these include: (i) sympathetic activity; (ii) metabolite production; and (iii) perivascular innervation [bib_ref] Sympathetic regulation of cerebral blood flow in humans: a review, Ter Laan [/bib_ref] [bib_ref] Autonomic Neural Control of Dynamic Cerebral Autoregulation in Humans, Zhang [/bib_ref] [bib_ref] Autonomic neural control of cerebral hemodynamics, Mitsis [/bib_ref] [bib_ref] Perivascular nerves and the regulation of cerebrovascular tone, Hamel [/bib_ref] [bib_ref] Regulation of cerebral blood flow, Peterson [/bib_ref] [bib_ref] Cerebral Blood Flow Regulation by Nitric Oxide: Recent Advances, Toda [/bib_ref]. Our data do not allow us to comment on the role that these factors played in disrupting autoregulatory processes during maximal apnoea. Further investigations are required to elucidate the precise mechanisms responsible for impairing dCA during prolonged, voluntary breath holding.
## Implications for the cerebrovascular health of breathhold divers
The observation that PhSI had increased by the breakpoint of maximal apnoea begs the question: what is the impact of impaired autoregulation on cerebrovascular health during breath-hold diving? Certainly, there are numerous reports of serious brain damage and neurological impairment in habitual breath-hold divers [bib_ref] Repetitive breath-hold diving causes serious brain injury, Tamaki [/bib_ref] [bib_ref] Medical problems associated with underwater diving, Melamed [/bib_ref] [bib_ref] Neurological accidents caused by repetitive breath-hold dives: two case reports, Kohshi [/bib_ref] [bib_ref] Neurological manifestations in Japanese Ama divers, Kohshi [/bib_ref] [bib_ref] Neurological disorders after repetitive breath-hold diving, Gempp [/bib_ref] [bib_ref] Apnea diving: long-term neurocognitive sequelae of repeated hypoxemia, Ridgway [/bib_ref]. However, the primary cause of these brain injuries is often attributed to cerebral arterial gas embolism, resulting from pulmonary barotrauma and/or shunting of venous gas emboli formed in peripheral tissues at depth. In a state of impaired dCA, the cerebral vessels behave more like a ''pressure passive'' system, wherein low to very-low frequency variations in arterial pressure (i.e., 0.01-0.15 Hz) are passively transmitted through the cerebrovasculature unabated, resulting in potentially severe fluctuations in cerebral blood flow and intracranial pressure. Following the above, one may speculate that arterial hypertension induced by apnoea [bib_ref] Cerebral and peripheral hemodynamics and oxygenation during maximal dry breath-holds, Palada [/bib_ref] [bib_ref] Cardiovascular Regulation During Apnea in Elite Divers, Heusser [/bib_ref] [bib_ref] Diving response and arterial oxygen saturation during apnea and exercise in breath-hold..., Andersson [/bib_ref] , and concurrent impairment of cerebral autoregulation, may together promote the development of intracranial hypertension, and thus heighten the risk of cerebrovascular insult during breath-hold diving. Further research is warranted in order to determine whether impaired dCA during maximal breath holding leads to cerebrovascular insult in trained divers.
# Methodological considerations
We used transcranial Doppler ultrasound of the right middle cerebral artery (MCA) to estimate bulk cerebral blood flow during maximal breath holding [bib_ref] A comparison of transcranial Doppler and cerebral blood flow studies to assess..., Dahl [/bib_ref] [bib_ref] The Effects of Involuntary Respiratory Contractions on Cerebral Blood Flow during Maximal..., Cross [/bib_ref]. This technique does not measure volumetric blood flow per se, rather it quantifies the velocity of blood passing through the insonated artery [bib_ref] Validity of cerebral arterial blood flow calculations from velocity measurements, Kontos [/bib_ref]. Changes in the radius of the insonated artery may therefore alter blood velocity, independent of volumetric flow through the MCA. Importantly, however, evidence suggests that MCA diameter is stable over a wide range of MAP and arterial blood gases [bib_ref] Indexes of Flow and Cross-sectional Area of the Middle Cerebral Artery Using..., Poulin [/bib_ref] [bib_ref] MRI Measures of Middle Cerebral Artery Diameter in Conscious Humans During Simulated..., Serrador [/bib_ref]. With this point in mind, we are confident that our values of cerebral blood flow-velocity is a valid marker of bulk cerebral blood flow. It must be acknowledged that only trained breath-hold divers were recruited in this study, and that all apnoeas were performed under ''dry'' laboratory conditions. Therefore, we caution that our findings may not apply to untrained/naïve participants, or to the conditions imposed on subjects when breath holding under water at depth. On this latter point, it was not feasible to use transcranial Doppler ultrasound to measure CBFV under water. Future studies may overcome this limitation via computational modelling of the pressure-flow characteristics of peripheral and cerebral vessels [bib_ref] Theoretical modeling of micro-scale biological phenomena in human coronary arteries, Wong [/bib_ref] [bib_ref] Modelling of blood flow resistance for an atherosclerotic artery with multiple stenoses..., Wong [/bib_ref].
That the trained diver in [fig_ref] Figure 1: also displays the changes in DW and PhSI during maximal apnoea for... [/fig_ref] demonstrated a fall in PhSI within the first 60-s of apnoea suggests dCA transiently improved after the beginning of breath holding. This improved dCA may have been related to our observation that subjects, on average, were mildly hypocapnic immediately prior to breath holding. If this hypocapnia persisted throughout the first 60-s of apnoea, it is conceivable that the lowered arterial CO 2 tension mediated an improvement in dCA -at least for the individual displayed in [fig_ref] Figure 1: also displays the changes in DW and PhSI during maximal apnoea for... [/fig_ref]. However, it was not feasible to measure PETCO 2 during apnoeic efforts and, in turn, we cannot comment on whether arterial hypocapnia modulated dCA during the early phase of maximal apnoea. In relation to the above, it is of note that other investigators have reported that arterial hypercapnia does not develop to any great extent within the first 60 s of voluntary breath holding [bib_ref] Alveolar Gas Exchanges and Cardiovascular Functions during Breath Holding with Air, Hong [/bib_ref] [bib_ref] Alveolar gas composition and exchange during deep breath-hold diving and dry breath..., Ferretti [/bib_ref] [bib_ref] Alveolar gas exchange during simulated breath-hold diving to 20 m, Liner [/bib_ref].
# Conclusions
The present study clearly demonstrates that the phase dynamics of cerebral autoregulation are acutely impaired during maximal ''dry'' apnoea in trained divers. Moreover, we provide evidence that the degree of impairment in dCA is related to the magnitude of rise in PETCO 2 observed during apnoea. The potential for this impaired dCA to heighten the risk of cerebrovascular injury during breath-hold diving should be further explored. PhSI: phase synchronisation index between the MAP and CBFV time series; PETCO 2 : end-tidal partial pressure of CO 2 ; PETO 2 : end-tidal partial pressure of O 2 ; D: absolute difference in magnitude between resting values and those observed at the breakpoint of apnoea. This figure demonstrates that those individuals who displayed the largest rise in PETCO 2 also presented with the greatest impairment in dynamic cerebral autoregulation during apnoea. The fall in PETO 2 , and thus magnitude of arterial hypoxaemia, was not related to the impaired autoregulatory response observed during maximal apnoea in trained divers. doi:10.1371/journal.pone.0087598.g003
[fig] Figure 1: also displays the changes in DW and PhSI during maximal apnoea for representative trained diver. [/fig]
[fig] Figure 2: displays the group-averaged values for DW and PhSI obtained before, during and after maximal voluntary apnoea. PhSI had increased to values higher than resting values by the breakpoint of maximal voluntary apnoea (P,0.05). The mean change in PhSI from rest until the apnoea breakpoint (D) was 0.1860.03. PhSI had returned to baseline by the end of the post-apnoea recovery [/fig]
[fig] Figure 3: The influence of CO 2 and O 2 on the dynamic cerebral autoregulation during maximal voluntary apnoea. BH: breath-hold; [/fig]
[table] Table 1: Cardiovascular and cerebrovascular parameters before, during and after maximal voluntary apnoea. [/table]
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Bony fusion of the maxilla and mandible as a sequelae of noma: A rare case report
[fig_ref] Figure 1: A [/fig_ref] [fig_ref] Figure 1: A [/fig_ref] [fig_ref] Figure 1: A [/fig_ref] [fig_ref] Figure 2: A panoramic radiograph re veals the fusion of the maxilla and mandible... [/fig_ref]
## A b c
was postprocessed in the coronal and axial planes. Three dimensional volumerendered images were generated from the acquired data. The multidetector computed to mography scan showed a bony fusion of the right lateral half of the maxilla with the mandible, extending medially up to the right second premolar and laterally to the base of the proximal aspect of the coronoid process. The length of the fusion was approximately 5 cm. The central portion of the fused area was mildly radiolucent (low density), and the outer margins were dense. (Figs. 3 and 4) Based on these findings and the patient's history of illness, the final diagnosis was fusion of the maxilla and mandible on the right side as a sequelae of noma.
Surgical treatment was performed to completely excise the bony fusion of the right arch through an extraoral approach. The patient was operated on under general an aesthesia. A single extraoral incision was made from be hind the ear and angle of the mandible, splitting the lower lip on the right side. The bony fusion was removed and the right side was closed with a galea flap that was intro duced intraorally by making a tunnel over the zygomatic arch and sutured to make buccal mucosa. A lateral tongue flap was used to make the buccal and lingual vestibules. The decayed anterior and posterior teeth, along with bone and calculus, were removed. Considerably more opening between the arches was noted [fig_ref] Figure 5: Postoperative followup after 3 month reveals 25 mm mouth opening [/fig_ref]. The recovery was uneventful, and the patient was satisfied with the surgical goal of increasing the size of the pathway for eating.
# Discussion
Cancrum oris or noma is still a major health problem in many developing countries. The triad of malnutrition, poor oral hygiene, and debilitating disease contributes to
## A b c
its continued presence. Although the precise prevalence of noma is unknown, it ranges from 30,000 to 140,000 cases. [bib_ref] Noma: a neglected enigma, Marck [/bib_ref] In highincome countries, noma is now extremely rare. In contrast, noma still persists in the poorest develop ing countries, predominantly in Africa. [bib_ref] Risk factors for noma disease: a 6year, prospective, matched casecontrol study in..., Barattimayer [/bib_ref] The epidemiology of noma shows a similar pattern, although the mortality rate has been drastically reduced from 90% to approxima tely 8%10% due to modern antibiotics. [bib_ref] A survey of cases of cancrum oris seen in IleIfe, Nigeria, Oginni [/bib_ref] Although noma can affect infants, children, and adults, 1 a slight predilec tion may exist for females over males. [bib_ref] Noma: report of a case resulting in bony ankylosis of the maxilla..., Deeb [/bib_ref] The direct cause of noma remains a mystery. [bib_ref] Noma: a neglected enigma, Marck [/bib_ref] It has been postulated that the disease is triggered by a consortium of microorganisms, of which Fusobacterium necrophorum is a key component. [bib_ref] Cancrum oris, Wazir [/bib_ref] The most common predisposing fac tors are poor oral hygiene, dehydration, malnutrition, and concomitant illnesses, such as measles, scarlet fever, or tuberculosis, malignancies, especially leukaemia, and im munodeficiency disorders, such as AIDS. [bib_ref] Radiologic examination of trismus as a complication of cancrum oris, Lagundoye [/bib_ref] The infection may begin as acute necrotizing ulcerative gingivitis. [bib_ref] Noma: report of a case resulting in bony ankylosis of the maxilla..., Deeb [/bib_ref] The lesion may then ulcerate and spread to the buccal or lingual sulcus and to the mucosa of the lip or cheek. The progression of the destructive process results in extension to the skin. The greater involvement of the interior of the oral cavity with lesser involvement of the overlying skin has resulted in the lesion being described as a 'cone gangreneux'. Once the slough separates, seque stration of the exposed bone and teeth occurs. The process of defect formation is rapid and is typically welldefined. [bib_ref] Cancrum oris a 35year retrospec tive study, Lazarus [/bib_ref] Gangrene of the soft tissues may then be grad ually follow ed by scar tissue repair of the resulting defect. The appear ance of false fibrosis occurs. Fibrous ankylosis often sets in before the oral wound is completely healed, due to the contraction of scar tissue in the periphery of the defect. The fibrosis progresses in some patients to true bony fu sion between the mandible and maxilla, and sometimes also to the temporomandibular joint. [bib_ref] Ankylosis of the mandible from cancrum oris, Oluwasanmi [/bib_ref] The facial bones, jaws, and teeth may be affected to varying degrees. [bib_ref] Cancrum oris, Wazir [/bib_ref] The presenting clinical features are similar to those reported by our patient. The site of the cancrum oris ulcer can be identified as a dimple in the smooth contour of the cheek due to ulcer scarring. [bib_ref] Radiologic examination of trismus as a complication of cancrum oris, Lagundoye [/bib_ref] Oluwasanmi et al. [bib_ref] Ankylosis of the mandible from cancrum oris, Oluwasanmi [/bib_ref] reported 15 patients with cheek defects, of whom eight had a telltale cheek scar overlying the area of the defect, similar to what was found in our patient.
Several studies have shown that the clinicopathological features of cancrum oris are complicated by trismus. If the
## A b c
ankylosis of the jaws is intraarticular, the bony fusion is situated at the temporomandibular joint, and if it is extra articular, the bony fusion involves the jaws. [bib_ref] Radiologic examination of trismus as a complication of cancrum oris, Lagundoye [/bib_ref] However, the ankylosis in our case involved the maxilla and man dible rather than the temporomandibular joint, which is a rare finding in noma cases. Bony fusion of the mandible is serious and is the most disabling type of fusion. Diffi culty with mastication, impairment of speech, poor oral hygiene, and facial asymmetry result in physical and psy chological disabilities, particularly in young children with a complete inability to open the mouth. 7,13 These problems were observed in our case. Oluwasanmi et al. [bib_ref] Ankylosis of the mandible from cancrum oris, Oluwasanmi [/bib_ref] reported changes in the temporo mandibular joint in two patients who had diminished joint space, whereas in two patients, widening was observed in one plane and narrowing in another plane. In our case, no fusion of the condyles was observed, but a reduced joint could be clearly seen in the coronal section of the com puted tomography scan.
Diseases occasionally confused with noma that could have been considered in the differential diagnosis of this case include visceral leishmaniasis, yaws, gangosa, syph ilis, midline lethal granuloma, tuberculosis, leprosy, lupus erythematous, mucormycosis, agranulocytic ulceration, clostridia gangrene, some tropical fungal infections, oral cancer, and physical trauma. However, these diseases do not generally occur in young children, do not have a rapid progression that would fit the history described by our patient, or do not produce the extent of tissue destruction seen here. [bib_ref] Noma: report of a case resulting in bony ankylosis of the maxilla..., Deeb [/bib_ref] Eating is a major problem for patients with ankylosis. In a series of 17 patients, 57% were found to be anaemic, with haemoglobin levels of under 75% of normal (12 g/ dL); levels as low as 19%30% of normal were not un common. Malnutrition and undernutrition occur in many cases; growth retardation may be so severe that an adoles cent of 15 years may have the stature of a child of eight years. [bib_ref] Ankylosis of the mandible from cancrum oris, Oluwasanmi [/bib_ref] This dynamic was observed in our patient. Al though the age of our patient was 28 years, she weighed 37.1 kg, had a height of 146 cm, and had a very low basal metabolic rate of 975 calories.
In extraarticular ankylosis due to cancrum oris, the bony changes are associated with the destruction of the maxilla, the malar bone, and a portion of the mandible. The remaining bone is characterized by areas of sclerosis or rarefaction and the distortion of the normal configura tion with loss of the trabecular pattern. [bib_ref] Ankylosis of the mandible: analysis of 76 cases, Adekeye [/bib_ref] Further threedimensional computed tomography imag ing in our case was a step forward in being able to clearly visualize the exact architecture of the area of ankylosis prior to surgery, making a much better surgical plan pos sible.
Antibiotics are mainly indicated for the acute and fulmi nating early stage of the disease, while surgical interven tion is required for the chronic and disfiguring late stage. The eventual plastic and reconstructive operations are de signed to reflect individual needs. In order to improve the overall health status of the patients during the acute stage, vitamins can be administered and ionic and acidbase imbalances can be corrected. The surgical correction of noma deformities can be challenging due to the unique patterns of destruction in each case. Hence, no standard surgical procedure is common to all noma cases. [bib_ref] NOMA: a preventable "Scourge" of African children, Ogbureke [/bib_ref] [bib_ref] Free flap transfer for closure and interpositionarthroplasty in noma defects of the..., Giessler [/bib_ref] The fused bridges are identified and excised by extraoral ap proaches, and scars on the mucosa and the skin are also excised and removed using the same approach. [bib_ref] Surgical treatment of noma, Marck [/bib_ref] This patient presented to us after 23 years of suffering. Her history showed that she was from a remote area. She could not preserve data relevant to the diagnosis of her illness or information that would establish which infec tion led to complete trismus and the bony fusion of the maxilla and mandible. We were not able to establish the nature of the infection that she suffered from. However, based on the history, oral examination, radiologic exami nation, and the literature, bony fusion of the maxilla and mandible was considered to be a sequelae of noma. We hypothesized that the massive soft tissue necrosis probably extended into the periosteum and bone, leading to bony fusion of the maxilla and mandible, which is a rare seque lae of noma. Even in a developing country like India, cases of noma may arise in remote areas, and complications in volving fusion may occur due to a lack of awareness, mis diagnosis, and negligence. The oral diagnostician should take the necessary steps after diagnosing cases of noma and provide a level of followup appropriate for avoiding complications like those observed in this case.
[fig] Figure 1: A. A frontal extraoral photograph shows the scars on both right and left cheeks. B. An extraoral photograph on the right side reveals the scar. [/fig]
[fig] Figure 2: A panoramic radiograph re veals the fusion of the maxilla and mandible extending from the right upper second premolar to the tip of coronoid process of mandible. [/fig]
[fig] Figure 3: A. An axial CT image shows the continuous fusion between the maxilla and mandible on the right side. B. A coronal CT image shows the similar features of axial section. C. A coronal CT image shows no fusion of the condylar head of both sides. [/fig]
[fig] Figure 5: Postoperative followup after 3 month reveals 25 mm mouth opening. [/fig]
[fig] Figure 4: Threedimensional CT images. A. Anterior facial area reveals the fused bone. B. Right oblique anterior view indicates bony fusion extending from the right premolar region to the anterior border of ramus of mandible. C. Left oblique anterior view shows no bony fusion. [/fig]
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Immunogenomic determinants of tumor microenvironment correlate with superior survival in high-risk neuroblastoma
# Background
The presence of effector T cells in the tumor microenvironment has been associated with improved survival in adults with many types of cancer.Several studies of melanoma and other solid tumors have demonstrated that expression of dendritic cell (DC) and CD8 + T cell-associated genes, or a T cell-inflamed gene signature, is correlated with favorable prognosis and response to immunotherapy with checkpoint blockade therapy or tumor vaccines.T cell-inflamed tumors are characterized by type I interferon (IFN) activation, immune potentiating chemokines, antigen presentation, cytotoxic effector molecules, and activated CD8 + T cells.The inflamed tumor microenvironment is additionally characterized by IFN-induced inhibitory pathways such as programmed death-ligand 1 (PD-L1) and indoleamine-2, 3 dioxygenase, and higher proportions of FOXP3 + regulatory T cells.Other known predictors of response to immunotherapy include, but are not limited to, a high tumor mutational burden (TMB)and a high neoantigen load.While TMB and neoantigen load often highly correlate with each other,previous studies have demonstrated both markers have low correlation with the presence of T cell inflammation, and TMB (or neoantigen load) and T cell-inflamed gene expression may represent non-redundant predictive biomarkers of immune checkpoint inhibitors efficacy.In contrast, resistance to immunotherapy has been correlated with tumors that lack the T cell-inflamed signature. There is increasing evidence that signaling pathways intrinsic to the neoplastic cells may impair the local immune response in tumors. Tumor cellintrinsic activation of the WNT/β-catenin pathway has been associated with a lack of T Open access cell infiltration in melanoma, bladder cancer, and more broadly across cancer.Activation of the phosphoinositide 3-kinase (PI3K) signaling pathway through loss-offunction mutations in phosphatase and tensin homolog (PTEN) can likewise mediate a non-T cell-inflamed tumor microenvironment in melanoma,and inactivation of LKB1 can have a similar effect in lung adenocarcinoma.Further, in lymphoma, diminished activation and recruitment of T cells have been reported with MYC activation, largely through inhibition of macrophage activation.MYC and several other activated transcriptional pathways have more broadly been associated with non-T cell-inflamed tumors across cancer types.In contrast to adult cancers, pediatric neoplasms have low mutational burden and most are non-T cell-inflamed, with scarce tumor-infiltrating lymphocytes (TILs) among anti-inflammatory M2 tumor-associated macrophages (TAMs). Although the response to immune checkpoint inhibition is poor for many pediatric cancers, post-consolidation immunotherapy with monoclonal antibodies targeting the GD2 ganglioside combined with cytokines significantly improves survival for children with high-risk neuroblastoma.Further, high response rates were also reported in newly diagnosed patients in a single institutional study with induction chemotherapy combined with anti-GD2 antibody, 27 and significant antitumor immunity was observed in a Children's Oncology Group (COG) clinical trial testing irinotecan and temozolomide combined with anti-GD2 antibody and GM-CSF in patients with relapsed/refractory neuroblastoma. The immunobiology of the neuroblastoma microenvironment is an emerging field. To increase our understanding about how immunogenomic determinants influence neuroblastoma phenotype, we analyzed the correlation between patient survival and T cell-inflamed gene expression and neoantigen load in tumor. We demonstrate that both biomarkers are prognostic in children with high-risk neuroblastoma and identify tumor-intrinsic oncogenic signaling pathways activated in neuroblastomas with a non-T cell-inflamed phenotype. These findings enhance a framework, whereby T cellinflamed expression and neoantigen load can provide new prognostic information to inform treatment decisions, and may also lead to the development of future immune therapeutic interventions.
# Methods
## Study cohorts and datasets
Two neuroblastoma cohorts were analyzed. The discovery cohort included patients from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program (n=149; 123 high-risk) (dbGAP accession ID phs000218.v22.p8) (online supplemental table 1). RNAseq paired-end (PE) FastQ files, whole exome sequencing (WES) alignment BAM files, somatic mutation MAF (Mutation Annotation Format) files, and clinical data were downloaded from Genomic Data Commons (GDC)(https:// portal. gdc. cancer. gov) (accessed 07/2017). The validation cohort included patients with clinical information in the International Neuroblastoma Risk Group (INRG) Data Commons 31 and tumor RNAseq data in the Gabriella Miller Kids First (GMKF) program (n=198; 48 high-risk) (online supplemental table 2). Universal system identification (USI) numbers were used to link the datasets. Access to RNAseq PE FastQ files in GMKF could not be obtained at the time of study, and therefore, preprocessed gene expression TSV files from the GMKF data portal (https:// kidsfirstdrc. org/) (accessed 08/2020) were used for analysis. Of the 209 patients identified, 11 were determined by USI number to also be included in the discovery cohort, hence were excluded from the validation cohort; 198 were kept for validation (online supplemental table 2).
## Rnaseq gene expression quantification
The quality of raw sequencing reads was assessed by FastQC 32 (V.0.11.5) for the tumor samples in the discovery cohort. Read counts were quantified at transcript level using Kallisto 33 (V.0.44.0) with human reference assembly GRCh38 and Gencode gene annotation (V.28), summarized into gene level using tximport
## Identification of t cell-inflamed and non-t cell-inflamed tumor groups
Using a defined T cell-inflamed gene expression signature, the tumors in the discovery cohort were categorized into three groups (T cell-inflamed, non-T cell-inflamed, and intermediate) using consensus clustering methods following previous protocols.In brief, an expression matrix consisting of the 160 genes from the T cell-inflamed signature 21 was subset from the TMMnormalized and log 2 -transformed RNAseq gene expression quantification matrix and was used to cluster tumors into 12 clusters by ConsensusClusterPlus (V.1.42.0) using hierarchical clustering with Euclidean distance and Ward.D2 linkage (2000 bootstraps and 80% usage of gene features). Tumors were then assigned with each of the three immune groups based on high, low, or intermediate expression of the T cell-inflamed signature. The number of clusters was determined using the elbow method.
## Mapping of t cell-inflamed and non-t cell-inflamed tumor groups between discovery and validation cohorts
The assignment of tumor groups in the discovery cohort cannot be migrated directly to that of tumors in the validation cohort due to the relative nature of gene expression data without spike-in controls. To address this issue, we projected T cell-inflamed gene expression of the validation cohort to the space of the discovery cohort using 11 patients that overlap between the two cohorts. First, we normalized and log 2 -transformed gene expression within each cohort. We calculated a T cell-inflamed score for each tumor, defined as the mean expression of all Open access genes from the signature.Then, we fit a linear regression model on the T cell-inflamed scores of the discovery cohort and validation cohort using tumors from the 11 overlapping patients, Score T = −0.7798 + 1.2418 × Score G (adjusted R 2 =0.967), where ScoreT represents T cellinflamed score of the discovery cohort (T as TARGET), and Score G represents T cell-inflamed score of the validation cohort (G as GMKF). We used this model to convert all T cell-inflamed scores of tumors in the validation cohort ( Score G ) to values comparable to that of the discovery cohort ( ScoreT ), then sorted all tumors by T cellinflamed scores lower to higher. Lastly, we assigned new tumor groups to the validation cohort based on existing tumor groups from the discovery cohort, employing the rules as follows: for all tumors harboring a score less than or equal to that of the last non-T cell-inflamed tumor on the sorted list, assign as non-T cell-inflamed; for all tumors harboring a score greater than or equal to that of the first T cell-inflamed tumor on the sorted list, assign as T cell-inflamed; otherwise, assign as intermediate.
## Differential gene expression detection and pathway activation prediction
For the discovery cohort analysis, we focused on 19,883 protein-coding genes defined in Gencode annotation (V.28) and followed the protocol established in our previous work.In brief, after removing genes with low expression (defined as CPM (counts per million of mapped reads) ≤3), 15,580 genes with CPM>3 in at least 30 tumors were TMMnormalized and log 2 -transformed. Differentially expressed genes (DEGs) comparing non-T cell-inflamed with T cellinflamed groups were identified using Linear Models for Microarray Data (limma) voom 35 method with precision weights (V.3.36.2) and filtered by false discovery rate (FDR)adjusted p<0.05, and fold change ≥1.5 or ≤−1.5. Upstream transcriptional regulators and change of direction (activation or inhibition) as a result of target molecules (encoded by DEGs) were predicted using Ingenuity Pathway Analysis (IPA) (QIAGEN, Germany) causal network analysiswith the curated Ingenuity Knowledge Base (accessed 12/2017). Transcriptional programs activated in non-T cell-inflamed relative to T cell-inflamed tumors were filtered at overlap p<0.05 (measuring the enrichment of target molecules in the dataset) and z-score ≥2.0 (measuring the predicted activation level of the pathways). For the validation cohort analyses, preprocessed RNAseq expression data downloaded from the GMKF data portal was quantified using Kallisto 33 (V.0.44.0), and the per-tumor gene expression files were downloaded and aggregated into cohort level, TMMnormalized, and log 2 -transformed for further analysis.
Somatic mutation detection, HLA genotyping, and neoantigen prediction For the discovery cohort, the somatic mutations were harmonized using four somatic variant callers (MuTect2, VarScan2, SomaticSniper, and MuSE).After rigorous filtering following GDC's guidelines (https:// docs. gdc. cancer. gov/ Data/ File_ Formats/ MAF_ Format), somatic variants that were detected by at least two callers and passed all the filters were selected for further analysis. Total TMB was defined as the total number of nonsynonymous somatic mutations (NSSMs), those that were predicted to alter protein sequence in tumor (insertions/deletions, missense/nonsense/stopgain mutations, and those that modify splicing sites). Putative neoantigens were predicted from NSSMs using netMHCpan(V.4.0), filtered by gene expression from the RNAseq data described as follows. Patients' major histocompatibility complex (MHC) class I haplotypes were predicted from WES of germline DNA using Optitype (V.1.3.1). Nine-mer peptides were generated from the mutated site through a sliding window approach using in-house python scripts. Our previous work had suggested that peptides of SYFPEITHI 38 mutant score >25 or delta score (mutant -wildtype)>5 bind to MHC class I molecules.In this study, we used netMHCpan that covers more human leukocyte antigen (HLA) genotypes than SYFPEITHI. To select neoantigens that are likely to have strong binding affinity to HLA-A molecules and expressed in tumor, we filtered for 9-mer peptides of netMHCpan mutant score >0.638 (equivalent to IC 50 nmol, strong binding) or delta score >0.070 (correlated with SYFPEITHI delta score 5) and derived from genes upregulated compared with the median of its expression across all tumors.
T cell-inflamed gene expression and pathway score calculation For each tumor, a T cell-inflamed score was computed as the mean expression of the 160 genes involved in the signature after scaling and centering across all tumor samples.A pathway activation score was calculated to each tumor following our published protocol, requiring at least 50% of the pathway-specific target molecules to be upregulated in a tumor sample (relative to its median expression across all tumor samples) in non-T cell-inflamed relative to T cell-inflamed group. For pathways in which less than 10 target molecules were present, 5 or more molecules were required to be upregulated to classify the pathway as activated. For pathways in which less than 5 target molecules were present, only pathways with all molecules upregulated were classified as activated. In addition, for each pathway identified in this study (MYCN, ASCL1, SOX11, and KMT2A), the expression level of a pathway was defined by the mean expression of all target molecules from this pathway, which was then used to correlate with the T cell-inflamed gene expression across all tumors by Spearman's correlation.
# Survival analysis
Cox proportional hazards (PH) models were used to test the association between the tumor group (T cell-inflamed, non-T cell-inflamed, and intermediate) and the survival outcome (event-free survival (EFS); overall survival (OS)) in the discovery (n=118 high-risk patients diagnosed between 2000 and 2010) and validation cohorts (n=17 high-risk patients with survival data available) using R Open access package survival (function coxph) (V.2.41.3). Univariable and multivariable Cox PH models were used to assess the significance of tumor group as a single predictor or after adjusting for covariates including age, MYCN status, and ploidy. In addition, Kaplan-Meier (KM) estimator with log-rank test was performed using R package survminer (V.0.4.2).
Immunohistochemistry immunofluorescence staining Immunofluorescence (IF) staining on human neuroblastoma tumors was performed by the Human Immunologic Monitoring Core Facility at The University of Chicago using tissue from 17 intermediate or high-risk neuroblastomas (5 MYCN-amplified and 12 MYCN-non-amplified). Briefly, slides were baked, cleared, and rehydrated. After heat-inducted epitope retrieval, the slides were placed in a humidity chamber, blocked by 10% donkey serum for 1 hour, incubated with anti-CD8 Ab (Dako, M7103) at 1:100 dilution for 1 hour, followed by Cy3 donkey anti-Mouse IgG (Jackson Immunological Research Lab, 715-165-150) at 1:500 dilution for 1 hour. The slides then incubated with anti-Batf3 Ab (Novus, AF7437) at 1:40 dilution for 1 hour, followed by Cy5 donkey anti-Rabbit IgG (Jackson Immunological Research Lab, 711-175-152) at 1:200 dilution for 1 hour. After thorough wash, slides were incubated in DAPI and mounted with Fluoromount (Sigma, F4680). Images of the slides were taken using a Leica SP8 laser scanning confocal microscope at Integrated Light Microscopy Core Facility. A pathologist (PP) scored tumors for intensity and distribution of CD8 + cells and Batf3 + cells in a blinded fashion.
# Statistical analysis
For analysis of contingency tables including comparison of tumor sample frequency between groups, Fisher's exact test was used. Differential gene expression analysis between groups were performed using empirical Bayes regression models in limma voom with precision weights. For multiple comparisons, p-value was adjusted using Benjamini-Hochberg FDR correction for multiple testing.Spearman's correlation ρ was used for measuring statistical dependence between normalized and log 2 -transformed expression level of different genes and between gene expression of the T cell-inflamed signature and pathways. p<0.05 was considered statistically significant. Statistical analysis was performed using R (V.3.5.2) and Bioconductor (release 3.8).
# Results
A T cell-inflamed gene expression signature defines three distinct groups in neuroblastoma Using a defined T cell-inflamed gene expression signature, we categorized the 149 primary neuroblastoma tumors from the discovery cohort (TARGET) into three subsets. High expression of T cell signature genes (T cell-inflamed) was detected in 57 (38.3%) tumors, low or no expression (non-T cell-inflamed) was identified in 45. In the discovery cohort, 123 of 149 patients were classified as high-risk, whereas 48 of 198 patients in validation cohort have high-risk neuroblastoma.In analyses restricted to high-risk patients, 53 (43.1%) and 23 (47.9%) were categorized as T cell-inflamed in the discovery and validation cohorts, respectively, and 33 (26.8%) and 18 (37.5%) were categorized as non-T cell-inflamed.
In the discovery cohort, MYCN amplification was significantly more prevalent in the non-T cell-inflamed tumors In the discovery cohort, the T cell-inflamed and intermediate groups showed similar probabilities of survival.
## Open access
Therefore, we combined these two groups and compared probability of survival to patients with non-T cell-inflamed tumors (OS: p=0.0076, EFS: p=0.10, log-rank test) (KM estimator shown in. Similar associations between T cell-inflamed/intermediate tumors and improved survival were observed in the 17 high-risk patients with available survival data from the validation cohort (OS: p=0.016, EFS: p=0.0098). No significant association with survival outcome was detected for age, MYCN status, or ploidy. Patients were not selected by diagnosis year 2010 or earlier due to small sample size. However, EFS and OS were not significantly different
## Open access
Neoantigen load is a prognostic marker independent of the T cell-inflamed expression signature Neoantigens are mutant antigens that are only expressed on tumor cells and not normal cells. Neoantigen-derived epitopes (neoepitopes) are recognized by antigenspecific CD8 + T cells.To evaluate the neoantigen load in neuroblastoma tumors, WES data from 198 matched tumor/normal pairs of the discovery cohort carrying one Open access or more somatic single nucleotide variants (SNVs) were analyzed. After combining calls of four somatic callers and rigorous quality filtering, 4235 somatic SNVs were identified in 3369 genes. Each tumor harbors a median of 17 somatic SNVs (range, 1-168 SNVs), with 15 somatic SNVs predicted to alter protein sequences (range, 1-162), which is consistent with the somatic mutation profile previously reported in high-risk neuroblastoma.To investigate if neoantigen load was associated with outcome in high-risk patients, of 118 high-risk patients diagnosed between 2000 and 2010 in the discovery cohort, we analyzed tumors from 89 patients with both WES and RNAseq data available. The total number of neoantigens in tumor was determined by filtering for those predicted to bind to MHC class I molecule HLA-A. We focused on HLA-A molecule because the prediction algorithm for this allele is the most reliable. A median of 4 (range 1-30) candidate neoantigens were identified in 78 of 89 tumors. Seventy-four patients diagnosed between year 2000 and 2010 were included in survival analysis. We found that the neoantigen load was significantly associated with OS (p=0.00022, log-rank test) (figure 3A) and EFS (p=0.0044) (figure 3B), although there was no significant difference in neoantigen load between non-T cell-inflamed and T cell-inflamed groups (p=0.22, two-sided Wilcoxon rank-sum test) (figure 3C). We defined four patient groups (hereafter referred as, quadrants (Q)) split by the threshold of T cell-inflamed (Tinfl) gene expression in non-T cell-inflamed tumors and median of neoantigen load (Neo) (Spearman's correlation coefficient ρ=0.053, p=0.65) (figure 3D): Q1 (n=8), Tinfl low Neo high ; Q2 (n=19), Tinfl low Neo low ; Q3 (n=20), Tinfl high Neo low ; Q4 (n=27), Tinfl high Neo high . OS and EFS were significantly different according to quadrant assignment (OS: p=0.00083; EFS: p=0.0061, logrank test) (figure 3E,F). Patients in Q1 and Q4, who had tumors harboring high level of neoantigens, had superior outcome compared with those in Q2 and Q3 (figure 3E,F).
## Tumor-intrinsic oncogenic transcriptional programs associated with a non-t cell-inflamed phenotype
To investigate if tumor-intrinsic transcriptional programs may play a role in inhibiting T cell infiltration in non-T cell-inflamed neuroblastomas, we first analyzed tumors from the discovery cohort for signaling pathways intrinsic to the neoplastic cells that were previously reported to impair the local immune response in other tumor types. This includes somatic activation mutations in CTNNB1 or damaging mutations in repressors of the pathway (APC/ APC2/AXIN1/AXIN2), somatic copy number loss in PTEN or activation mutations in PIK3CA,activation mutations in VEGF-A,and loss of function mutations in B2M,STK11/LKB1, 19 IDH1/2,and NRAS/KRAS/ HRAS.Only three tumors harbored a missense mutation in AXIN2 (p.A113T), and two tumors had PIK3CA missense (p.K111N) or nonsense mutations (p.E888X), but none occurred at the known PIK3CA activation mutation positions.
We next took an unbiased approachto identify transcriptional programs that are activated in non-T cell-inflamed tumors by comparing the whole transcriptome RNAseq expression of 33 non-T cell-inflamed to 53 T cell-inflamed tumors from the high-risk patients in the discovery cohort. A total of 1730 genes were identified that were significantly differentially expressed between the two tumor groups, with 230 upregulated in non-T cell-inflamed group and 1500 upregulated in the T cell-inflamed group (FDR-corrected p<0.05, fold change ≥1.5 or ≤−1.5). Causal network analysisimages of all IHC slides are provided at https:// github. com/ riyuebao/ NBL-TME-Immunogenomics) demonstrated lower infiltration with CD8 + T cell and Batf3 + DCs in MYCN-amplified tumors compared with MYCNnon-amplified neuroblastomas (figure 4B), although the difference did not reach statistical significance in this small cohort (p=0.22, two-sided Fisher's exact test). In both the discovery and validation cohorts, the DC genes are highly correlated with the T cell-inflamed gene expression (p<0.05, Spearman's correlation) (online supplemental.
To determine if activation of transcriptional programs other than MYCN signaling is associated with the non-T cell-inflamed phenotype, we repeated the differential gene expression and causal network analyses using only MYCN non-amplified tumors (n=91). Genes significantly upregulated in 16 non-T cell-inflamed neuroblastomas compared with 49 T cell-inflamed tumors were used to predict upstream regulators. Three pathways (ASCL1, SOX11, and KMT2A) were identified to be activated in non-T cell-inflamed tumors without MYCN amplification (activation z-score ≥2.0, p<0.05) (figure 4C). We next calculated an activation score for each pathway using previously described methods. The results showed that the three pathways operate in a partially exclusive manner (online supplemental, with activation of SOX11, KMT2A, and ASCL1 signaling detected in 66%, 30%, and 30% of non-T cell-inflamed tumors, respectively, compared with less than 5% of the T cell-inflamed tumors. Taking together, the activation of one or more pathways regulated by MYCN, ASCL1, SOX11, or KMT2A was found in 85% of the non-T cell-inflamed tumors. The inverse correlation between the expression of T cell-inflamed gene signature and the four pathways (MYCN, ASCL1, SOX11, KMT2A) was confirmed in the validation cohort (online supplemental, providing strong evidence that the activation of the four Open access Open access transcriptional programs was significantly associated with a non-T cell-inflamed phenotype.
# Discussion
Although improved survival and response to immunotherapy have been observed in adults with cancers showing T cell infiltration, the immunobiology of neuroblastoma tumors and its association with outcome had been poorly understood. In this study, we categorized 149 clinically annotated primary neuroblastoma tumors in the TARGET program as T cell-inflamed, non-T cell-inflamed, and intermediate using a defined T cellinflamed gene expression signature. The gene signature also identified the same three tumor groups in an independent cohort comprised of publicly available tumor genomic data housed in the GMKF program linked to clinical information in the INRG Data Commons. In both cohorts, MYCN amplification was significantly more prevalent in the non-T cell-inflamed tumors compared with the T cell-inflamed tumors. Interestingly, we also found that patients in both cohorts diagnosed at age <18 months had tumors that were enriched in the non-T cell-inflamed tumor group.
In analyses restricted to high-risk patients in the TARGET cohort, OS was significantly better for those with T cell-inflamed tumors compared with those with non-T cell-inflamed tumors. A similar trend was observed for EFS, although statistical significance was not reached. Further, the T cell-inflamed signature maintained independent statistical significance for OS in multivariable analysis adjusted for age, MYCN status, and ploidy. This association between T cell-inflamed tumors and superior outcome was validated in the clinically annotated GMKF cohort of tumors. Because neoantigens are recognized by the immune system and can be targeted to increase anti-tumor immunity,we also analyzed neoantigen load in the neuroblastoma tumors. Although no significant difference in neoantigen load was detected among T cell-inflamed, non-T cell-inflamed, or intermediate groups, superior OS was seen in the cohort of patients with tumors harboring a high neoantigen load. Taken together, these results suggest that T cell-inflamed gene expression and high neoantigen load may independently
## Open access
impact the clinical behavior of neuroblastoma tumors, resulting in improved survival.
The lack of correlation between the T cell-inflamed expression signature (also known as an IFN-γ-associated expression signature) and TMB, which is highly correlated with neoantigen load, 51 has been reported in many adult cancers, including melanoma,head and neck, 7 and pan-cancer.It is well established in the literature that TMB (or neoantigen load) and T cell-inflamed expression are both prognostic but seemly have little correlation. In particular, the pan-cancer study reports four groups of patients determined by high/low IFN-γ-associated expression signature and high/low TMB.Only patients possessing high levels of both signatures had the best response rate, and a significant number of patients only showed high levels of one of the signatures.The mechanism underlying the decoupling of T cell-inflamed expression signature and TMB and neoantigen load remains to be explored.
Others have evaluated inflammatory cell infiltrates in neuroblastoma tumors using different methodologies and markers. Asgharzadeh and colleagues 53 assessed the relationship between TAMs and the clinical behavior of metastatic MYCN-non-amplified neuroblastoma. Using IHC, significantly greater numbers of infiltrating macrophages with CD163 staining, which identifies alternatively activated M2 macrophages, were observed in metastatic neuroblastomas compared with locoregional tumors. Thus, TAMs may promote aggressive growth in neuroblastoma, as reported in Hodgkin's lymphoma 54 and breast cancer.Further, expression studies using a TaqMan low-density array assay demonstrated higher levels of inflammation-related genes (CD14, CD33, FCGR3 (CD16), interleukin-6 receptor, and interleukin-10) in tumors from patients diagnosed at ≥18 months compared with younger patients. These inflammatory genes are largely expressed in macrophages and can signify intratumor macrophage polarization to the anti-inflammatory M2-like phenotype, suggesting that TAMs contribute to the aggressive clinical behavior of neuroblastomas associated with older age. Age is an established prognostic marker in neuroblastoma, and more favorable outcome is associated with age <18 months, reflecting the unique biology of infant tumors.Although different inflammatory cells were evaluated in our studies, we observed a higher prevalence of T cell-inflamed tumors in patients diagnosed ≥18 months compared with infants. While specific inflammatory cells differentially influence tumor growth, the age-dependent differences in expression of tumor-associated inflammatory cell genes may contribute to underlying favorable tumor phenotype that is commonly seen in infants with neuroblastoma.
More recently, Wei and colleagues 56 analyzed gene expression signatures of TILs in neuroblastomas, immune cells that have previously been reported to be predictive of clinical outcomes for patients with cancers.Similar to our study, higher levels of cytotoxic TIL signature genes were observed in the MYCN-non-amplified tumors compared with tumors with amplification of MYCN.
Further, these investigators also reported improved survival in a cohort of patients with MYCN-non-amplified tumors with increased signatures for activated NK cells, CD8 + T cells, cytolytic activity, clonal expansion of T cell receptors, and exhaustion markers.
The inverse correlation between MYCN amplification and T cell-inflamed tumors seen in our study and others suggests that MYCN signaling inhibits T cell infiltration in neuroblastoma tumors. In support of MYCN's role in mediating exclusion of T cells from the microenvironment of neuroblastoma tumors, we identified activation of MYCN signaling in non-T cell-inflamed tumors (activation z-score ≥2.0, p<0.05) comparing expression profiles between non-T cell-inflamed and T cell-inflamed tumors. In addition, we identified three transcriptional programs, ASCL1, SOX11, and KMT2A, that were activated in non-T cell-inflamed tumors without MYCN amplification. ASCL1 (alias hASH1 in human) is a known proneural transcription factor essential for neurogenesis. However, in neuroblastoma ASCL1 represses genes involved in neuron differentiation.Recent studies have demonstrated that ASCL1 is a MYCN-dependent and LMO1dependent member of the adrenergic neuroblastoma core regulatory circuitry (CRC), an interconnected autoregulatory loop of transcription factors whose expression is driven by themselves and other members of the CRC.Interestingly, LMO1 and the CRC members bind to enhancer elements and directly upregulate the ASCL1 gene, resulting in promotion of cell growth and repression of neuronal differentiation.Activation of ASCL1 signaling is also predictive of poor prognosis in neuroendocrine lung cancers.In glioblastoma, ASCL1 is critical to the maintenance of stem cells through activation of WNT signaling. 61 SOX11 is a transcription factor essential for neuron survival and neurite outgrowth.In our study, the expression of MYCN and SOX11 pathways is positively correlated (Spearman's ρ=0.81 in TARGET and 0.83 in GMKF, respectively, p<0.0001), suggesting the two mechanisms may interact. Indeed, recent studies reported that SOX11 was a direct target of MYCN.However, 30% of MYCN non-amplified tumors showed SOX11 pathway activation, which may indicate other signaling routes independent of MYCN. KMT2A (alias MLL1 in human) is an epigenetic regulator of neuronal function. In pancreatic cancer where anti-PD1/PD-L1 immunotherapy is ineffective, MLL1 directly binds to the promoter of the checkpoint inhibitor PD-L1 and activates its transcription, and combinatorial therapy of anti-MLL1 and anti-PD1/PD-L1 was proven to suppress tumor growth in mouse models.Taken together, these transcriptional programs support a stem cell-like phenotype in neural tissues, which is a consistent theme with what has been observed in adult tumors for a state of epithelial-mesenchymal transition being associated with immuno-oncology resistance.In conclusion, the association of improved survival with T cell-inflamed neuroblastoma and high neoantigen load indicate that crosstalk between tumor cells and components of the microenvironment influence neuroblastoma Open access phenotype. Our studies also suggest that tumor-intrinsic MYCN, ASCL1, SOX11, or KMT2A signaling may mediate immune exclusion in neuroblastoma. Understanding the molecular mechanisms that drive the presence or absence of T cell infiltration and neoantigen load should enable more personalized treatment approaches and provide insight for the development of new therapies that may enhance response to immunotherapy and improve outcome. Clinical trials testing the efficacy of anti-GD2 antibodies and other modalities of immunotherapy in patients with neuroblastoma tumors that are T cellinflamed or harbor high neoantigen load are warranted. |
Patients' experiences of, and engagement with, remote home monitoring services for COVID‐19 patients: A rapid mixed‐methods study
Introduction: Remote home monitoring models were implemented during the COVID-19 pandemic to shorten hospital length of stay, reduce unnecessary hospital admission, readmission and infection and appropriately escalate care. Within these models, patients are asked to take and record readings and escalate care if advised.There is limited evidence on how patients and carers experience these services. This study aimed to evaluate patient experiences of, and engagement with, remote home monitoring models for COVID-19.Methods: A rapid mixed-methods study was carried out in England (conducted from March to June 2021). We remotely conducted a cross-sectional survey and semistructured interviews with patients and carers. Interview findings were summarized using rapid assessment procedures sheets and data were grouped into themes (using thematic analysis). Survey data were analysed using descriptive statistics.Results: We received 1069 surveys (18% response rate) and conducted interviews with patients (n = 59) or their carers (n = 3). 'Care' relied on support from staff members and family/friends. Patients and carers reported positive experiences and felt that the service and human contact reassured them and was easy to engage with. Yet, some patients and carers identified problems with engagement (e.g., hesitancy to self-escalate care). Engagement was influenced by patient factors such as health and knowledge, support from family/friends and staff, availability and ease of use of informational and material resources (e.g., equipment) and service factors. Health Expectations. 2022;25:2386-2404. 2386 | wileyonlinelibrary.com/journal/hexThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
# | introduction
In recent years, there has been a shift in healthcare delivery, 1 with services having adopted technology in different ways, including virtual consultations, [bib_ref] Real-world implementation of video outpatient consultations at macro, meso, and micro levels:..., Greenhalgh [/bib_ref] [bib_ref] Advantages and limitations of virtual online consultations in a NHS acute trust:..., Shaw [/bib_ref] [bib_ref] Guidance on the introduction and use of video consultations during COVID-19: important..., Wherton [/bib_ref] [bib_ref] Digital health and care in pandemic times: impact of COVID-19, Peek [/bib_ref] or remote monitoring models of healthcare. [bib_ref] Transformational improvement in quality care and health systems: the next decade, Braithwaite [/bib_ref] [bib_ref] Digital health and care in pandemic times: impact of COVID-19, Peek [/bib_ref] Within remote home monitoring models, patients and carers are asked to record health readings in one place (e.g., at home), and these readings are reviewed and responded to by professionals elsewhere. [bib_ref] Remote home monitoring-of patients with cancer during the COVID pandemic: a pilot..., Steimer [/bib_ref] [bib_ref] Understanding heart failure; explaining telehealth-a hermeneutic systematic review, Greenhalgh [/bib_ref] These changes in healthcare delivery potentially alter the landscape of 'care', as they accompany or even move away from traditional face-to-face care models, [bib_ref] New networked technologies and carers of people with dementia: an interview study, Powell [/bib_ref] and instead place further emphasis on formal or informal carers providing care at a distance and reviewing readings remotely.This shift in healthcare delivery is also consistent with recent moves towards self-management and patient activation within healthcare, whereby accountability for care has changed. [bib_ref] Rethinking the patient: using burden of treatment theory to understand the changing..., May [/bib_ref] [bib_ref] Flipped healthcare for better or worse, Vallo Hult [/bib_ref] [bib_ref] Why does patient activation matter? An examination of the relationships between patient..., Greene [/bib_ref] Patients are becoming more involved in self-management, for example, learning how to detect and manage their symptoms, and treatments, and escalation of care associated with their condition, 7,13-17 and healthcare tasks (e.g., managing medication, organizing care appointments, taking measurements). [bib_ref] Taxonomy of the burden of treatment: a multi-country web-based qualitative study of..., Tran [/bib_ref] While some patients may welcome this, [bib_ref] Lay and health care professional understandings of self-management: a systematic review and..., Sadler [/bib_ref] there have been concerns that self-management places a burden on patients and families, rather than facilitating shared care. [bib_ref] Rethinking the patient: using burden of treatment theory to understand the changing..., May [/bib_ref] [bib_ref] Lay and health care professional understandings of self-management: a systematic review and..., Sadler [/bib_ref] Additionally, the effectiveness of these concepts is not fully understood yet. [bib_ref] Why does patient activation matter? An examination of the relationships between patient..., Greene [/bib_ref] [bib_ref] Lay and health care professional understandings of self-management: a systematic review and..., Sadler [/bib_ref] [bib_ref] Patient and public involvement in chronic illness: beyond the expert patient, Greenhalgh [/bib_ref] [bib_ref] Self-management education programmes by lay leaders for people with chronic conditions, Foster [/bib_ref] [bib_ref] How effective are expert patient (lay led) education programmes for chronic disease?, Griffiths [/bib_ref] The COVID-19 pandemic further enhanced and accelerated the need for healthcare services to use technology in care delivery [bib_ref] Digital health and care in pandemic times: impact of COVID-19, Peek [/bib_ref] and escalated the need for patient self-management. Remote home monitoring models have previously been used to provide care for chronic conditions. [bib_ref] Determining the cost of implementing and operating a remote patient monitoring programme..., Peretz [/bib_ref] [bib_ref] Predictive performance and impact of algorithms in remote monitoring of chronic conditions:..., Castelyn [/bib_ref] [bib_ref] Remote home monitoring of older surgical cancer patients: perspective on study implementation..., Jonker [/bib_ref] During the pandemic, remote home monitoring models were used for acute conditions such as COVID with the aim of shortening length of stay in hospital, reducing unnecessary hospital admissions or readmission and infection transmission and escalating care as needed. [bib_ref] Remote home monitoring (virtual wards) for confirmed or suspected COVID-19 patients: a..., Vindrola-Padros [/bib_ref] Many different types of COVID-19 remote home monitoring models were implemented throughout England. Some models referred patients from community services (e.g., GPs, hot hubs and emergency departments), known as COVID Oximetry @home. [bib_ref] Remote home monitoring (virtual wards) for confirmed or suspected COVID-19 patients: a..., Vindrola-Padros [/bib_ref] Others referred patients onto the service as early discharges from hospital, known as COVID virtual wards. See Box 1 for a brief description of services. [bib_ref] Remote home monitoring (virtual wards) for confirmed or suspected COVID-19 patients: a..., Vindrola-Padros [/bib_ref] According to national eligibility criteria, patients were eligible to receive these services if they had a confirmed or suspected diagnosis of COVID-19 and were either BOX 1. Description of COVID-19 remote home monitoring services [bib_ref] Remote home monitoring (virtual wards) for confirmed or suspected COVID-19 patients: a..., Vindrola-Padros [/bib_ref] symptomatic with COVID-19 and aged 65 years or older, or younger than 65 years of age if clinically extremely vulnerable.
While remote home monitoring models may reduce the need for staff to assess patients in person, they place more responsibility, commitment and workload onto patients and carers. [bib_ref] Rethinking the patient: using burden of treatment theory to understand the changing..., May [/bib_ref] For example, in COVID-19 remote home monitoring services, patients and carers are expected to measure and record oxygen saturations and escalate care if readings drop below certain thresholds. 29, [bib_ref] The implementation of remote home monitoring models during the COVID-19 pandemic in..., Vindrola-Padros [/bib_ref] This increased responsibility may be appropriate and beneficial for some patients, but may not be suitable for everyone. [bib_ref] Digital transformation could increase the burden of treatment on patients, Mair [/bib_ref] Some people may be unable to meet expectations placed on them by healthcare services and experience negative impacts from treatment burden. [bib_ref] Rethinking the patient: using burden of treatment theory to understand the changing..., May [/bib_ref] Negative impacts may include health consequences faced by patients due to not adhering to treatment and patients' professional, social, emotional and financial situation. [bib_ref] Taxonomy of the burden of treatment: a multi-country web-based qualitative study of..., Tran [/bib_ref] Different individuals may tolerate different levels of treatment burden, and it has been suggested that this needs to be assessed regularly as tolerance changes over time. [bib_ref] Rethinking the patient: using burden of treatment theory to understand the changing..., May [/bib_ref] [bib_ref] Thinking about the burden of treatment, Mair [/bib_ref] Many factors worsen treatment burden, including situational factors (e.g., travel), personal factors (e.g., beliefs and relationships) and structural factors (e.g., treatment factors and access to resources). [bib_ref] Taxonomy of the burden of treatment: a multi-country web-based qualitative study of..., Tran [/bib_ref] Therefore, formal and informal support networks are needed to support patients. [bib_ref] Understanding heart failure; explaining telehealth-a hermeneutic systematic review, Greenhalgh [/bib_ref] [bib_ref] What is quality in assisted living technology? The ARCHIE framework for effective..., Greenhalgh [/bib_ref] Treatment burden may negatively impact on patient experience and levels of engagement. This is problematic, given that patient engagement with remote home monitoring is crucial. Patient engagement has been defined as patients understanding the information that they are given ('receipt') and being able to perform the required activities ('enactment'). [bib_ref] The assessment, monitoring, and enhancement of treatment fidelity in public health clinical..., Borrelli [/bib_ref] [bib_ref] Measures of fidelity of delivery of, and engagement with, complex, face-to-face health..., Walton [/bib_ref] While previous research indicates factors that may influence patient engagement with treatment models more generally, [bib_ref] Understanding heart failure; explaining telehealth-a hermeneutic systematic review, Greenhalgh [/bib_ref] [bib_ref] Rethinking the patient: using burden of treatment theory to understand the changing..., May [/bib_ref] [bib_ref] Taxonomy of the burden of treatment: a multi-country web-based qualitative study of..., Tran [/bib_ref] [bib_ref] What is quality in assisted living technology? The ARCHIE framework for effective..., Greenhalgh [/bib_ref] there is a lack of research on patient experience and engagement with remote home monitoring services for an acute condition such as COVID-19. 29, [bib_ref] The implementation of remote home monitoring models during the COVID-19 pandemic in..., Vindrola-Padros [/bib_ref] If patients do not engage with these services, they may be at risk of negative outcomes that the service aimed to prevent, for example, silent hypoxia (very low oxygen saturations, often without breathlessness) [bib_ref] Remote management of Covid-19 using home pulse oximetry and virtual ward support, Greenhalgh [/bib_ref] and/or delayed admission to hospital. [bib_ref] Retrospective cohort study of admission timing and mortality following COVID-19 infection in..., Alaa [/bib_ref] [bib_ref] Oxygen and mortality in COVID-19 pneumonia: a comparative analysis of supplemental oxygen..., Mansab [/bib_ref] Additionally, if engagement is limited, then it is not possible to evaluate whether or not the service influences key outcome measures such as any changes in mortality or hospital use. This study addresses this gap by evaluating patient experience of and engagement with COVID-19 remote home monitoring services.
This study aimed to explore what formal and informal support patients received as part of COVID-19 remote home monitoring services in England, UK (COVID Oximetry@home and virtual wards models), and patient experience of and engagement with these services. This manuscript addressed the following questions:
## | design
This study used a mixed-methods design, and included crosssectional survey data from patients and carers and qualitative data from semi-structured interviews with patients and carers. A mixedmethods study design was chosen as we sought to perform a comprehensive assessment of patients' views and experiences of these services, from a wide range of sites, and also to gain an indepth understanding of the factors influencing engagement with these services. The surveys helped to capture an overview of patient engagement and experience, and the interviews enabled an in-depth understanding of experience and engagement.
This was a rapid study (data collection period: March-June 2021). Detailed methods are reported in [fig_ref] 1: What types of formal and informal support did patients receive as part... [/fig_ref].
This study was part of a larger rapid mixed-methods evaluation of remote home monitoring for COVID-19 patients.
## | sample
We recruited patients and carers from 25 sites (COVID-19 remote home monitoring services delivered in National Health Service (NHS) trusts or primary care providers). We recruited sites from across six English regions, and these covered populations of <250,000 to over 1 million (see for details). Seventeen of the twenty-five sites participated in both surveys and in-depth interviews; the remaining were survey-only sites.
Patients who had received COVID-19 remote home monitoring services were recruited into the survey (aimed to recruit all onboarded patients between January 2021 and June 2021) and for the interviews (4-6 patients/carers from each of the 17 case study sites). If patients were unable to take part but wanted to participate, we invited their carer to complete the survey/interview on their behalf.
## | measures
We developed the survey and semi-structured topic guides specifically for this study. Questions (see [fig_ref] 1: What types of formal and informal support did patients receive as part... [/fig_ref] were informed by the relevant literature [bib_ref] Understanding heart failure; explaining telehealth-a hermeneutic systematic review, Greenhalgh [/bib_ref] [bib_ref] New networked technologies and carers of people with dementia: an interview study, Powell [/bib_ref] [bib_ref] Remote home monitoring (virtual wards) for confirmed or suspected COVID-19 patients: a..., Vindrola-Padros [/bib_ref] [bib_ref] What is quality in assisted living technology? The ARCHIE framework for effective..., Greenhalgh [/bib_ref] [bib_ref] The assessment, monitoring, and enhancement of treatment fidelity in public health clinical..., Borrelli [/bib_ref] [bib_ref] Measures of fidelity of delivery of, and engagement with, complex, face-to-face health..., Walton [/bib_ref] [bib_ref] Technology assessment and the sociopolitics of health technologies, Lehoux [/bib_ref] [bib_ref] The behaviour change wheel: a new method for characterising and designing behaviour..., Michie [/bib_ref] [bib_ref] Understanding ethnic and other socio-demographic differences in patient experience of primary care:..., Lyratzopoulos [/bib_ref] [bib_ref] Sociodemographic and health service organizational factors associated with the choice of the..., Pianori [/bib_ref] [bib_ref] Language proficiency among respondents: implications for data quality in a longitudinal face-toface..., Wenz [/bib_ref] (see Appendices S1 and S2).
Information sheets and the survey were available in six other languages (Polish, Bengali, Urdu, Punjabi, French and Portuguese).
The survey and interview guide were piloted with the members of the study public patient involvement (PPI) group and the general T A B L E 1 Detailed methods for the survey and interviews
## Survey interviews
Setting This study took place in England, within NHS trusts or primary care practices/Commisioning Groups (CCGs) that implemented COVID-19 remote home monitoring services.
Sample-site selection - Twenty-eight services were included in our national evaluation. 25/28 sites agreed to take part in the patient survey (reported in this manuscript). - Services were sampled using a range of criteria, including the setting (primary care or secondary care), type of model (prehospital, early discharge, both), mechanism for patient monitoring (paper-based, app, both), geographic location (across different areas of the country), timing of implementation (implemented since Wave 1 of the pandemic or recently implemented) and involvement in the evaluation with the other evaluation partners (Imperial and IAU). - Sites were recruited through an expression of interest process, whereby we presented our study at local and national meetings and asked sites to express interest in participating.
- A smaller sample of the overall study sites were included as case studies to conduct a more in-depth analysis of patient experiences. - Seventeen of the twenty-five sites were selected as indepth case study sites using a range of criteria (setting, type of model, mechanism for patient monitoring, timing of implementation, involvement in evaluation with other partners). Four of the seventeen sites were purposively selected by NHSX for a more in-depth analysis of patient experiences of tech-enabled models of care; sites using different tech-enabled platform were selected.
Sample-eligibility criteria
## Measures
- Patient surveys were developed specifically for this study using relevant service documentation, 27,28 theoretical frameworks [bib_ref] Understanding heart failure; explaining telehealth-a hermeneutic systematic review, Greenhalgh [/bib_ref] [bib_ref] New networked technologies and carers of people with dementia: an interview study, Powell [/bib_ref] [bib_ref] What is quality in assisted living technology? The ARCHIE framework for effective..., Greenhalgh [/bib_ref] [bib_ref] Technology assessment and the sociopolitics of health technologies, Lehoux [/bib_ref] [bib_ref] The behaviour change wheel: a new method for characterising and designing behaviour..., Michie [/bib_ref] and previous literature on engagement. [bib_ref] The assessment, monitoring, and enhancement of treatment fidelity in public health clinical..., Borrelli [/bib_ref] [bib_ref] Measures of fidelity of delivery of, and engagement with, complex, face-to-face health..., Walton [/bib_ref] - The survey included closed questions on the service that patients received, their experience with the service and their engagement with the service. We also asked questions about patients' experience of tech versus analogue models. Questions were followed by open ended questions to give participants the opportunity to share wider thoughts (see Appendix S1). - The survey also included questions about participants' sociodemographic characteristics (gender, age, ethnicity education, employment, disability, sexual orientation, first language and geographical region). [bib_ref] Understanding ethnic and other socio-demographic differences in patient experience of primary care:..., Lyratzopoulos [/bib_ref] [bib_ref] Sociodemographic and health service organizational factors associated with the choice of the..., Pianori [/bib_ref] [bib_ref] Language proficiency among respondents: implications for data quality in a longitudinal face-toface..., Wenz [/bib_ref] - Before use, and to ensure that the questions were appropriate, the survey was reviewed by the study clinical advisory group and reviewed by members of the study's PPI group and the public before use. The survey was amended before use (e.g., amending wording, increasing font size, adding definitions for key terms).
- Interview topic guides were developed specifically for this study using relevant service documentation, 27,28 theoretical frameworks [bib_ref] Understanding heart failure; explaining telehealth-a hermeneutic systematic review, Greenhalgh [/bib_ref] [bib_ref] New networked technologies and carers of people with dementia: an interview study, Powell [/bib_ref] [bib_ref] What is quality in assisted living technology? The ARCHIE framework for effective..., Greenhalgh [/bib_ref] [bib_ref] Technology assessment and the sociopolitics of health technologies, Lehoux [/bib_ref] [bib_ref] The behaviour change wheel: a new method for characterising and designing behaviour..., Michie [/bib_ref] and previous literature on engagement. [bib_ref] The assessment, monitoring, and enhancement of treatment fidelity in public health clinical..., Borrelli [/bib_ref] [bib_ref] Measures of fidelity of delivery of, and engagement with, complex, face-to-face health..., Walton [/bib_ref] - The topic guide included questions about their journeys of remote home monitoring, their experiences of being ill and monitored at home, experiences with escalation and discharge, their engagement with the service and recommendations for improving these models (see Appendix S2). - Interviews also included questions about participants' sociodemographic characteristics (gender, age, ethnicity, education, employment, disability, sexual orientation, first language, geographical region). - To determine whether questions were appropriate and relevant, we discussed the interview topic guides with our PPI members and the 70@70 nurses. The topic guides were amended accordingly.
(Continues)
[formula] T A B L E 1 (Continued) [/formula]
## Survey interviews
Procedure-recruitment - Both survey options (online and paper) included prefacing information with a background to the study, potential risks, indicating voluntary participation, anonymity and a description of how the data will be used. - This page also included boxes that patients/carers were asked to tick to indicate their consent to take part in the study. - NHS staff distributed surveys so researchers had no access to patient information. - NHS staff from participating services sent the patient survey to patients (or their carers if applicable) onboarded onto the service between 1st January 2021 and 11th June 2021. - Sites chose how to disseminate the survey (post or text/email). - Survey sites kept a record of the number of surveys sent out to determine the response rate. - If patients were not able/willing to take part in the survey, they were given the option to ask their carer or family member to complete the survey on their behalf, reflecting on the patient's experience with the service. - Patients/carers returned completed surveys directly to the study team for analysis, either electronically through REDCap or via post using pre-paid envelopes. - In addition to English, we also offered participants the opportunity to receive an information sheet and survey in six other languages (Polish, Bengali, Urdu, Punjabi, French and Portuguese).
- Participants were sent an information sheet before the interview and were asked to provide written consent before taking part in the interview. - At the start of the interview, researchers also confirmed verbally that participants were still happy to take part in the interview. - Study coordinators at each site purposively identified a sample of participants (a range of characteristics e.g., age, gender, ethnicity), and contacted them and asked if they were happy to be approached by a researcher. - The researcher then contacted them via telephone or email to discuss the study. - Participants were sent information sheets and consent forms and asked to complete these before the interview (either digitally or via post). - If patients were not able/willing to take part in the interview, they were asked by site coordinators if their carer (if they have one) could be approached to capture their perceptions of the patient's journey and overall experience with the service.
## Procedure-data collection
- Participants were approached by NHS staff at the place where they received their care (called 'study coordinators in this manuscript), to take part in one of two ways: an online survey or a paper survey sent through the post with a free-post envelope. - Surveys were mostly distributed at discharge from the service, but some sites distributed surveys at onboarding to the service. - Data collection took place between March and June 2021 (with surveys being sent retrospectively to patients who had received care from January 2021 onwards). - Surveys were returned to the research team either electronically via REDCap or by posting surveys in pre-paid envelopes to the team. - Data from patient surveys sent via post were inputted into REDCap by members of the study team. - All data were securely stored in the university Data Safe Haven via REDCap.
- A researcher arranged a time to carry out the interview.
- Each site had a different lead researcher, who conducted the interviews and liaised with sites on an on-going basis. Interviews were conducted by six researchers. - Interviews were carried out via telephone or an online platform (e.g., Zoom or MS Teams) as preferred by the participant. - Interviews were designed to last between 45 and 60 min. The length of interviews ranged from 05:51 to 67:38 min. - Data collection for interviews was conducted between February and June 2021. - All interviews were semi-structured, audio-recorded (subject to consent being given), transcribed verbatim by a professional transcription service (TP Transcription limited) and kept in compliance with the General Data Protection Regulation (GDPR) 2018 and Data Protection Act 2018. - Interview data and transcripts were securely stored on the university Data Safe Haven. - Quotes were fully anonymized before use in dissemination. - Although we offered translation services for interviews, all interviews took place in English.
# Analysis
- The quantitative survey data were analysed using SPSS statistical software (version 25).
## | data collection
Study coordinators working within each service distributed electronic or paper surveys to patients and carers.
Potential interview participants were approached by study coordinators from each site. If they were interested in taking part, they were contacted by a researcher, who sent them an information sheet and consent form. Participants were asked to return the consent form before the interview. Interviews were conducted by six researchers. Interviews were conducted over Microsoft Teams, Zoom or telephone.
# | analysis
Survey data were analysed using SPSS statistical software (version 25). Descriptive statistics were used to explore patient experience and engagement (see [fig_ref] 1: What types of formal and informal support did patients receive as part... [/fig_ref]. Open-text survey data were extracted into an Excel spreadsheet and coded inductively.
Interview data were analysed using rapid assessment procedure (RAP) sheets (see [fig_ref] 1: What types of formal and informal support did patients receive as part... [/fig_ref]. RAP sheets are tools that can be used to rapidly capture key findings from different data sources. [bib_ref] Carrying out rapid qualitative research during a pandemic: emerging lessons from COVID-19, Vindrola-Padros [/bib_ref] The data inputted into RAP sheets were inductively coded using thematic analysis by one researcher. We developed a framework
[formula] T A B L E 1 (Continued) [/formula]
Survey Interviews groups and service models (as reported by patients and carers). - In addition to the descriptive analysis presented in this manuscript, we also conducted further multivariate and univariate analyses on disparities and differences between different participant groups in relation to engagement and tech-vs analogue modes, but these findings are presented elsewhere.- For data relating to patient experience and engagement, all cases were analysed (whether carer, patient or unknown). 'Unknown' cases refer to cases in which it was not clear whether the patient or carer had completed the survey. Therefore, to avoid making assumptions, we have marked these cases as 'unknown' but included the data relating to engagement with the service as it was still correctly completed and included reflections on their experiences. - Where data were missing for specific questions, cases were excluded from the analysis and the denominator was reported. The denominators differ across questions as all questions were optional; therefore, if people decided not to complete them, this led to missing responses. Additionally, there was question routing included within our survey, which meant that not all questions were appropriate for each participant to complete. open-text responses). - We did not receive any surveys in any other languages other than English; therefore, all analyses were conducted in English.
- RAP sheets were developed per site to facilitate crosscase comparisons and per population (to make comparisons between subgroups). - The categories used in the RAP sheets were based on the questions included in the interview topic guide, maintaining flexibility to add categories as the study is ongoing. - Research leads from each site added notes and summaries of findings to the RAP sheet following each interview, for each site. - The data inputted into RAP sheets were inductively coded using thematic analysis by one researcher. - Themes and subthemes were developed, discussed and agreed by the research team. - We then developed a framework based on these themes and subthemes, and one researcher used this framework to extract quotes from all original transcripts. 50 - The coding framework included participants' views of the service, experiences of being referred, information received about the service and experiences performing remote home monitoring behaviours and barriers and facilitators to performing remote home monitoring behaviours. - Analysis was conducted in English (as no interviews were conducted in other languages). - Interview and survey data were triangulated. Interview and survey data were analysed separately initially, before being brought together to compare and contrast findings during analysis and interpretation. [bib_ref] Triangulation in Data Collection, Flick [/bib_ref] [bib_ref] Enhancing the quality and credibility of qualitative analysis, Patton [/bib_ref] Abbreviations: CCGs, clinical commisioning groups; NHS, National Health Service; PPI, public patient involvement; RAP, rapid assessment procedure.
[formula] WALTON ET AL. | 2391 [/formula]
based on the themes and subthemes that we developed, and one researcher used this framework to extract quotes from all original transcripts.
Survey and interview findings were analysed separately and then triangulated to compare the consistency of the findings. [bib_ref] Triangulation in Data Collection, Flick [/bib_ref] [bib_ref] Enhancing the quality and credibility of qualitative analysis, Patton [/bib_ref] 3 | RESULTS
## | participant characteristics
We received 1069 surveys (18% response rate) from patients (n = 936, 87.6%) and carers (n = 48, 4.5%) across 25 sites (see . In some surveys, it was unclear whether it was completed by the patient or the carer (n = 85, 8%).
We conducted 62 interviews with patients (n = 59) and carers (n = 3) across 17 sites (see for demographics). However, we were unable to recruit any participants who declined the service or disengaged from the service.
Most patients (70% n = 749/1069 survey and 71% n = 44/62
interview participants) were referred to the service via community methods (see . Patients and carers reported using a range of methods to record and report their readings to the service, including analogue (paper and phone) (49%, n = 522/1069 survey and 31% n = 19/ 62 interview participants) and tech-enabled methods (51% n = 547/1069 survey and 44% n = 27/62 interview participants) (see .
3.2 | What types of formal and informal support did patients receive as part of COVID-19 remote home monitoring services?
Below, we describe a summary of survey findings (see and
interview findings relating to formal and informal support.
## | formal support from staff
The 'care' on offer differed across sites and patients, with variation in the type and frequency of monitoring offered by services.
Responses from the patient survey indicated that the frequency with which patients had contact with a member of staff varied, but Sites were characterized with respect to their population size,the proportion in urban versus rural areas 55 and the proportion in the most and least deprived areas (with respect to national quintiles).For sites based on CCG areas, we calculated these characteristics using publicly available data at the lower super output area level mapped to CCGs, 57 while for trust-based sites, we used data derived from inpatient Hospital episode statistics admissions during the financial year 2019/20 (Nuffield trust analysis of Hospital episode statistics admitted patient care data set, 2019/20), in addition to web searches for the trust catchment populations. Ethnicity was also calculated using publicly available data.T A B L E 3 Demographic characteristics of patient and carer survey respondents A few patients and carers reported not speaking to staff at all (see .
[formula] n (%) [/formula]
This was supported by interview findings, which indicated that the frequency of taking and communicating readings to the service ranged from once a day to more than three times a day. Findings indicate that patients are supported by staff throughout different stages of the service, including providing information, monitoring (e.g., phone calls if patients and carers forget to submit readings and in some cases face-to-face visits to take readings), escalating care (e.g., providing advice on whether to seek help, calling ambulances for patients), signposting and comfort and reassurance.
## | burden of treatment on patients and carers in informal support roles
Survey findings indicated that almost all patients used an oximeter to record readings when receiving the service (95%, n = 1014/1069).
Many patients reported completing a diary (52%, n = 555/1069) and
providing readings over the phone (47%, n = 498/1069) or using technology-enabled methods (e.g., text 29%, n = 309/1069).
Escalation-related behaviours were reported less frequently by patients, with only a third of patients reporting seeking further help due to readings being lower than recommended thresholds (32%, n = 344/1069), and only a fifth of patients reporting checking their readings for issues (20%, n = 215/1069) (see .
Many patients were supported by family and friends to engage with the service. A quarter of survey respondents needed help to use equipment (25%, range 11%-50% across sites), and more than half of the interview participants were supported by family members.
Most patients and carers reported having informal support to help them use the oximeter and support with taking and recording readings. Only a small proportion of participants reported that they did not need support using the oximeter (10%, n = 107/1058) or taking and recording readings (19%, n = 201/1057) (see . Many patients and carers were not aware of the service before referral.
## T a b l e 3 (continued)
Some patients and carers also spoke about how the service helped them to monitor their own improvement and that it potentially improved their outcomes.
Patients and carers reported very positive views of the workforce and that they were helpful and put patients at ease, and were professional and potentially even lifesaving. Continuity of staff was thought to be important.
A few patients had negative experiences with individual staff members, for example, that they were dismissive, did not recognize that they needed help, were not interested or lacked clinical expertize to support patients or answer their queries.
3.4 | What are the factors influencing burden of treatment and ability to engage with COVID-19 remote home monitoring services?
Findings indicated that patients and carers generally found it easy or very easy to engage with the service and the resulting activities, including understanding information, monitoring using the oximeter, recording readings and providing readings and escalating care (see . Most survey respondents indicated that they did not experience problems with the service (72%; n = 771/1069) and did not report barriers to engagement with the service (80%, The most frequent challenges reported within the survey were returning the oximeter, contacting healthcare professionals when needed and seeking further help (see . While many survey respondents discussed problems with the team (35%, n = 87/249) or had their problems resolved (33%, n = 76/232), over half of these participants said that problems had not been resolved (54%, n = 126/232).
Findings from the surveys and interviews indicated three overarching themes that influenced burden of treatment and patients' ability to engage with COVID-19 remote home monitoring services: (i) patient factors, (ii) wider support and resources and (iii) factors relating to the service (see for details of example findings for each theme and subtheme and example quotes).
## | patient factors
Knowledge, memory, physical health, attitudes towards the service and having time to complete the required tasks influenced engagement (see knowing what to do helped them to engage with the service (55%, n = 583/1069). This was supported by interview findings that indicated that participants having the appropriate knowledge helped them to use the oximeter, monitor/record/communicate readings and escalate care. However, interview findings indicated that knowledge was a barrier for some participants (e.g., relating to understanding/interpreting information and equipment, language barriers, not knowing how to fill out diary/complete readings/escalate care or when to call for help). - Memory: Forgetting to do the readings was a barrier to engaging with the service for some survey (4%, n = 37/ 1069) and interview participants. However, phone calls helped participants to do readings, and some participants wrote readings on post-it notes to facilitate memory. - Physical health: A majority of survey respondents felt that their own health helped them to engage (54%, n = 581/ 1069). However, within the interviews, many barriers relating to physical health were identified, including feeling too poorly/not in the right frame of mind, sleeping a lot, having health conditions that made it difficult to monitor, difficulties hearing, difficulties getting to the telephone and difficulties with eyesight. These barriers were also reported within the survey (4%, n = 39/1069 reported own health as a barrier, and some participants wrote in open-text findings that they felt too poorly to engage). - Attitudes towards the service and behaviours: Survey and interview participants reported knowing why the service was important (e.g., 60%, n = 637/1069 survey respondents) and wanting to engage with the service (46%, n = 491/1069), and these positive views helped them to engage with the service. However, some participants reported a lack of interest in monitoring/ recording, or views that monitoring did not help or made them anxious if the reading was low. Additionally, barriers to seeking further support/escalating care were identified, including worries about going to hospital (due to COVID-19, lack of support, difficulties communicating). - Time: A fifth of survey participants felt that having time helped them to engage with the service (20%, n = 217/ 1069). Interview findings supported this by highlighting the importance of developing a routine. However, a few participants did not have enough time (e.g., those working from home). Wider support and resources
- Support from staff/service: Survey and interview participants spoke about how support from healthcare professionals helped them to engage with the service (46%, n = 488/1069). Interview findings highlighted that support helped to understand information, helped with monitoring, obtaining equipment, recording, communication and escalating care. Support was reassuring. However, a small number did highlight that they did not have enough support from healthcare professionals or that they could not get through to a member of the team. - Support from family/friends: A quarter of survey respondents (25%, n = 266/1069) felt that support from
- 'The nurse was very good, can't praise her really high enough. She was a friendly voice to speak to. Fair enough, you know, I've got a bit of a support system, but for somebody who hasn't got that much of a support system around them, I think that friendly voice would go a long way just to, you know, easing their minds'. Site D, interviewee 5 - 'And then near the end when I was getting a bit complacent, I was sort of almost well, a couple of times I didn't put them in and they would phone and say, "Are you okay? You've not submitted you reading." So that was just really supportive, and I said it certainly reassured me'.
## | wider support and resources
Support from staff/service, support from family members/ friends, accessibility and availability of materials, equipment and technology influenced engagement (see . For example, support from staff members (e.g., 46% [n = 488/1069] of survey respondents) and family/friends (e.g., 25% [n = 266/1069] of survey respondents) was crucial in helping many patients to use the service.
## | service factors
Monitoring characteristics, service characteristics, scope of service and availability of treatment influenced engagement (see . For example, some participants felt that the inconsistent timing of calls was a barrier and some felt that calls were too frequent, whereas others felt that they were not frequent enough. Additionally, some patients and carers felt that the scope of the service was a barrier to engagement, in that it did not cover wide symptoms of COVID-19
and was not holistic. However, a small number of participants reported that lack of support from family/friends was a barrier to engagement. - Accessibility and availability of materials: The amount of information received was sometimes reported as a barrier in the interviews (e.g., too much, too little or contradictory and confusing information). - Equipment: Some participants already had their own equipment, which facilitated engagement, and a few participants reported not having the right equipment (e.g., faulty oximeters/not having thermometers) - Technology: Some participants felt that reminder texts or alerts from an app helped them to engage, and that the technology was easy to use. However, other participants experienced difficulties with the app, oximeter and technology systems.
- 'So my dad was initially involved in I think it was nine days, so the first nine days he took full care of mum to be honest clinically I was involved in a lot of the calls because I think my dad's getting quite stressed.
[…] so yes, he did the physical side of it. He would do the observations. And then he'd call me first thing in the morning, or he'd drop a text to say these are the observations. I'd call and have a quick chat, knowing the nurse was going to call us. So I guess it was a bit of a joint effort between us'. (Site F,
Service factors - Monitoring characteristics: Participants identified barriers relating to inconsistency of call timing, amount of calls, not being able to see progress and frequency of monitoring and recording. - Service characteristics: Some participants felt that there were problems relating to delays in enrolment, limited hours of service and not being able to continue monitoring following discharge. - Scope of service: Scope of service was a barrier to escalation as some participants did not know whether to ring to ask for help. Some participants reported that the service was not holistic (did not cover all symptoms of COVID). - Availability of treatment: Some participants mentioned problems contacting their GP, and inability to receive oxygen in their own home if needed as barriers.
- 'I found, to start with I found the text messages useful but the longer they went on the more irritating. I was, I felt like I was chained to the phone and you know and to my equipment.
## | strengths and limitations
Integration of mixed-methods data helped to provide in-depth perspectives on experiences of, and engagement with, COVID-19 remote home monitoring services. A large team of researchers (from a range of disciplines, with extensive expertize in qualitative and quantitative methods) was involved, thus strengthening the interpretation of findings. Findings were shared with clinical and academic stakeholders. Our study sampled a large range of sites with a range of characteristics, thus enhancing the generalizability of the findings.
Compared with patient onboarding data, our patient sample was underrepresentative of some groups (e.g., older patients, Black, Asian and minority ethnic communities and most deprived) and overrepresentative of other groups.The response rate for the survey was fairly low (17.5%). Additionally, we were unable to recruit interview or survey participants who had declined the service, dropped out from the service and those who were unable or did not want to take part in surveys and interviews. Therefore, findings may not be representative of all patient groups and experiences.
While we did include carers within our sample, the focus of our research was on patient experiences of remote home monitoring services. Therefore, it is possible that we have not captured carers' experiences in detail. However, some carers shared their own experiences during the interviews and in responding to the survey.
## | implications
Burden of treatment may not only affect those with multimorbidity or chronic conditions but can also affect those with acute conditions.
Findings indicate that remote monitoring may increase treatment burden for some patients and families.
COVID-19 remote home monitoring services aimed to target patient groups at higher risk from COVID-19, and yet, many of these groups appear more likely to report difficulties in engagement with these services, for example, older patients and patients with health problems. Remote monitoring may not be appropriate for everyone (e.g., those without support). Services need to gauge a person's support network and any concerns surrounding remote home monitoring when assessing eligibility for these services. Services must then tailor the healthcare offer to enable patients to engage (e.g., providing further support for those from at-risk groups or who do not have informal support, or linking patients with care networks if needed). All patients should be provided contact details to contact the service, should problems arise. Face-to-face support (e.g., for monitoring) from staff and families has implications for infection transmission.
# | future research
Further research is needed to explore the experiences of those who decide not to use remote home monitoring services or disengage from these services. Further research should explore the burden of treatment for chronic conditions compared with acute conditions.
Additionally, it would be helpful to further explore which groups are able to tolerate burden associated with remote home monitoring pathways and the impact of treatment burden from informal caring responsibilities on families.
# | conclusions
COVID-19 remote home monitoring services place a large responsibility on patients and carers in relation to monitoring and escalating care.
While patients and carers found the service reassuring and a positive experience, many factors influenced their ability to engage with the service. This indicates that the service may be conditional on a range of factors relating to the patient (e.g., knowledge and memory), their support and resources (e.g., support from family, friends and staff) and service factors (e.g., scope of the service and frequency of monitoring).
# Author contributions
All authors were responsible for the study conception, design and data collection throughout the study. Holly Walton, Cecilia Vindrola-Padros and Nadia Crellin led the data analysis. Holly Walton and Cecilia Vindrola-Padros drafted the manuscript with contribution from all authors. All authors commented on drafts of the manuscript
[fig] •: Descriptive statistics were calculated to compare patient experiences of the service across patient For patient interviews, data collection and analysis were carried out in parallel and facilitated through the use of RAP sheets as explained in Vindrola-Padros et al. 49 public, through the following activities: (a) workshop with the PPI group, (b) pilot interview with one PPI member and (c) survey reviewed by the PPI member and members of the public. Suggested amendments relating to accessibility and wording of questions were incorporated before use. [/fig]
[table] 1: What types of formal and informal support did patients receive as part of COVID-19 remote home monitoring services? What was the burden of treatment on patients and carers in informal support roles? 2. What are patients' and carers' experiences of engaging with COVID-19 remote home monitoring services? 3. What are the factors influencing burden of treatment and ability to engage with COVID-19 remote home monitoring services? [/table]
|
Central serous chorioretinopathy treatment with a systemic PDE5 and PDE6 inhibitor (sildenafil)
# Introduction
Central serous chorioretinopathy (CSCR) is a disease characterized by macular serous retinal detachment, first described by Von Graefe in 1866.It has had numerous definitions of causation and may be accompanied by a detachment of the pigment epithelium (PED).
The prognosis is often quite good with no treatment and resolution is common within 3-6 months although visual disturbances that interfere with reading tasks may persist. More severe or recurrent cases can produce significant visual symptoms. Yannuzzi and colleagues reported a subset of CSCR patients where 25% had visual acuity of 20/200 or worse. [bib_ref] Peripheral retinal detachment and retinal pigment epithelial atrophic tracts secondary to central..., Yannuzzi [/bib_ref] The two main pathophysiological changes in CSCR relate to a primary disease of the RPE or a psychogenic component that ultimately affects, among other things, the choriocapillaris. Clearly the disease is multifactorial, and stress appears to be a significant risk factor. Treatment is presently quite varied and includes mineralocorticoid antagonism, beta adrenergic antagonism, or laser therapy with variable results. A landmark summary of the clinical characterization and the treatment panorama is provided in a recent paper on evidence based treatment guidelines.The present report describes a treatment using sildenafil to increase blood flow to the choroid via production of phosphodiesterase 5 and 6 (PDE5/PDE6) inhibition. PDE6 inhibition decreases Warburg-like glycolysis in rod photoreceptors and promotes oxidative metabolism while PDE5 inhibition increases nitric oxide perfusion and waste removal. [bib_ref] Rod metabolic demand drives progress in retinopathies, Lin [/bib_ref] This last reference specifically describes improvement in photoreceptor neuronal function possibility due to enhanced mitochondrial ATP production.
This case describes a long standing CSCR which was treated with a good result. When treatment stopped, CSCR recurred; when treatment restarted, CSCR again rapidly resolved (challenge, dechallenge, rechallenge). Sildenafil citrate (Viagra®, Pfizer, New York, NY, USA) is primarily indicated and FDA-approved for erectile dysfunction (ED), and has become one of the most popular drugs globally. Through selective inhibition of the cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase types 5 inherently present in all vascular tissues, the medication improves erectile function in men with ED by enhancing the smooth muscle relaxant effects of nitric oxide. [bib_ref] Effects of sildenafil on the relaxation of human corpus cavernosum tissue in..., Ballard [/bib_ref] From this increased blood flow effect, sildenafil has also demonstrated use for other conditions such as cardiac ischemia 6 and pulmonary hypertension. [bib_ref] Sildenafil in severe pulmonary hypertension associated with chronic obstructive pulmonary disease: a..., Vitulo [/bib_ref] Sildenafil also inhibits PDE6 in rod and cone photoreceptors. PDE6 is a homolog of PDE5.
This case report is part of an IRB-approved study of sildenafil treatment of macular degenerations and dystrophies.
## Case report
A 50 year old male noted recurrent metamorphopsia of the central vision in his right eye occurring during periods of stress in his writing career. Both his ability to function and his ability to maintain concentration on his work were seriously affected. Optical coherence tomography (OCT) showed serous elevation of the para-macular area. He agreed to a trial of sildenafil as part of a Columbia University Medical Center Institutional Review Board (IRB) approved study of sildenafil for treatment of macular degenerations and dystrophies. Sixty milligrams of oral sildenafil was prescribed (40 mg in the morning and 20 mg at bedtime).
Despite his symptoms, the visual acuity remained 20/20 in both eyes prior to and throughout treatment. Vision returned to normal with no serous fluid on OCT within 2 weeks and sildenafil was discontinued (dechallenge). Three weeks later, he again became symptomatic, serous fluid recurred, and sildenafil was resumed (rechallenge). Within 2 weeks serous fluid disappeared and he remained symptom-free while on sildenafil for 5 months [fig_ref] Figure 1: Central serous chorioretinopathy [/fig_ref].
Serous fluid was also observed superior to the optic nerve in the fellow (left) eye, although no visual symptoms were described for this eye [fig_ref] Figure 2: Central serous chorioretinopathy [/fig_ref]. In this eye fundus hypoautofluorescence (hypo-AF) was followed by hyperautofluorescence (hyper-AF) after treatment.
# Discussion
Despite evidence of increased choroidal blood flow with sildenafil intake in normal, healthy patients, 7 , 8 , 9 very few investigations regarding potential therapeutic effects of this medication in eyes with macular disease, such as central serous chorioretinopathy (CSCR) have been done. Reasons include reports of CSCR linked to sildenafil usage within large databases, [bib_ref] Central serous chorioretinopathy associated with sildenafil, Fraunfelder [/bib_ref] and rare instances of other possible ophthalmic toxicities. [bib_ref] Bilateral posterior ischemic optic neuropathy associated with the use of sildenafil for..., Coca [/bib_ref] Several cases analyzed by Fraunfelder and Fraunfelder [bib_ref] Central serous chorioretinopathy associated with sildenafil, Fraunfelder [/bib_ref] suggested that CSCR activity was temporally linked with the medication, either with subretinal fluid present during sildenafil intake (positive challenge) or resolution with its cessation (positive dechallenge). However, a post-marketing surveillance survey demonstrated lack of a significant association between sildenafil usage and CSCR, [bib_ref] Central serous chorioretinopathy and phosphodiesterase-5 inhibitors: a case-control postmarketing surveillance study, French [/bib_ref] and toxicity has instead primarily been found in the setting of excessive dosage or medication impurity from non-regulated sources. [bib_ref] Sildenafil citrate induced retinal toxicityelectroretinogram, optical coherence tomography, and adaptive optics findings, Yanoga [/bib_ref] Sildenafil is not recommended for patients on nitrates as it may reduce blood pressure significantly.
Our hypothesis is that select patients with macular disease including CSCR may benefit from increased choroidal blood flow afforded by sildenafil. [bib_ref] Age-related macular degeneration: choroidal ischaemia?, Coleman [/bib_ref] [bib_ref] Treatment of macular degeneration with sildenafil: results of a two-year trial, Coleman [/bib_ref] [bib_ref] Vascular response to sildenafil citrate in aging and age-related macular degeneration, Yui [/bib_ref] We anticipate that sildenafil, administered with attention to "challenge" and "dechallenge" settings -that is, continued treatment beyond the initial resolution of serous fluid -should produce clinical improvement for these patients. The use of spironolactoneand eplerenone to a lesser extent -most closely parallels the present treatment but has shown slower treatment response and greater side effects. [bib_ref] Central serous chorioretinopathy: recent findings and new physiopathology hypothesis, Dariuch [/bib_ref] [bib_ref] Eplerenone for chronic central serous chorioretinopathy in patients with active, previously untreated..., Lotery [/bib_ref] We note that in short-wavelength fundus autofluorescence (SW-AF) images [fig_ref] Figure 2: Central serous chorioretinopathy [/fig_ref] the serous retinal detachment associated with CSCR initially presented with a focal area of hypo-SW-AF superior to the optic disc in the fellow eye. The reduced autofluorescence (AF) is likely due to blockage of the transmission of fundus AF by subretinal fluid, as previously suggested. [bib_ref] Infrared fundus autofluorescence and central serous chorioretinopathy, Sekiryu [/bib_ref] [bib_ref] Fundus autofluorescence in acute and chronic central serous chorioretinopathy, Dinc [/bib_ref] [bib_ref] Near-infrared and shortwavelength autofluorescence imaging in central serous chorioretinopathy, Ayata [/bib_ref] [bib_ref] Fundus autofluorescence in acute and chronic-recurrent central serous chorioretinopathy, Framme [/bib_ref] Subsequently, however, this site was marked by increased SW-AF presenting with a speckled pattern. This aberrant SW-AF signal could represent a window defect created by reduced photopigment in the CSCR lesion. Alternatively, the SW-AF intensity at this location could reflect an actual elevation in SW-AF due to increased bisretinoid lipofuscin formation within damaged photoreceptor cells.
# Conclusions
This case represents a positive response to PDE5/6 inhibition of central serous chorioretinopathy using an oral medication with minimal to no adverse effects. The use of the challenge-dechallenge-rechallenge paradigm strengthens the validity of the use of sildenafil as a viable treatment option in these patients.
# Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Consent for publication
Consent to publish this report has been obtained from this patient. This report does not contain any personal identifying information.
# Funding
This study was support, in part, by grants from the National Eye Institute/NIH (Bethesda, Maryland) EY024091, Triad Foundation, Ithaca, New York and the St. Giles Foundation, New York, New York. Jonas Children's Vision Care is supported by the National Institute of
## Conflict of interest
The authors do not have any proprietary interests in the materials described in the article.
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
## Intellectual property
We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.
## Research ethics
We further confirm that any aspect of the work covered in this manuscript that has involved human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.
IRB approval was obtained (required for studies and series of 3 or more cases).
Written consent to publish potentially identifying information, such as details or the case and photographs, was obtained from the patient(s) or their legal guardian(s).
## Authorship
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The International Committee of Medical Journal Editors (JCMJE) recommends that.
Authorship be based on the following four criteria:
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# Funding
Supported by the National Eye Institute/National Institutes of Health Grant EY024091 and unrestricted grants from the Triad Foundation, Ithaca, New York and St. Giles Foundation, New York, New York.
## Declaration of competing interest
The following authors have no financial disclosures: DJC, WL, SD, MPB, JS, SHT.
[fig] Figure 2: Central serous chorioretinopathy. (Left) Short wavelength fundus autofluorescence images of the fellow eye showing subretinal fluid superior to the optic nerve at baseline and after treatment. (Right) Macular thickness measurements acquired superior to the optic disk. D.J. Coleman et al. [/fig]
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Binary Fingerprints at Fluctuation-Enhanced Sensing
We have developed a simple way to generate binary patterns based on spectral slopes in different frequency ranges at fluctuation-enhanced sensing. Such patterns can be considered as binary "fingerprints" of odors. The method has experimentally been demonstrated with a commercial semiconducting metal oxide (Taguchi) sensor exposed to bacterial odors (Escherichia coli and Anthrax-surrogate Bacillus subtilis) and processing their stochastic signals. With a single Taguchi sensor, the situations of empty chamber, tryptic soy agar (TSA) medium, or TSA with bacteria could be distinguished with 100% reproducibility. The bacterium numbers were in the range of 2.5 × 10 4 -10 6 . To illustrate the relevance for ultra-low power consumption, we show that this new type of signal processing and pattern recognition task can be implemented by a simple analog circuitry and a few logic gates with total power consumption in the microWatts range.
# Introduction
Bacterium detection and identification has an important role in medical, agricultural, environmental, defense, etc. applications. Analyzing their odor [bib_ref] Implications of aerobiology in respiratory allergy, Chanda [/bib_ref] [bib_ref] Bioaerosols and occupational lung disease, Lacey [/bib_ref] has good prospects because of high speed, low cost, wide availability, good sensitivity and selectivity, while solid-state electronic noses [bib_ref] Distributed sensor system for quantification of individual components in a multiple gas..., Eklov [/bib_ref] [bib_ref] System identification of electronic nose data from cyanobacteria experiments, Searle [/bib_ref] [bib_ref] The electronic nose applied to dairy products: a review, Ampuero [/bib_ref] [bib_ref] Medical applications of odor-sensing devices, Persaud [/bib_ref] can be applied.
Recently, we have carried out an experimental study [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref] with commercial Taguchi sensors to test the shape of the power density spectrum of the stochastic component of their signal as a pattern to recognize bacteria. The power density spectrum S( f ) of the spontaneous fluctuations of the sensor signal is one of the easiest and natural tools for Fluctuation-Enhanced Sensing (FES) of chemicals [bib_ref] Extracting information from noise spectra of chemical sensors: single sensor electronic noses..., Kish [/bib_ref] [bib_ref] Nanocrystalline tungsten oxide thick films with high sensitivity to H 2 S..., Solis [/bib_ref] [bib_ref] On the sensitivity, selectivity, sensory information and optimal size of resistive chemical..., Kish [/bib_ref] [bib_ref] Detecting harmful gases using fluctuation-enhanced sensing, Kish [/bib_ref] [bib_ref] Fluctuation-enhanced multiple-gas sensing, Solis [/bib_ref] [bib_ref] Comparison of classical and fluctuation-enhanced gas sensing with Pd x WO 3..., Ederth [/bib_ref] [bib_ref] Advanced agent identification at fluctuation-enhanced sensing, Kwan [/bib_ref] [bib_ref] Fluctuation-enhanced sensing: Status and perspectives, Schmera [/bib_ref] [bib_ref] Modelling on oxygen chemisorption induced noise in metallic oxide gas sensors, Gomri [/bib_ref] [bib_ref] Adsorption-desorption noise in gas sensors: Modelling using langmuir and wolkenstein models for..., Gomri [/bib_ref]. While it is reasonably simple to generate it from the measured data, it contains significant sensing information and it has been shown to enhance sensitivity by a factor of 300, or more [bib_ref] Detecting harmful gases using fluctuation-enhanced sensing, Kish [/bib_ref] [bib_ref] Comparison of classical and fluctuation-enhanced gas sensing with Pd x WO 3..., Ederth [/bib_ref]. It is also relatively straightforward to construct a theory to explain its behavior [bib_ref] Fluctuation-enhanced sensing: Status and perspectives, Schmera [/bib_ref] [bib_ref] Modelling on oxygen chemisorption induced noise in metallic oxide gas sensors, Gomri [/bib_ref] [bib_ref] Adsorption-desorption noise in gas sensors: Modelling using langmuir and wolkenstein models for..., Gomri [/bib_ref].
In the present paper, we show a new method to generate binary patterns from measured spectra, an ultra-low power implementation of such a system including a simple Boolean logic circuit as a microprocessor-free pattern recognizer, see Sections 2, 3 and 6, respectively.
In order to demonstrate the feasibility of the method and the nature of binary patterns, we conducted relevant experimental tests/evaluations where we have used some of the spectra published in paper [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref] and spectra from new measurements.
## Binary patterns for low power consumption
To achieve ultra-low power consumption, we must avoid the usage of microprocessors and extensive data processing. The sensor signal must be processed in the simplest possible way, presumably with analog circuitry, and the pattern recognition must be a deterministic process based on a few simple logic decisions. Let us make the following notations: n α (local slope) is the average local slope of the power density spectrum S( f ) in the n-th frequency sub-band and β = α n / N ∑ is the average of n α over the entire measurement band, see [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref] as an illustration for logarithmically equidistant sub-band boundaries. The boundaries of sub-bands can be equidistant or any convenient settings. These quantities can easily be generated by a low number of operational amplifiers and filters, see Figures 2 and 3.
The deviation n Δ of the local slope is defined for each sub-band as the difference between n α and β in the following equation:
[formula] n n Δ = − β α (1) [/formula]
The sign n σ of the local deviation n Δ will be binary bit related to that sub-band:
[formula] ) signum( n n Δ = σ (2) [/formula]
The quantity σ n is a binary pattern that indicates if α n is larger or smaller than β in the n-th subband of the spectrum. The advantage of the quantity σ n is that it provides a single bit information about the spectral pattern. In the case of N non-overlapping frequency bands, the n σ ( N n ,..., 1 = ) quantities represent N bit information obtained from a single sensor. Then a simple, deterministic, fast and low-power pattern recognizer can easily be constructed by applying a Boolean logic rule to identify/distinguish the particular spectral patterns with their relevant set of the n σ bits.
All these tasks can be realized without the use of power hungry devices such as microprocessors, other types of sequential logic, or analog-digital converters. In Section 3, we show a simple realization with analog circuitry of power consumption in the microWatt range and in Section 7, based on experimental patterns, a Boolean pattern recognition logic in the nanoWatt/picoWatt regime, see also Section 8.
## An ultra-low-power realization of the scheme
The proposed system for ultra-low-power consumption, see [fig_ref] Figure 2: Major building blocks of the low-power sensing system [/fig_ref] , includes three major parts: Sensor, Analog circuits to generate the binary patterns and a Boolean Logic circuit. The system described here uses equidistant sub-band boundaries: uniform bandwidths with non-overlapping sub-bands. In [fig_ref] Figure 3: Details of the Analog Unit of the sensing system with 6 bit... [/fig_ref] , the outline of the Analog Unit is shown. The preamplifier amplifies the stochastic component of sensor signal. The spectral slope estimator is the combination of a small number of amplifiers, filters and rectifiers. The binary patterns are generated by a set of comparators. A simple realization of the analog electronics to estimate the local slope n α and global slope β can be seen in the Spectral slope estimator part of [fig_ref] Figure 3: Details of the Analog Unit of the sensing system with 6 bit... [/fig_ref]. In this realization, for fingerprints of N bit resolution, we will need N + 1 sub-bands. To obtain the local slope α n of each sub-band, first we rectify and then smooth the noise by a low-pass filter at the output of each band-pass filter in order to estimate the mean-square amplitude U n 2 (t) there. Then we estimate the average slope in the
[formula] frequency band ( f n − f n−1 ) / 2 { ; ( f n + f n +1 ) / 2 } as: α n = U n 2 (t) − U n−1 2 (t) ( f n − f n−1 ) 2(3) [/formula]
To estimate n α from the mean-square voltages, see Equation, differential amplifiers are used, see
## Experiments with bacteria and heated semiconducting metal oxide sensors
To demonstrate the feasibility of the new method, the number of bits required for the identification, the simplicity, and the low power consumption of such a system, we carried out experiments with bacteria and commercial Taguchi sensors. Note, commercial Taguchi sensors must be heated thus they consume a lot of power, however, nanoparticle film alternatives are often much more sensitive and do not need heating during sensing [bib_ref] Gas-sensing properties of nanocrystalline WO3 films made by advanced reactive gas deposition, Solis [/bib_ref] [bib_ref] Conduction invasion noise in nanoparticle WO3/Au thin-film devices for gas sensing application, Hoel [/bib_ref].
Thus, whenever the new method described in this paper is to be used for ultra-low power applications, nanoparticle sensors or similar room-temperature devices will be needed to fully utilize the low power consumption of the electronics.
## Sample preparation
Here we briefly summarize the sample preparation steps. For more details, see [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref]. As Gram negative pathogenic vegetative bacterium surrogate, mid-log phase cultures of E. coli K12 MG1655 (E. coli Genetic Resources at Yale CGSC, New Haven, NE) were grown in Luria Bertani (LB) mediumat 37 °C. The cells were harvested at 2,880 g for 9 minutes and resuspended in 5% Phosphate Buffer Saline (PBST, pH 7.4) to 10 9 CFU/milliLiter concentration. Aliquots (100 μL) of the E. coli cell suspension were spread in appropriate dilution on Difco Tryptic Soy Agar (TSA) plates (Becton Dickinson Co., Sparks, MD), and incubated overnight at 37 °C.
As Gram positive (Anthrax) surrogate, 50 mg of lyophilized Bacillus atrophaeus (aka Bacillus globigii, BG) (U.S. Army Edgewood Proving Ground, Edgewood, MD) was resuspended in 5 mL of sterile deionized water and centrifuged at 2,880 g for 9 min to remove traces of the culture medium. The supernatant was aspired and the pellet was resuspended in 10 mL of sterile deionized water. Aliquots (100 μL) of the stock Bacillus subtilis were spread in appropriate dilution on TSA plates and incubated overnight at 30 °C.
As reference, sterile TSA plates containing identical (27 mL) amounts of the medium without bacteria were also prepared. [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref] The data shown below were measured on a single commercial Taguchi sensor SP32 (Figaro Inc.) after the new sensor was preheated ("burned-in") in laboratory air for several days with the nominal heating voltage until its stochastic signal component (resistance fluctuations) developed a stable power density spectrum S r ( f ) . In addition, to enhance the sensitivity and selectivity of the sensor, the Sampling-and-hold (SH) method, see [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref] , was used. At the SH protocol the sensor is heated for a short time, and then the heating (and gas flow) is turned off and, after the sensor has cooled down, the measurement is done [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref].
## Experimental setup
The measurement system [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref] is shown in [fig_ref] Figure 4: Outline of the experimental setup [/fig_ref]. The sensor and the sample were in a grounded stainless steel sensor chamber of one Liter volume. The sample in a Petri-dish was located 5 cm below the sensor. A DC bias through the sensor converted its resistance fluctuations into voltage fluctuations that were amplified by a low-noise preamplifier and their power density spectra were evaluated by a dynamic signal analyzer in the frequency range of 100 Hz-100 kHz. The voltage spectra were transformed back to power density spectra S r ( f ) of the fluctuations of sensor resistance [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref] [bib_ref] Extracting information from noise spectra of chemical sensors: single sensor electronic noses..., Kish [/bib_ref].
## Types of samples
The situations tested in these experiments were: empty chamber; TSA; TSA + E. coli at different bacterium numbers; TSA + Anthrax-surrogate; finally TSA + E. coli + Anthrax-surrogate. Here empty means no sample; TSA represents the culture medium: tryptic soy agar; E. coli means the harmless laboratory strain MG1655 as a surrogate for the pathogenic vegetative bacteria Escherichia coli; and Anthrax stands for the Anthrax-surrogate bacterium, the spore forming Bacillus subtilis. The samples with maximal bacterium number had one million bacteria.
Between different sample measurements, the chamber was flushed by synthetic air for 3 minutes. To see the reproducibility of the spectra, the measurements with each sample were repeated at least twice.
## Binary pattern extracted from experiments
In the figures below, for the sake of better visibility of the differences by the naked eye, the normalized power density spectrum γ( f ) is used:
[formula] 2 ) ( ) ( s r R f S f f × = γ (4) [/formula]
where R s is the actual sensor resistance. Note, for the binary patter generation describe in Section 2, both the original and the normalized spectra yield the same result.
For simplicity, we measured the average slopes in six sub-bands with logarithmically equidistant width by fittings of the ) ( f γ plotted as a log-log plot by Origin software. The sub-bands were 100−333 Hz for bit B1, 0.333-1 kHz for bit B2, 1−3.3 kHz for bit B3, 3.3−10 kHz for bit B4, 10−33 kHz for bit B5 and 33-100 kHz for bit B6. The binary pattern used for driving the logic circuit is found to have the following characteristics: (1) Good reproducibility: Examples are shown in [fig_ref] Figure 5: Figure 5 [/fig_ref] : measurement data obtained with independently prepared samples at different dates. The spectra in [fig_ref] Figure 5: Figure 5 [/fig_ref] yield identical patterns shown in [fig_ref] Figure 8: The spectra inFigures 5-7yield the same 6-bits pattern [/fig_ref]. Normalized power density spectra of the resistance fluctuations of the sensor SP32 measured in the sampling-and-hold [bib_ref] Fluctuation-enhanced sensing of bacterium odors, Chang [/bib_ref] working mode. Each sample had one million bacteria. The alias "Anthrax" stands for Anthrax surrogate Bacillus subtilis. (2) Inability to differentiate between the two types of bacteria: the applied sensor and the simple 6 bit pattern generation we used for these tests were unable to differentiate between the two bacteria, while they were able to differentiate between all the other cases (empty, TSA, bacteria). This fact originates from the particular settings of pattern generation because the differences between spectra with different bacteria could be distinguished by naked eye. However, we find this situation satisfactory because our goal was not to present a fully featured/optimized system but to show how much can be achieved with just a simple, ad-hoc, demo version of a 6 bits system.
(3) Robustness against variations of the bacterium number, see . This characteristic was unexpected with Taguchi sensors, which are nonlinear devices, but it could be expected with linear sensors. The most probable reason why we still experienced this property with our sensor is the linear response of nonlinear systems against small perturbations; a situation relevant for Taguchi sensors.
The measurement conditions to test the impact of bacterium numbers were as follows. Six different bacterium numbers of E. coli were used: 2.5 × 10 4 , 5 × 10 4 , 10 5 , 2.5 × 10 5 , 5 × 10 5 and 10 6 . The normalized power density spectra and the binary patterns are shown in [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref]. Variations of the normalized power density spectrum at different bacterium numbers. [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref]. Variations of the binary pattern at different bacterium numbers. Bit B5 is not reliable therefore that bit should not be used for pattern recognition, see the Boolean logic in Section 6.
The binary pattern in [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref] can also identify three conditions: empty, TSA and TSA + bacteria (E. coli). When the bacterium number decreases from 10 6 to 2.5 × 10 4 , the bits remained the same except bit B5 (relevant to sub-band 10 k~33 kHz). As a consequence, bit B5 should not be used in the Boolean logic for pattern recognition, except perhaps as extra information about the bacterium number. However, the rest of the bits provide sufficient information to identify the different types of samples: empty, TSA and TSA + bacteria (E. coli).
## Boolean logic circuit for pattern recognition with ultra-low power need
The obtained bit patterns are shown in . As we have shown above bit B5 is not robust against variations in the bacterium number thus we can consider it as an invalid bit unless we want to use that for the indication of bacterium number of E. coli. Thus a simple 5-bits Boolean logic circuit driven by pattern bits 1-4 and 6 can identify the different situations. The Boolean logic circuit in [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref] has a two-bits output where only 3 of the four possible states are used to display the recognized pattern: empty, TSA, or TSA + bacteria (either E. coli or Anthrax). . The logic input of the Boolean logic circuit generated from [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref]. Zero stands for σ n = −1 and 1 stands forσ n = 1.
## Bit b1
Bit B2 Bit B3 Bit B4 Bit B5 Bit B6
Empty
[formula] 0 1 1 1 N/A N/A TSA 1 0 0 0 N/A 0 TSA+bacteria 1 0 0 0 N/A 1 [/formula]
The output of the Boolean logic at the different situations is shown in [fig_ref] Table 2: The output of the Boolean logic circuit at the different situations [/fig_ref]. The output 1,0 is not shown because it is invalid. From [fig_ref] Table 2: The output of the Boolean logic circuit at the different situations [/fig_ref] , the following Boolean logic equations can be extracted:
[formula] 4 3 2 1 1 Bit B B B B ⋅ ⋅ ⋅ =(5)Bit 2 = B 1 ⋅ B 2 ⋅ B 3 ⋅ B 4 ⋅ B 6(6) [/formula]
The Boolean logic circuit to realize Equations 5, 6 is shown in [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref]. [fig_ref] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries [/fig_ref]. The Boolean logic circuit to realize the binary pattern recognition for the sampling-and-hold sensor SP32.
When this Boolean logic circuit is realized by CMOS logic gates, its static power consumption is in the nano/picoWatt regime. In addition, this circuit consumes some extra power for a short time (nanosecond) while it switches when the situation of agents change. Due to the rarity of such events, the power consumption in the Binary logic is basically due to the leakage current of transistors. At practical situations, these powers required by the logic circuitry are negligible.
## Power consumption of the whole sensing system
As a result of avoiding digital computation and using analog processing with the simple logic decisions instead, the main advantage of our system is its simplicity and ultra low power consumption. The analog circuits dominate the power consumption of this system and that is relatively small due to the low-frequency operation (<100 kHz). Ina more sophisticated analog circuitry for a different sensing approach is shown, including a wireless unit (known to be power hungry), with only 3 μW total power dissipation. Thus we can safely claim that the power dissipation of our analog circuitry is in the μW range or below. In comparison, a laptop computer based pattern recognizer, which would be able to run the same task, would dissipate around 20−50 Watts.
## Summary
In this work we have reported an exploratory study to generate and test highly distinguishable and robust types of binary patterns from power density spectra obtained at fluctuation-enhanced sensing of bacterial odors. We have shown a way how these binary patterns can be generated by an analog circuitry of ultra-low power consumption and used to drive a Boolean logic based pattern recognizer with negligible power consumption. We demonstrated these findings by single-sensor experiments recognizing bacteria with 100% success rate and zero false alarm rates.
Concerning an important question asked by a Referee about discriminating gram-positive and gramnegative bacteria, the answer is that this method is sensing the odor of the bacteria. If these sets represent some characteristic odor components then classification can be possible. Otherwise, the only way is to teach the system (construct the binary logic) to recognize each specific bacterium.
[fig] Figure 1: Illustration of n α and β for logarithmically equidistant sub-band boundaries. S( f ) is the power density spectrum of the fluctuations of the sensor signal. [/fig]
[fig] Figure 2: Major building blocks of the low-power sensing system. [/fig]
[fig] Figure 3: Details of the Analog Unit of the sensing system with 6 bit resolution shown inFigure 2, see the text for explanation. [/fig]
[fig] Figure 4: Outline of the experimental setup. [/fig]
[fig] Figure 5: Figure 5. Normalized power density spectra of the resistance fluctuations of the sensor SP32 measured in the sampling-and-hold [8,10] working mode. Each sample had one million bacteria. The alias "Anthrax" stands for Anthrax surrogate Bacillus subtilis. [/fig]
[fig] Figure 6: Reproducibility of the experimental data shown in Figures 5 with new samples. [/fig]
[fig] Figure 7: Reproducibility of the experimental data shown inFigures 5-6with new samples. [/fig]
[fig] Figure 8: The spectra inFigures 5-7yield the same 6-bits pattern. [/fig]
[table] Table 2: The output of the Boolean logic circuit at the different situations. [/table]
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Adenosine Triphosphate Release and P2 Receptor Signaling in Piezo1 Channel-Dependent Mechanoregulation
Organs and tissues and their constituent cells are physiologically submitted to diverse types of mechanical forces or stress, one common sequence of which is release of intracellular ATP into extracellular space. Extracellular ATP is a well-established autocrine or paracrine signaling molecule that regulates multiple cell functions and mediates cell-tocell communications via activating the purinergic P2 receptors, more specifically, ligandgated ion channel P2X receptors and some of the G-protein-coupled P2Y receptors. The molecular mechanisms that sense mechanical and transduce forces to trigger ATP release are poorly understood. The Piezo1, a newly identified mechanosensing ion channel, shows widespread expression and confers mechanosensitivity in many different types of cells. In this mini-review, we briefly introduce the Piezo1 channel and discuss the evidence that supports its important role in the mechanoregulation of diverse cell functions and, more specifically, critical engagement of ATP release and subsequent P2 receptor activation in Piezo1 channel-dependent mechanoregulation. Such ATP release-mediated coupling of the Piezo1 channel and P2 receptors may serve a signaling mechanism that is more common than we currently understand in transducing mechanical information to regulation of the attendant cell functions in various organs and tissues.
# Introduction
Adenosine triphosphate (ATP), while it is best known for its intracellular role as the cellular energy source, gains increasing recognition as an extracellular signaling molecule when it is released into extracellular spaces. In mammalian cells, the ATP-based signaling system comprises of three principal components: release of intracellular ATP into the extracellular space, activation of the ligand-gated ion channel P2X receptors and/or G-protein-coupled P2Y receptors for extracellular ATP, and removal of extracellular ATP to terminate its action by a broad family of ATP-scavenging ecto-nucleotidases that convert ATP to ADP, adenosine monophosphate, or adenosine . This system represents one of the most common signaling mechanisms regulating cell functions and mediating cell-to-cell communications and plays a critical role in a wide range of physiological processes, such as hearing, tasting, nociception, immune responses, muscle contraction, learning, and memory. There exists a large volume of evidence that alterations in such an ATP-based signaling system contribute in the pathogenesis and progression of diverse conditions, ranging from hearing loss, pain, inflammatory diseases, hypertension, neurodegenerative diseases, and psychotic disorders to cancer metastasis.
It is conceivable that ATP easily leaks from damaged or dying cells as a danger signal alerting tissue damage and inflammation. However, decades of studies provide clear evidence to show that many types of cells can release ATP without compromise in cell viability and a variety of physical and chemical signals or stimuli can induce non-lytic release of ATP. Two general release pathways, namely, vesicular and diffusion, have been proposed for efflux of intracellular ATP. However, the molecular mechanisms mediating ATP release are still not fully elucidated, in part due to that such mechanisms appear to be diverse and cell-type specific. Furthermore, many types of cells are equipped with multiple ATP release mechanisms and deploy them according to the nature of the incoming stimuli. Vesicular release via exocytosis represents the major mechanism by which neurons release ATP into the synaptic cleft in the peripheral and central nervous systems. Vesicular ATP release via exocytosis has been also described in astrocytes, urothelial cells, neutrophils, and pancreatic β-cells. In this regard, it is worth mentioning that the vesicular nucleotide transporter (VNUT) plays a critical role in mediating vesicular storage and thereby subsequent release of ATP(for more details, see. On the other hand, several distinctive types of ion channels have been suggested to act as conduits permitting diffusion of ATP out of cells. The volumeregulated anion channel (VRAC) has been identified to mediate non-synaptic release of ATP from axons in response to action potential-induced swelling. The pannexin hemi-channels, calcium homeostasis modulator 1 (CALHM1), cystic fibrosis transmembrane conductance regulator (CFTR), maxi-anion channel, and P2X7 receptor as well as the VRAC have been reported to mediate or regulate ATP efflux from a variety of non-neuronal cells. For detailed discussion of these ATP release mechanisms, the readers can consult recently published reviews (e.g.,.
Cells are physiologically submitted to diverse types of mechanical forces or stress and virtually all types of cells exhibit a mechanosensitivity. They can sense external or "outside-in" mechanical forces, for example, fluid flow-induced shear stress, osmotic stress, and pressure-induced membrane stretch. Cells can also generate traction forces via actin-myosin interactions at the focal adhesion zones and apply such "inside-out" mechanical forces to survey the mechanical and geographical properties of extracellular matrix and cellsupporting substrates. Importantly, cells are able to convert mechanical forces into intracellular signals and even integrate mechanical information into the genomic blueprint, indicating that mechanical stimulation can have long-term effects as well as short-term effects on cell functions. Mechanical stimuli are long known as a potent trigger for non-cytolytic release of ATP both in vivo and in vitro, and accumulating evidence supports that FIGURe 1 | Schematic illustration of the adenosine triphosphate (ATP)-based signaling system in mammalian cells. The ATP-based signaling system comprises of the following three principal components. (A) Release of intracellular ATP, which occurs via exocytosis (vesicular) and/or diffusion through many different types of ion channels. (B) Extracellular ATP as an autocrine or paracrine signal activating ligand-gated ion channel P2X receptors and/or G-protein-coupled P2Y receptors. ATP gates all P2X receptor ion channels, allowing extracellular Ca 2+ influx. Alternatively, ATP activates the P2Y receptors, mainly P2Y 1 , P2Y 2 , and P2Y 11 , leading to sequential activation of G α,q/11 , phospholipase C (PLC), conversion of membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to inositol triphosphate (IP 3 ) and diacylglycerol (not depicted), activation of the IP 3 receptor (IP 3 R), and Ca 2+ release from the endoplasmic reticulum (ER). (C) Termination of the actions of ATP by converting to ADP, AMP, and adenosine (Ade) by ecto-nucleotidases, including ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), and by ecto-5'-nucelotidase (E-NT).
ATP release and subsequent activation of the P2 receptors act as a crucial signal transduction mechanism in the mechanoregulation of cell functions. However, the molecular identity of the mechanosensor that directly detects the mechanical forces and triggers ATP release remained elusive. The Piezo1 ion channel has emerged as an intrinsically mechanically activated Ca 2+ -permeable cation channel that confers cells with an ability of sensing diverse forms of mechanical stimuli. Furthermore, a large number of recent studies have shown an important role of the Piezo1 channel in the mechanoregulation of a wide range of physiological and pathological functions. Of interest, accumulating evidence supports that ATP release and P2 receptor signaling are important in mediating Piezo1 channel-dependent mechanoregulation. The two separate domains of investigation thus need to join forces in order to develop a full and mechanistic understanding of mechanoregulation. The aim of this minireview is to introduce the Piezo1 channel and discuss the recent studies that provide evidence to support its crucial role in several types of mechanosensitive cells in the induction of ATP release and subsequent activation of the P2X or P2Y receptors and the mechanoregulation of the attendant cell functions. With increasing evidence to show their overlapping expression in many different types of mechanosensitive cells, the Piezo1 channel and P2 receptors, via coupling by ATP, may serve as a signaling mechanism that is more common than we currently understand in transducing the mechanical information to functional regulation.
## A brief introduction to the piezo1 channel
The Piezo1 protein (also known as Fam38a) was identified to form the mechanically activated ion channel mediating pressureinduced ionic currents in mouse neuroblastoma Neuro2A cells. In the same seminal study, a homologue protein, Piezo2 (also known as Fam38b), was found to express in a subset of mouse dorsal root ganglia neurons and can also form a mechanically activated ion channel with comparatively faster inactivation kinetics. The Piezo proteins are large in size, being ~2,500-2,800 amino acid residues long and with predicted molecular weights of ~290-320 kDa for the mouse and human proteins. They are predicted to have a unique membrane topology composed of 38 transmembrane segments and intracellular N-and C-termini . Several structures containing the core parts of the mouse Piezo1 channel have been recently determined by cryo-electron microscopy. These structures reveal a trimeric assembly and a three-bladed propeller-like architecture of the Piezo1 channel. For further structural details, the readers can consult recently published reviews (e.g.,.
Studies have demonstrated wide expression of the Piezo1 channel that enables many different types of cells to sense a diversity of "outside-in" mechanical forces, including indentation, membrane stretch, shear stress, osmotic stress, ultrasound, and compression. There is also compelling evidence to suggest that the Piezo1 channel can be activated by traction forces. Thus, two different, so-called "force-from-lipids" and "force-from-filaments, " mechanisms have been proposed for mechanical activation of the Piezo1 channel. In the "force-from-lipids" mechanism, mechanical forces introduce membrane tension that leads to reorganization of lipids within and surrounding the channel proteins. The resultant alterations in the membrane lipid-channel protein interactions induce the channel to open. This gating mechanism has gained support from a recent study. The "force-from-filaments" mechanism proposes that the interactions between the channel and extracellular matrix or intracellular cytoskeletal proteins provoke conformational changes leading to the channel opening.
The mechanically activated ion channels are less amenable to electrophysiological studies as compared to the ion channels activated by other modalities, such as changes in membrane potential, temperature, or chemical ligands. This is in part because of the unease of applying mechanical stimuli to cells under the experimental settings and the challenge of accurately determining the mechanical forces inducing the channel activation. Yoda1, a synthetic chemical, selectively activates the Piezo1 channel with an EC 50 (the concentration evoking 50% of the maximal response) of 2.5-27 µM, determined by measuring Yoda1-induced Ca 2+ responses in cells expressing the recombinant mouse and human Piezo1 channels. The discovery of Yoda1 has made it technically more approachable to the study of the Piezo1 channel under in vitro conditions. Grammostola spatulata mechanotoxin 4 (GsMTx4), a 34-amino acid peptide isolated from the venom of a tarantula spider and known to block mechanically activated currents, has been shown to inhibit the Piezo1 channel in the low micromolar concentrations. Mechanistically, GsMTx4 acts on the extracellular side as a channel gating modifier to modulate the arrangements of membrane lipids in the surroundings of the channel protein and thereby decreases the efficiency of force transduction from the lipid bilayer to the channel. Ruthenium red (RR), a polycationic ion, can also inhibit the Piezo1 channel-mediated mechanically activated currents with an IC 50 (the concentration causing 50% inhibition of the response) of 5.4 µM, which was shown at the Drosophila Piezo channel, and RR is thought to be an open channel blocker. Gadolinium ion (Gd 3+ ) in the micromolar concentrations is known to inhibit the Piezo1 channel. These negative allosteric modulators or inhibitors are lack of the specificity towards the Piezo1 channel. Nonetheless, they provide useful pharmacological tools, in combination with genetic means, to better understand the role of the Piezo1 channel in physiological and pathological processes.
The expression of the Piezo1 channel has been shown in an increasing number of cell types in various tissues and organs, including neurons, astrocytes, smooth muscle cells, endothelial cells, epithelial cells, red blood cells, immune cells, periodontal ligament cells, neural progenitor cells, mesenchymal stem cells, and embryonic stem cells (e.g.,. The Piezo1 channel is mainly located in the plasma membrane. Some evidence suggests that the Piezo1 channel is also present in the membrane of endoplasmic reticulumand in the cytoplasmic compartments near the nucleusand nuclear envelope. A large number of recent studies have disclosed a critical role for the cell surface Piezo1 channel or, more specifically, Piezo1-mediated Ca 2+ influx, in the regulation of a multiple of cell functions (e.g.,; see a recent review by Xiao, 2019). As discussed next, accumulating evidence supports that ATP release and subsequent activation of the P2X and/or P2Y receptors are critical in mediating Piezo1 channel-dependent mechanoregulation.
## Stretch-induced piezo1-dependent adenosine triphosphate release from urothelial cells and regulation of bladder function
It is known that as the urinary bladder distends, the urothelial cells become stretched and, as a result, release ATP, which in turn excites the innervating pelvic nerve afferents. The P2X3 receptor is expressed in the pelvic nerves, and the excitability of the pelvic nerve afferents induced by bladder distension was strongly attenuated in the P2X3-knockout mice, supporting a major role of the P2X3 receptor in transducing ATP release from urothelial cells to excitation of the pelvic nerve afferents. Both VNUT-dependent vesicular ATP release via exocytosis and ATP efflux through the CALHM1 and pannexin-1 hemi-channels have been shown to mediate ATP release from urothelial cells in response to mechanical forces. Furthermore, transient receptor potential (TRP) channels, particularly the TRPV4 channel, were suggested to sense mechanical stretch to induce ATP release from urothelial cells. However, compelling evidence indicates that mechanical activation of the TRPV4 channel is indirect, depending on mechanical induction of phospholipase A2-mediated generation of arachidonic acid and/or P450 epoxygenase-mediated generation of 5′,6′-epoxyeicosatrienoic acid from arachidonic acid. Thus, the molecular mechanism that directly senses mechanical stimuli to trigger ATP release from urothelial cells remained elusive. A recent study has shown expression of the Piezo1 channel in urothelial cells from both human and mouse bladders. In addition, membrane stretch induced a Ca 2+ influx-dependent increase in the [Ca 2+ ] i in mouse urothelial cells, and such Ca 2+ response was strongly attenuated by treatment with GsMTx4 or small interference RNA (siRNA)-mediated knockdown of the Piezo1 expression. The same study has further found that stretch stimulated ATP release from mouse urothelial cells. Importantly, stretch-induced ATP release was dependent of extracellular Ca 2+ and was suppressed by treatment with GsMTx4 or by siRNAmediated knockdown of the Piezo1 expression. This recent study, taken together with the previous study identifying the P2X3 receptor in coupling urothelial ATP release to pelvic nerve afferent activation, supports the notion that the Piezo1 channel in urothelial cells sense the bladder distension and triggers ATP release from urothelial cells and that ATP in turn acts as a paracrine signal excites the pelvic nerve afferents via activation of the P2X3 receptor .
## In other words, the piezo1 channel in urothelial cells and p2x3
FIGURe 2 | Adenosine triphosphate (ATP) release and activation of P2 receptors in Piezo1 channel-dependent mechanoregulation. (A) The Piezo1 channel in urothelial cells senses mechanical forces resulting from bladder distension and induces urothelial cells to release ATP, which in turn acts as a paracrine signal to excite the pelvic nerve afferents by activating the P2X3 receptor. Such signaling mechanism is critical for maintaining the normal bladder function. (B) The Piezo1 channel in endothelial cells mediates blood flow-induced release of ATP that serves an autocrine signal acting on the P2Y2 receptor to regulate vascular function and blood pressure. (C). The Piezo1 channel in red blood cells mediates shear stress-induced release of ATP as an autocrine signal to activate yet identified P2 receptor(s) to regulate cell volume. See text for various molecular mechanisms that are known to mediate ATP release. receptor in sensory neurons are important duo players, linked by ATP release from urothelial cells, to maintain the normal bladder function.
## Shear stress-induced piezo1-dependent adenosine triphosphate release from endothelial cells and regulation of vascular function
The vascular endothelium experiences dynamic blood flowinduced shear stress. It is well recognized that the ability of endothelial cells to sense and respond to shear stress is vital for development, function, and disease of the vascular system. ATP release from endothelial cells in response to shear stress has been well documented, and there is compelling evidence to support a critical role of the pannexin-1 hemi-channel in mediating shear stress-induced ATP release. A recent study has shown that ATP released from endothelial cells upon exposure to shear stress serves as a paracrine signal that activates the P2Y2 receptor and downstream signaling pathways, including endothelial nitric oxide synthase to generate nitric oxide (NO), to induce vasodilation . Consistently, endothelium-specific deletion of the P2Y2 receptor expression in mice led to loss of blood flow-induced vasodilation, resulting in hypertension . A more recent study from the same group has examined the role of the Piezo1 channel in mediating shear stress-induced ATP release from endothelial cells. Exposing endothelial cells to shear stress or Yoda1 induced robust Ca 2+ responses and ATP release, both of which were significantly attenuated by siRNAmediated knockdown of the Piezo1 expression. ATP release induced by shear stress or Yoda1 was also suppressed by siRNAmediated reduction in the expression of pannexin-1 or pannexin-2, indicating that shear stress-induced Piezo1-dependent ATP release is at least in part mediated by the pannexin hemi-channels. Perfusion of the mouse mesenteric arteries or exposure to Yoda1 induced vasodilation, which was impaired by endothelium-specific deletion of the Piezo1 expression. Furthermore, endothelium-specific and conditional knockout of the Piezo1 expression led to elevated blood pressure in mice, as observed for endothelium-specific and conditional knockout of the P2Y2 receptor . Collectively, these studies provide compelling evidence to support a vital role of the Piezo1 channel in mediating blood flow-induced release of ATP from endothelial cells as an autocrine signal to regulate the vascular function via activating the P2Y2 receptor .
## Shear stress-induced piezo1-dependent adenosine triphosphate release from red blood cells and regulation of cell volume
Like endothelial cells, red blood cells in circulation are exposed to considerable flow-induced shear stress. Hereditary stomatocytosis and hereditary xerocytosis are rare genetic disorders characterized by red blood cell dehydration. Several gain-of-function mutations in the Piezo1 channel have been shown to be causatively associated these conditions, highlighting a crucial role of the Piezo1 channel in maintaining the normal red blood cell homeostasis. Both human and mouse red blood cells are reported to express the Piezo1 channel on the cell surface. Interestingly, membrane stretch elicited strong Ca 2+ influxdependent increase in the [Ca 2+ ] i in red blood cells isolated from wild-type mice, but not from mice with conditional knockout of the Piezo1 expression. Similarly, exposure to Yoda1 induced Piezo1-dependent Ca 2+ entry in mouse red blood cells. Fluid flow-induced shear stress also elicited robust Ca 2+ influx in human red blood cells, which was significantly suppressed by treatment with GsMTx4, RR or Gd 3+. Furthermore, genetic deletion of the Piezo1 expression led to red blood cell over-hydration and increased mechanical fragility both in vitro and in vivo. Conversely, Yoda1induced activation of the Piezo1 channel caused red blood cell dehydration. These findings demonstrate an indispensable role of the Piezo1 channel in regulating red blood cell function and reveal the Piezo1 channel as a promising target for the development of therapeutics to treat hereditary stomatocytosis and hereditary xerocytosis.
It is long known that red blood cells release ATP in response to mechanical stimuli, such as osmotic stressand shear stress. A previous study showed that ATP release under in vitro conditions remained constant in response to shear stress below a certain threshold, but increased significantly above the threshold, which was accompanied with cellular deformation. A subsequent study provides evidence to suggest that the pannexin-1 hemi-channel is the main pathway mediating ATP release induced by shear stress both above and below the threshold, whereas the CFTR is engaged in deformation-dependent ATP release. A recent study shows that shear stress-induced ATP release was strongly correlated with extracellular Ca 2+ concentration. Shear stress-induced ATP release as well as Ca 2+ influx in human red blood cells was attenuated by treatment with GsMTx4, RR or Gd 3+. These results suggest that the Piezo1 channel is important in mediating induction by shear stress of ATP release from red blood cells . Several ATP-sensitive P2 receptors, including P2X1, P2X7, and P2Y1, P2Y11 are expressed in red blood cells, and evidence exists to support that activation of these P2 receptors in red blood cells stimulates a number of signaling pathways that is critical for cell functions, including cell volume regulation. However, it has not been ascertained which P2 receptor(s) participate(s) in Piezo1-dependent regulation of red blood cell functions.
## Piezo1-dependent adenosine triphosphate release from mesenchymal stem cells and regulation of cell migration
Mesenchymal stem cells (MSCs), which have promising applications in tissue regeneration and cell-based therapies, are highly mechanosensitive. It is well recognized that MSCs release ATP in response to mechanical stimulation both in vitro and in vivo. It is also known that several P2X and P2Y receptors are expressed in MSCs and mediate ATP-induced regulation of cell proliferation, migration, and differentiation. A previous study using bone marrow-derived MSCs suggests that fluid flow-induced ATP release via the pannexin hemi-channels and subsequent activation of the ATP-sensitive P2Y receptors increased cell proliferation. A more recent study shows that shockwave-induced ATP release via undefined release mechanisms and subsequent activation of the P2X7 receptor stimulated osteogenic differentiation. The expression of the Piezo1 channel has been documented in several very recent studies using MSCs from different species and tissues. Our recent study shows that Yoda1-induced activation of the Piezo1 channel in human dental pulp MSC promoted migration, which was suppressed by siRNA-mediated knockdown of the Piezo1 expression. More importantly, Yoda1-induced activation of the Piezo1 channel stimulated ATP release from human dental pulp MSCs. Yoda1-induced Piezo1-dependent increase in cell migration was inhibited by treatment with apyrase, a scavenger of extracellular ATP, and also with PPADS, a P2 receptor generic antagonist. Taken together, these results support the notion that activation of the Piezo1 channel enhances MSC migration via inducing release of ATP as an autocrine signal that activates the P2 receptors. Our previous study has identified P2X7, P2Y1, and P2Y11 as the major P2 receptors that participate in mediating ATP-induced stimulation of human dental pulp MSC migration. It is highly interesting to examine the role of ATP release and the P2 receptors in Piezo1-dependent mechanoregulation of MSC functions such as differentiation and migration.
Adenosine Triphosphate Release and P2 Receptor as a Common Signaling Mechanism in Piezo1 Channel-Dependent Mechanoregulation?
As mentioned above, recent studies demonstrate expression of the Piezo1 channel in many different types of mechanosensitive cells with an important role in the mechanoregulation of attendant cell functions. The majority, if not all, of these cells, are known to express the P2X/P2Y receptors that are important in mediating ATP-induced regulation of their functions. This raises the perspective that ATP release integrates the Piezo1 channel and P2 receptor as a more common signaling mechanism in the mechanoregulation of cell functions.
The expression of the Piezo1 channel is required for alignment of endothelial cells in response to shear stress. Similarly, the P2Y2 receptor in endothelial cells plays an important role in mediating shear stress-induced cell alignment. It is interesting to investigate whether shear stress-induced ATP release couples the Piezo1 and the P2Y2 receptor in the regulation of vascular development. Another recent study shows that shear stress induces ATP release from red blood cells, on one hand, and an increase in the [Ca 2+ ] i and NO generation in endothelial cells and formation of inter-endothelial junctions, on the other. These shear stress-induced responses or effects both in red blood cells and endothelial cells were prevented by pharmacological inhibition and genetic depletion of the pannexin-1 channel on red blood cells. It is unknown whether shear stress-induced Piezo1-dependent ATP release from red blood cells acts as a paracrine signal to induce Ca 2+ signaling in endothelial cells via activating the P2X/P2Y receptors.
As discussed above, ATP release coupling of the Piezo1 channel in urothelial cells and the P2X3 receptor in the pelvic nerve afferents is important in maintaining the normal bladder function. Such a signaling mechanism may also play an important role in mediating dentinal pain. It is known that dentinal fluidinduced odontoblast deformation can evoke dentinal pain. A recent electrophysiological study shows that pressure-induced odontoblast deformation elicited inward currents that caused membrane depolarization and induced action potentials in co-cultured isolectin IB4-negative medium-sized trigeminal ganglion neurons. Furthermore, such inward currents were significantly attenuated by treatment with NF110, a P2X3 receptor antagonist, or with GsMTx4 as well as with a cocktail of TRP channel inhibitors. It is thus hypothesized that Piezo1/TRP-dependent ATP release from odontoblasts in response to mechanical stimulation excites myelinated Aδ neurons via activating the P2X3 receptor, thereby forming a signaling mechanism generating dentinal pain.
Cancer cells in the metastasis process encounter mechanical forces such as compression from the surrounding extracellular matrix and cells in the primary site, invasion into neighboring tissues, intravasation and extravasation through endothelial cells, micro-metastasis at target tissues or organs. They also experience blood flow-induced shear stress during circulation in the blood stream. It is conceivable that mechanical forces influence cancer cell migration, invasiveness, and metastasis. Consistently, several recent studies provide increasing evidence to show that activation of the Piezo1 channel stimulates cell proliferation in gastric cancer cells , and enhances cell migration in gastric cancer cellsand malignant MCF-7 breast cancer cells but reduces non-small cell lung cancer progression and cell migration. Compelling evidence already exists to support that extracellular ATP can regulate cancer cell migration, invasiveness, and metastasis via activating the P2X7, P2Y2 or P2Y11 receptors. Particularly, it was shown that ATP released from platelets bound to the circulating cancer cells and consequently activates the P2Y2 receptor on endothelial cells to promote formation of inter-endothelial junctions for cancer cell migration. As discussed above, shear stress can induce ATP release from red blood cells. It is attractive to speculate that shear stress-induced ATP release from red blood cells acts as a paracrine signal to induce formation of inter-endothelial junctions via activating the P2X/P2Y receptors in endothelial cells and thereby facilities intravasation and extravasation of cancer cells.
## Concluding remarks
It is evident from the discussion above that accumulating evidence supports an important role of ATP release as an autocrine and/or paracrine signal and subsequent activation of the P2 receptors in Piezo1 channel-dependent mechanoregulation of cell functions and associated physiological processes . As illustrated by hereditary stomatocytosis and hereditary xerocytosis, alterations in such signaling mechanisms resulting from mutations in the Piezo1 channel in red blood cells can lead to cell dysfunction and severe human diseased conditions. Studies so far support the Piezo1 channel as an intrinsic mechanosensor to trigger ATP release in response to mechanical stimulation. However, it remains unknown how activation of the Piezo1 channel regulates the ATP release mechanisms. Finally, more investigations are required to determine whether the Piezo1 channel and P2 receptors coupled by ATP release form a common signaling mechanism in transducing mechanical force information to regulation of cell functions.
# Author contributions
All authors contributed to the development of the concept. L-HJ wrote the manuscript. All the authors commented and approved the manuscript.
# Acknowledgments
The research works from the authors' laboratory were supported by the Disciplinary Group of Psychology and Neuroscience Xinxiang Medical University (2016PN-KFKT-06) and visiting professorship from University of Tours to LHJ, and a PhD studentship from Kuwait High Commission to FM. |
Brain Activation in Response to Low-Calorie Food Pictures: An Explorative Analysis of a Randomized Trial With Dapagliflozin and Exenatide
Background and Aim: Sodium-glucose cotransporter-2 inhibitors (SGLT2i) induce less weight loss than expected. This may be explained by SGLT2i-induced alterations in central reward and satiety circuits, contributing to increased appetite and food intake. This hyperphagia may be specific to high-calorie foods. Glucagon-like peptide-1 receptor agonists (GLP-1RA) are associated with lower preferences for high-calorie foods, and with decreased activation in areas regulating satiety and reward in response to high-calorie food pictures, which may reflect this lower preference for energy-dense foods. To optimize treatment, we need a better understanding of how intake is controlled, and how [(un)healthy] food choices are made. The aim of the study was to investigate the effects of dapagliflozin, exenatide, and their combination on brain activation in response to low-calorie food pictures.Methods:We performed an exploratory analysis of a larger, 16-week, double-blind, randomized, placebo-controlled trial. Sixty-eight subjects with obesity and type 2 diabetes were randomized to dapagliflozin, exenatide, dapagliflozin plus exenatide, or double placebo. Using functional MRI, the effects of treatments on brain responses to low-calorie food pictures were assessed after 10 days and 16 weeks.Results: Dapagliflozin versus placebo decreased activity in response to low-calorie food pictures, in the caudate nucleus, insula, and amygdala after 10 days, and in the insula after 16 weeks. Exenatide versus placebo increased activation in the putamen in response to low-calorie food pictures after 10 days, but not after 16 weeks. Dapagliflozin plus exenatide versus placebo had no effect on brain responses, but after 10 days dapagliflozin plus exenatide versus dapagliflozin increased activity in the insula and amygdala in response to low-calorie food pictures.
# Introduction
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) are recently introduced drug classes for the treatment of type 2 diabetes (T2D). Besides their effects on glucose regulation, both reduce body weight.
Weight loss induced by SGLT2i is attributable to urinary glucose excretion. However, observed weight loss is less than expected based on the amount of calories excreted. Since energy expenditure remains unchanged with SGLT2i, the discrepancy between the observed and expected weight loss implies an increase in calorie intake [bib_ref] Energy Balance After Sodium-Glucose Cotransporter 2 Inhibition, Ferrannini [/bib_ref]. Previously, we showed that dapagliflozin affects central satiety and reward centers, as we found that dapagliflozin increased regional brain responses to highly palatable food cues [bib_ref] Effects of Dapagliflozin and Combination Therapy With Exenatide on Food-Cue Induced Brain..., Van Ruiten [/bib_ref]. Together with an observed increased appetite and carbohydrate intake, this could contribute to less weight loss than expected based on urinary calorie excretion. Other studies also suggested a specific increase in (craving for) high calorie/sweet foods with SGLT2i treatment [bib_ref] Increased Sugar Intake as a Form of Compensatory Hyperphagia in Patients With..., Horie [/bib_ref] [bib_ref] Canagliflozin Increases Calorie Intake in Type 2 Diabetes Without Changing the Energy..., Matsuba [/bib_ref].
The weight loss induced by GLP-1RAs is mainly attributable to suppressed appetite signaling in the brain and increased satiety, which leads to a reduced food intake via direct and indirect actions in the CNS. Interestingly, GLP-1 receptor activation selectively reduced intake of highly palatable, energy-dense food without affecting intake of standard diets in animals [bib_ref] Glucagon-Like Peptide-1 Receptor Activation in the Nucleus Accumbens Core Suppresses Feeding by..., Mietlicki-Baase [/bib_ref] [bib_ref] The Food Intake-Suppressive Effects of Glucagon-Like Peptide-1 Receptor Signaling in the Ventral..., Mietlicki-Baase [/bib_ref] [bib_ref] Endogenous Glucagon-Like Peptide-1 Suppresses High-Fat Food Intake by Reducing Synaptic Drive Onto..., Wang [/bib_ref]. In humans, GLP-1RA treatment is also associated with lower preferences for fatty, energy-dense foods [bib_ref] Effects of Once-Weekly Semaglutide on Appetite, Energy Intake, Control of Eating, Food..., Blundell [/bib_ref] [bib_ref] Proof of Concept: Effect of GLP-1 Agonist on Food Hedonic Responses and..., Brindisi [/bib_ref] [bib_ref] GLP-1 Analog Modulates Appetite, Taste Preference, Gut Hormones, and Regional Body Fat..., Kadouh [/bib_ref] [bib_ref] The Effect of Semaglutide 2.4 Mg Once Weekly on Energy Intake, Appetite,..., Friedrichsen [/bib_ref]. In addition, GLP-1RA treatment was associated with decreased activation in the insula, putamen, and amygdala in response to palatable food cues [bib_ref] GLP-1 Receptor Activation Modulates Appetite-and Reward-Related Brain Areas in Humans, Van Bloemendaal [/bib_ref] [bib_ref] Liraglutide Reduces CNS Activation in Response to Visual Food Cues Only After..., Ten Kulve [/bib_ref] , which may reflect a lower preference for energy-dense foods. However, it is unknown if GLP-1RA treatment is associated with an increased CNS activation to low-calorie food pictures, which may reflect a preference for healthy low-calorie foods.
Given their unique mechanisms of lowering glucose and body weight, the combination of SGLT2i and GLP-1RA appears promising [bib_ref] Exenatide Once Weekly Plus Dapagliflozin Once Daily Versus Exenatide or Dapagliflozin Alone..., Frias [/bib_ref] [bib_ref] Semaglutide Once Weekly as Add-on to SGLT-2 Inhibitor Therapy in Type 2..., Zinman [/bib_ref] [bib_ref] Dulaglutide as Add-on Therapy to SGLT2 Inhibitors in Patients With Inadequately Controlled..., Ludvik [/bib_ref]. Recently we showed that in combination therapy, exenatide blunted the increased CNS activation observed with dapagliflozin in response to palatable food cues [bib_ref] Effects of Dapagliflozin and Combination Therapy With Exenatide on Food-Cue Induced Brain..., Van Ruiten [/bib_ref]. However, the response to low-calorie foods has not been investigated.
Most studies investigated brain responses to high-calorie foods, as high-calorie foods induce greater hedonic responses in the brain. Interestingly, some studies also investigated brain responses to low-calorie food pictures and suggested that highand low-calorie foods may be (partially) differently processed by the brain [bib_ref] Developmental Changes in the Functional Brain Responses of Adolescents to Images of..., Killgore [/bib_ref] [bib_ref] Differential Activation of the Dorsal Striatum by High-Calorie Visual Food Stimuli in..., Rothemund [/bib_ref] [bib_ref] Distinct Modulatory Effects of Satiety and Sibutramine on Brain Responses to Food..., Fletcher [/bib_ref]. In order to optimize treatments for obesity, we need a better understanding of how the intake of different nutrients is controlled, and how food choices are made. Therefore, the aim of the current study was to explore the effects of dapagliflozin, exenatide, and their combination on CNS activation in response to low-calorie food pictures.
# Methods
## Study design
This was an exploratory analysis of the Dapagliflozin plus Exenatide on Central REgulation of Appetite in diabeteS typE 2 (DECREASE) study [bib_ref] Effect of Exenatide Twice Daily and Dapagliflozin, Alone and in Combination, on..., Van Ruiten [/bib_ref] (NCT03361098). The study was approved by the Medical Ethics Committee of the Amsterdam University Medical Center, location Vumc, the Netherlands, and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. All patients provided informed consent.
## Participants
Participants were recruited from our outpatient clinic database and by advertisement in local newspapers. We included men and postmenopausal women with type 2 diabetes, aged 18 to 75 years, with a stable body weight (<5% reported change during the previous 3 months), a BMI >25 kg/m 2 who used metformin with or without sulfonylurea (stable dose for ≥ 3 months). HbA1c levels for participants treated with metformin monotherapy were 7%-10% and for metformin plus sulfonylurea 7.5%-10%. Exclusion criteria were a history of severe cardiovascular, renal, or liver disease, malignancies (excluding basal cell carcinoma), uncontrolled thyroid disease, the use of any centrally acting agent or oral glucocorticoids, substance abuse, neurological or psychiatric disease including eating disorders and depression, and MRI contra-indications. Written informed consent was obtained from all participants.
## General experimental protocol
The design of this randomized double-blind placebo-controlled trial has been described in detail previously [bib_ref] Effect of Exenatide Twice Daily and Dapagliflozin, Alone and in Combination, on..., Van Ruiten [/bib_ref]. In summary, patients were randomized 1:1:1:1, performed by an independent trial pharmacist using computer-generated numbers, to 1. dapagliflozin 10 mg plus exenatide-matched placebo; 2. exenatide twice-daily 10 µg plus dapagliflozin-matched placebo; 3. dapagliflozin plus exenatide; or 4. placebo dapagliflozin plus placebo exenatide for 16 weeks. Exenatide (or placebo) was injected twice daily 15 to 30 minutes before breakfast and dinner, and was initiated at a dose of 5 µg, followed by a dose increase to 10 µg after 4 weeks, which was maintained until the end of the study. Dapagliflozin (or placebo) was taken once daily at 8 PM during the 16-week treatment period. To maintain blinding throughout the study, participants were treated in a double-dummy design. There was no difference in appearance between exenatide and placebo injections or dapagliflozin or placebo tablets. There were three endpoint visits, at baseline, after 10 days, and 16 weeks of treatment. On these three visits, fMRI measurements and measurements of anthropometrics, blood pressure, and body composition were performed, and blood was drawn. The 10-day measurement was chosen to assess the effects of treatments on the CNS responses, independent of changes in body weight. The 16week follow up time point was chosen because the largest reductions in HbA1c and body weight occur in the first~16 weeks of treatment, and remain more or less stable thereafter (2, 24).
## Fmri protocol
The fMRI measurements were performed as described previously [bib_ref] GLP-1 Receptor Activation Modulates Appetite-and Reward-Related Brain Areas in Humans, Van Bloemendaal [/bib_ref] [bib_ref] Liraglutide Reduces CNS Activation in Response to Visual Food Cues Only After..., Ten Kulve [/bib_ref] [bib_ref] Brain Reward Responses to Food Stimuli Among Female Monozygotic Twins Discordant for..., Doornweerd [/bib_ref]. Briefly, pictures were presented in three runs comprising six blocks each: two blocks consisting of high-calorie food (sweet and savoury), two blocks of low-calorie food (fruits and vegetables), and two blocks of non-food items (e.g., trees, flowers, rocks, and bricks). Within each block seven pictures were presented for 2.5 s each, separated by a 0.5 s blank screen. Each block was followed by 9 s of grey blank screen with a fixation cross. Across each block and session, pictures were matched for shape and color Imaging data were acquired using a 3.0 Tesla GE Signa HDxt scanner (General Electric, Milwaukee, WI, USA). Structural MRI was obtained using a T1-weighted sequence. fMRI data were acquired using an echo planar imaging T2-weighted blood oxygenation level-dependent sequence with 40 ascending slices per volume (3 mm thickness, 0 mm gap), which resulted in whole-brain coverage. Functional images were pre-processed with fMRIprep v1.2.3 (26), as described previously (3). T1-coregistered volumes were normalized to Montreal Neurological Institute space. Pre-processed data were analysed in the context of the general linear model with SPM12 (Wellcome Trust Centre for Neuroimaging, London, U.K.). At the first (single-subject) level, high-calorie food, low-calorie food, and non-food blocks were modeled. To assess if the effect of treatment on CNS activation related to viewing food pictures, and was food-cue specific, we computed the following contrasts at each time point: low-calorie vs non-food and high-calorie vs. non-food. To test our hypotheses, dapagliflozin, exenatide, and dapagliflozin plus exenatide were compared with placebo after 10 days and 16 weeks of treatment. In additional analyses, dapagliflozin was compared with dapagliflozin-exenatide.
A priori ROIs were determined based on previous studies (i.e., insula [including adjacent opercula], striatum [i.e., putamen and caudate nucleus], amygdala, and orbitofrontal cortex [OFC]), as these regions are consistently shown to be involved in responses to food cues and are part of the central reward circuits (27)). CNS activations were reported as significant when these survived family-wise error (FWE) correction for multiple comparisons (P FWE < 0.05) at the voxel level using small volume correction within the predefined ROIs, using 10-mm radius spheres (5-mm sphere for the amygdala) as described previously [bib_ref] GLP-1 Receptor Activation Modulates Appetite-and Reward-Related Brain Areas in Humans, Van Bloemendaal [/bib_ref] [bib_ref] Endogenous GLP-1 Mediates Postprandial Reductions in Activation in Central Reward and Satiety..., Ten Kulve [/bib_ref].
# Results
## Baseline characteristics
Of 68 patients, 65 patients completed the study protocol between September 2017 and March 2020 (3). One patient in the combination group discontinued treatment due to nausea, and one patient in the placebo group discontinued treatment due to personal reasons. One patient in the exenatide group experienced claustrophobia during baseline MRI measurements; this patient continued treatment without follow-up MRI measurements. Baseline characteristics were well balanced between treatment groups (
## Anthropometrics and glycemic control
As previously published, compared with placebo, dapagliflozin reduced body weight by -2.5 ± 0.5 kg (p<0.001), exenatide by -1.4 ± 0.5 kg (p<0.01), and the combination by -2.8 ± 0.5 kg (p<0.001), after 16 weeks of treatment (3). In addition, compared with placebo, dapagliflozin reduced HbA1c by 0.5 ± 0.19% (5 mmol/mol, p<0.01), exenatide by 0.8 ± 0.18% (8.4 mmol/mol, p<0.001), and the combination by -1.2 ± 0.19% (11.9 mmol/mol, p<0.001) after 16 weeks (3).
## Cns responses to food pictures
## Dapagliflozin compared with placebo
After 10 days dapagliflozin compared with placebo decreased activity in the bilateral caudate nucleus (right, T 3.5, P-FWE 0.035; left, T 3.7, P-FWE 0.023), left insula (T 3.6, P-FWE 0.027), and left amygdala (T 3.1, P-FWE 0.021) in response to low-calorie pictures (Table 2; [fig_ref] FIGURE 1 |: Average differences in CNS activation between dapagliflozin [/fig_ref]. After 16 weeks of treatment, dapagliflozin compared with placebo reduced activity in the right insula (T 3.5, P-FWE 0.041) in response to low-calorie pictures (Table 2; [fig_ref] FIGURE 1 |: Average differences in CNS activation between dapagliflozin [/fig_ref].
## Exenatide compared with placebo
After 10 days exenatide compared with placebo increased activity in the right putamen (T 3.6, P-FWE 0.027) and tended to increase activity in right insula (T 3.3, P-FWE 0.057) in response to lowcalorie pictures (Table 2; [fig_ref] FIGURE 2 |: Average differences in CNS activation after 10 days of treatment [/fig_ref]. After 16 weeks exenatide compared with placebo had no effect on brain activation in response to low-calorie pictures.
## Dapagliflozin-exenatide compared with placebo
Dapagliflozin-exenatide compared with placebo had no effect on brain activation in response to low-calorie pictures. [fig_ref] FIGURE 2 |: Average differences in CNS activation after 10 days of treatment [/fig_ref]. After 16 weeks combination therapy tended to increase activity in the left putamen (T 3.1, P-FWE 0.065) in response to low-calorie pictures [fig_ref] TABLE 2 |: Effects of dapagliflozin, exenatide and the combination of dapagliflozin and exenatide on... [/fig_ref].
# Discussion
This is the first study investigating CNS activation in response to low-calorie food pictures with dapagliflozin, exenatide, or This table describes the areas where significant differences in activations were observed with dapagliflozin, exenatide, and combination of dapagliflozin plus exenatide compared with placebo treatment. For each comparison, the contrast (activation during low-calorie > non-food pictures) is presented. The areas with significant differences are listed, including the cluster size of this effect, the T value, and the FWE corrected p-value after small volume correction. The last column describes the coordinates of the peak voxel of the observed difference in MNI space. For completeness non-significant results in regions of interest are showed in grey. Combination, dapagliflozin plus exenatide; T, T-value; P-FWE , P-value family-wise error corrected for multiple comparisons on the basis of cluster extent (small volume correction); R, right; L, left; MNI, Montreal Neurological Institute coordinates in mm, which represents the exact three dimensional location [x=horizontal, y=horizontal, z= vertical axis in mm distance from the origin (which is the intersection of the three axis)] in the brain of the significant activation; NS, indicating that there were no statistical significant results for this comparison. Here, we demonstrated that the neural responses to food pictures with dapagliflozin may be food cue specific, as dapagliflozin was associated with reduced activation in response to low-calorie (non-hedonic) food pictures after short (10 days) and longerterm (16 weeks) treatment. In addition, exenatide was associated with increased CNS responses to low-calorie food pictures, while the combination of dapagliflozin and exenatide showed no effect on low-calorie food pictures.
Previously, we showed that the presumed SGLT2i-induced hyperphagia (1, 2, 29) may be specific for high-calorie/sweet foods, as we and others found an increase in carbohydrate intake [bib_ref] Sodium-Glucose Cotransporter 2 Inhibition and Glycemic Control in Type 1 Diabetes: Results..., Perkins [/bib_ref] , or sugar intake [bib_ref] Increased Sugar Intake as a Form of Compensatory Hyperphagia in Patients With..., Horie [/bib_ref]. In line with this, we previously observed that dapagliflozin increased CNS activation in response to high-calorie foods, which may reflect the neurophysiological correlate for the observed preference for high-calorie/sweet foods. Interestingly, this is the first study that also demonstrated decreased CNS activation in response to lowcalorie food pictures, which may reflect a specific decreased preference for low-calorie foods. This may have clinical consequences as a specific dislike for low-calorie foods in . Horizontal slice showing average differences between dapagliflozin (black bar) and placebo (white bar) after 16 weeks of treatment in the right insula (left panel: P FWE < 0.05, T = 3.5), and non-statistically significant in the right insula (right panel: P FWE = 0.074, T = 3.2) in response to the viewing of low-calorie food pictures versus non-food pictures. The left side of the brain slices is the left side of the brain. The color scale reflects the T value of the functional activity. Results are presented at the threshold of P < 0.05, familywise error (FWE) corrected on cluster extent within the regions of interest using small volume correction (10 mm sphere; 5 mm sphere for the amygdala). In the graphs, blood oxygen level-dependent (BOLD) signal intensity (effect size) is plotted (arbitrary unites), mean and SEM. The numbers on the y-axes of the bar graphs are the x,y,z, coordinates of the peak voxel of the observed difference in Montreal Neurological Institute (MNI) space. R, right; L, left. combination with a preference for high-calorie foods and may hamper SGLT2i-induced weight loss. Some studies suggested a specific dislike for calorie-dense/ sweet foods with GLP-1RA treatment [bib_ref] Effects of Once-Weekly Semaglutide on Appetite, Energy Intake, Control of Eating, Food..., Blundell [/bib_ref] [bib_ref] Proof of Concept: Effect of GLP-1 Agonist on Food Hedonic Responses and..., Brindisi [/bib_ref] [bib_ref] GLP-1 Analog Modulates Appetite, Taste Preference, Gut Hormones, and Regional Body Fat..., Kadouh [/bib_ref] [bib_ref] The Effect of Semaglutide 2.4 Mg Once Weekly on Energy Intake, Appetite,..., Friedrichsen [/bib_ref]. Here, we are the first to demonstrate that exenatide increased CNS activation in response to low-calorie food pictures, while we previously demonstrated that liraglutide (16) and exenatide (3) reduced CNS activation to high-calorie food pictures. Together, these findings suggest an increased preferential response to low-calorie foods. Interestingly, in line with these results, a recent observational study found a positive correlation between endogenous circulating GLP-1 and dorsal striatal responsiveness to low-calorie food cues, and a negative correlation between GLP-1 and dorsal striatal responding to high-calorie food cues [bib_ref] Obesity and Dietary Added Sugar Interact to Affect Postprandial GLP-1 and Its..., Jones [/bib_ref].
After combining dapagliflozin with exenatide, the dapagliflozin-induced decrease in CNS activation in response to low-calorie food pictures had disappeared, presumably due to activation increasing effects of exenatide. These findings complement the findings of CNS responsiveness to highcalorie food pictures, as we found no effect of dapagliflozin plus exenatide, while dapagliflozin increased responses and exenatide decreased CNS responses to high-calorie food pictures [bib_ref] Effects of Dapagliflozin and Combination Therapy With Exenatide on Food-Cue Induced Brain..., Van Ruiten [/bib_ref]. Together, this suggests that exenatide can (partially) alter the food preferences induced by dapagliflozin, which may contribute to more weight loss with the combination. "Effects of treatments compared with placebo were observed in putamen, caudate nucleus, insula and amygdala. These brain regions are part of a complex reward circuitry [bib_ref] A Core Eating Network and Its Modulations Underlie Diverse Eating Phenomena, Chen [/bib_ref]. The insula receives gustatory and visceral afferents, is involved in taste memory and is also involved in the rewarding aspects of food [bib_ref] Food Related Processes in the Insular Cortex, Frank [/bib_ref]. The insula modulates the activity of the other brain regions, based on homeostatic signals, and translates these internal signals into subjective feelings such as the urge to eat [bib_ref] How Do You Feel-Now? The Anterior Insula and Human Awareness, Craig [/bib_ref]. The putamen is engaged in the reward processing and conditioning, and activation in the putamen is associated with predicting future weight gain [bib_ref] Greater Striatopallidal Adaptive Coding During Cue-Reward Learning and Food Reward Habituation Predict..., Burger [/bib_ref]. The amygdala is involved in the association of cues with reward and emotional learning [bib_ref] The Amygdala and Reward, Baxter [/bib_ref].
"SGLTs are glucose transporters in the intestine, kidney, and also in the brain [bib_ref] Biology of Human Sodium Glucose Transporters, Wright [/bib_ref] [bib_ref] Regional Distribution of SGLT Activity in Rat Brain In Vivo, Yu [/bib_ref] [bib_ref] Sodium/ glucose Cotransporter 2 Is Expressed in Choroid Plexus Epithelial Cells and..., Chiba [/bib_ref]. The expression of SGLT2 in brain is low, and therefore its physiological function remains questionable [bib_ref] Glucose Transporters in Brain in Health and Disease, Koepsell [/bib_ref]. Dapagliflozin has a higher affinity for SGLT2 [bib_ref] SGLT2 Inhibition in the Diabetic Kidney-From Mechanisms to Clinical Outcome, Van Bommel [/bib_ref] , but could potentially also affect SGLT1. SGLT1 is expressed in neurons throughout the brain, showing high expression in regions that are involved in learning, regulation of feeding behavior, energy expenditure and glucose homeostasis [bib_ref] Glucose Transporters in Brain in Health and Disease, Koepsell [/bib_ref]. A study in rats demonstrated a direct effect of SGLT2i on the CNS as central administration of SGLT2i increased food intake [bib_ref] Central Administration of Sodium-Glucose Cotransporter-2 Inhibitors Increases Food Intake Involving Adenosine Monophosphate-Activated..., Takeda [/bib_ref]. Metabolic adaptions may also play a role to compensate for the chronic calorie loss. "Further research to investigate the exact mechanism of how SGLT2 inhibition affects the CNS is needed."
Although the design of the trial is one of its major strengths, the study was not specifically designed for this exploratory analysis and should therefore be considered as hypothesis generating. We assessed appetite scores, but we did not administer questionnaires determining preferences for specific (aspects of) food (i.e., sweet, fatty, savory, salty). Food intake was measured with an ad libitum lunch buffet, but daily food intake was not measured. However, measuring food intake is a major challenge in free-living individuals, and people with obesity are known to underreport their intake [bib_ref] People With a Body Mass Index 30 Under-Report Their Dietary Intake: A..., Wehling [/bib_ref]. Although we investigated changes in CNS activation in predefined ROIs which are consistently shown to be involved in responses to food cues, and are part of the central reward circuits, it would have been of interest to include regions for homeostatic control of feeding such as the nucleus tractus solitarii in the brain stem and the hypothalamus. However, visualization of both using fMRI is challenging due to their size and location. Further research primarily investigating the neurophysiological correlates for food preferences, with sufficient participants, and stringent measurements of food intake (i.e., weighed food intake) should be performed to confirm our results.
In conclusion, previously we showed that dapagliflozin treatment increased activation in feeding regulation areas in responses to high-calorie foods, which suggests that the hyperphagia with dapagliflozin treatment may be specific for high-calorie/sweet foods. Here, we also demonstrated a decreased activation in response to low-calorie foods in people with obesity and T2D, which may reflect a specific decreased preference for low-calorie foods. This may have clinical consequences as a specific dislike for low-calorie foods in combination with a preference for high-calorie foods may hamper the weight loss of SGLT2i. In contrast, exenatide treatment resulted in an increased activation in response to low-calorie foods. The combination of dapagliflozin plus exenatide may lead to more favorable brain responses to food cues, as we observed that the dapagliflozin-induced decrease to low-calorie food pictures had disappeared. These findings provide further insight in the appetite and weight lowering effects with SGLT2i and GLP-1RAs alone and in combination and may contribute to optimizing weight loss.
# Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
# Ethics statement
The studies involving human participants were reviewed and approved by Medisch etische toetsings commissie VUmc (Metc VUmc). The patients/participants provided their written informed consent to participate in this study.
# Author contributions
CR designed the study, conducted the experiments, performed the data analysis, and wrote the article. DV designed the fMRI paradigm, performed the data analysis, and contributed to writing the article. MN contributed to writing the article. RI designed the study and the fMRI paradigm, performed the data analysis, and wrote the manuscript. All authors have seen and approved the final version of the manuscript. CR and RI are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed to the article and approved the submitted version.
# Funding
This work was funded by an investigator initiated grant from AstraZeneca (ESR-16-11865). The funder had no role in the study design, data analyses or interpretation, or drafting of the manuscript, nor in the decision to submit the manuscript for publication.
[fig] FIGURE 1 |: Average differences in CNS activation between dapagliflozin (black bar) and placebo (white bar). (A) Axial slice showing average differences between dapagliflozin (black bar) and placebo (white bar) after 10 days of treatment in the left caudate nucleus (P FWE < 0.05, T = 3.7) (upper left panel), the right caudate nucleus (P FWE < 0.05, T = 3.5) (upper right panel), the left amygdala (P FWE < 0.05, T = 3.1) (middle left panel), and the left insula (P FWE < 0.05, T = 3.6) (middle right panel) in response to the viewing of low-calorie versus non-food pictures. (B) [/fig]
[fig] FIGURE 2 |: Average differences in CNS activation after 10 days of treatment. (A) Axial slice showing average differences between exenatide (black bar) and placebo (white bar) in the right putamen (P FWE < 0.01, T=3.2) (upper panel) in response to the viewing of low-calorie versus non-food pictures. (B) Horizontal slice showing average differences between dapagliflozin plus exenatide (combi) (black bar) and dapagliflozin (white bar) in the left insula (P FWE < 0.01, T = 4.3) (middle left panel), right insula (P FWE < 0.01, T = 4.3) (middle right panel), and right amygdala (P FWE < 0.05, T = 2.8) (bottom panel) in response to the viewing of low-calorie food pictures versus non-food pictures.The left side of the brain slices is the left side of the brain. The color scale reflects the T value of the functional activity. Results are presented at the threshold of P < 0.05, familywise error (FWE) corrected on cluster extent within the regions of interest using small volume correction (10 mm sphere; 5 mm sphere for the amygdala). In the graphs, blood oxygen level-dependent (BOLD) signal intensity (effect size) is plotted (arbitrary unites), mean and SEM. The numbers on the y-axes of the bar graphs are the x,y,z, coordinates of the peak voxel of the observed difference in Montreal Neurological Institute (MNI) space. Combi, dapagliflozin plus exenatide; R, right; L, left. [/fig]
[table] Table 1: (3). [/table]
[table] TABLE 2 |: Effects of dapagliflozin, exenatide and the combination of dapagliflozin and exenatide on brain responses to low-calorie food pictures. [/table]
|
Redox-active charge carriers of conducting polymers as a tuner of conductivity and its potential window
[bib_ref] Conducting Polymer Hydrogels as 3D Electrodes: Applications for Supercapacitors, Ghosh [/bib_ref] [bib_ref] Solid electrolytic capacitor with highly stable conducting polymer as a counter electrode, Kudoh [/bib_ref] [bib_ref] Hierarchical nanostructured conducting polymer hydrogel with high electrochemical activity, Pan [/bib_ref] [bib_ref] Poly(3,4-ethylenedioxythiophene) and Its Derivatives: Past, Present, and Future, Groenendaal [/bib_ref] [bib_ref] Electrooxidation of aromatic oligomers and conducting polymers, Diaz [/bib_ref] [bib_ref] Conductive and Magnetic Properties of 3,4-Dimethoxy-and 3,4-Ethylenedioxy-Capped Polypyrrole and Polythiophene, Zotti [/bib_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref] [bib_ref] High-Conductivity Poly(3,4-ethylenedioxythiophene): Poly(styrene sulfonate) Film and Its Application in Polymer Optoelectronic Devices, Ouyang [/bib_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref] [bib_ref] Conductivity, morphology, interfacial chemistry, and stability of poly(3,4-ethylene dioxythiophene)-poly(styrene sulfonate): A photoelectron..., Crispin [/bib_ref] [fig_ref] Figure 2 |: Increase of the amount of redox-active charge carriers of PEDOT [/fig_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref] [fig_ref] Figure 2 |: Increase of the amount of redox-active charge carriers of PEDOT [/fig_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref] [fig_ref] Figure 2 |: Increase of the amount of redox-active charge carriers of PEDOT [/fig_ref] [bib_ref] Charge carrier mobility in doped semiconducting polymers, Arkhipov [/bib_ref] [bib_ref] Doping-induced change of carrier mobilities in poly(3-hexylthiophene) films with different stacking structures, Jiang [/bib_ref]
[formula] s!nm!nn b~nbz1~nbð2Þ [/formula]
[bib_ref] Charge carrier mobility in doped semiconducting polymers, Arkhipov [/bib_ref] [bib_ref] Doping-induced change of carrier mobilities in poly(3-hexylthiophene) films with different stacking structures, Jiang [/bib_ref] [fig_ref] Figure 3 |: Electrochemical impedance spectra of PEDOT [/fig_ref] [bib_ref] The effect of pore size distribution on the frequency dispersion of porous..., Song [/bib_ref] [bib_ref] Electrochemical impedance spectroscopy of porous electrodes: the effect of pore size distribution, Song [/bib_ref] [bib_ref] Electrochemical porosimetry, Song [/bib_ref] [fig_ref] Figure 4 |: Electrochemically detected signals of three different electroactive species [/fig_ref] [fig_ref] Figure 4 |: Electrochemically detected signals of three different electroactive species [/fig_ref] [fig_ref] Figure 4 |: Electrochemically detected signals of three different electroactive species [/fig_ref] [fig_ref] Figure 4 |: Electrochemically detected signals of three different electroactive species [/fig_ref]
# Discussion
In this work, we reported that the solvent anealing not only increases electric conductivity of PEDOT:PSS but also widens conductive potential window of the conducting polymer. Also, evidences were provided, which support the hypothesis that electroactive portion of charge carriers could determine the conductivity of conducting polymers. Even if total number of charge carriers or doping level were unchanged, the solvent annealing would induce the increase of the electroactive carriers. The widened conductive potential window of conducting polymers was shown to be utilized as electrodes of chemical sensors.
# Methods
Materials. Three different PEDOT:PSS solutions (1.0 , 1.7 wt.%) were purchased from Heraeous: AI4083, PH and PH1000. The other reagents were purchased from Sigma-Aldrich.
Solvent annealing. 50 mL dimethyl sulfoxide (DMSO) were added to 1 mL of PEDOT:PSS solution, followed by stirring over 2 h.
Film formation. The bare or DMSO-containing polymer solutions were casted as a form of film on substrates. They were drop-casted on screen printed carbon electrodes (Zensor R&D, Area 5 19.6 mm 2 ) for electrochemistry or spin-coated on glass substrates for conductivity measurement. The same amount of PEDOT were loaded on the electrodes independent of its kind. The thicknesses of the films were around 3.5 um with less than 0.5 um deviation.
Conductivity measurement. Electric conductivities were measured by using a fourpoint-probe technique (CMT-SR1000N, Advanced Instrument Technology). 1 mL of aqueous solutions of PEDOT:PSS with or without 5 vol.% DMSO were coated on glass substrates (1.5 cm by 1.5 cm) at 1,500 to 2,000 rpm for 60 s and then dried at room temperature for 1 day or 140uC for 10 min.
Electrochemical measurement. Cyclic voltammograms and impedance spectra were measured by using a potentiostat (VMP3, Bio-Logic). Electrochemical measurements were configured with three electrode systems: screen-printed carbon electrodes as working electrodes, Pt flag as a counter electrode and Ag/AgCl (0.197 V vs. NHE) as a reference. 0.1 M H 2 SO 4 (aq) was used as electrolyte for measuring redox activity of PEDOT:PSS. Three different electroactive species were used to confirm the conductive windows of -DMSO and 1DMSO: 1 mM K 3 Fe(CN) [bib_ref] The origin of the high conductivity of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) plastic electrodes, Crispin [/bib_ref] [fig_ref] Figure 1 |: Conductive potential widow widening of PEDOT [/fig_ref]. The net currents were calculated by subtracting the current obtained in A-absent electrolyte (-A) from the current obtained in Apresent electrolyte (1A). K 3 Fe(CN) 6 (FeCN, 1 mM, (a)), indigo carmine (IC, 0.25 mM, (b)) and flavin adenine dinucleotide (FAD, 1 mM, (c)) were selected as A. To identify the species responsible for peaks, the cyclic voltammograms obtained on glassy carbon disk electrodes (GC, area 5 3.14 mm 2 ) were also shown with indicated magnifications of current. The raw cyclic voltammograms were provided in [fig_ref] Figure 2 |: Increase of the amount of redox-active charge carriers of PEDOT [/fig_ref]
[fig] Figure 1 |: Conductive potential widow widening of PEDOT:PSS induced by DMSO-annealing. Cyclic voltammograms (a) to (c), (e) to (g), (i) to (k) and scan-rate (v) dependency of peak currents (i p ) (d), (h), (l) of three different PEDOT:PSS. The first, second and third rows correspond to AI4083, PH and PH1000, respectively. The first and second columns are for bare (2DMSO) and solvent-annealed (1DMSO) PEDOT:PSS, respectively. The cyclic voltammograms at 100 mV sec 21 are compared between -DMSO and 1DMSO of the same PEDOT:PSS in the third column. The values of anodic peak current were used for d to l because cathodic peak current showed similar behaviors in terms of v-dependency. The lines in figures of the fourth column show the linear relationship with a unity slope indicating that faradaic processes responsible for currents are surface-confined with facile kinetics. [/fig]
[fig] Figure 2 |: Increase of the amount of redox-active charge carriers of PEDOT:PSS induced by DMSO-annealing. (a) and (b), X-ray photoelectron spectra (XPS) of PH1000 (2DMSO and 1DMSO) potentiostatically conditioned at 10.5 V, 20.5 V and 20.8 V. Intensity was normalized by the maximum value of T peak. The peak locations of doublets for T, S1 and S2 were indicated on the abscissa. (c), Spectral change of potentiostatically controlled samples from their fully oxidized states at 10.5 V. Squared brackets indicate the spectrum of PH1000 potentiostatically controlled at the potential indicated within it. (d), 2DMSO/1DMSO ratio of the redox-active fraction of total charge carriers estimated spectroscopically from XPS and electrochemically from cyclic voltammograms (CV) in Fig. 1. www.nature.com/scientificreports SCIENTIFIC REPORTS | 3 : 2454 | DOI: 10.1038/srep02454 [/fig]
[fig] Figure 3 |: Electrochemical impedance spectra of PEDOT:PSS. (a) and (b), AI4083. (c) and (d), PH. (e) and (f), PH1000. The left and right columns are for PEDOT:PSS prepared in absence and presence of DMSO (2DMSO and 1DMSO), respectively. Impedance was measured at 10.5 V and 20.5 V as biased potentials every sample from 100 kHz to 0.5 Hz. www.nature.com/scientificreports SCIENTIFIC REPORTS | 3 : 2454 | DOI: 10.1038/srep02454 [/fig]
[fig] Figure 4 |: Electrochemically detected signals of three different electroactive species (A) on PH1000-filmed electrodes (2DMSO and [/fig]
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Extensive Deep Vein Thrombosis after Electrophysiology Study
Images in Cardiovascular Medicine Extensive Deep Vein Thrombosis after Electrophysiology Study
2020 Dec
MDMDImages in Cardiovascular Medicine Extensive Deep Vein Thrombosis after Electrophysiology Study
Korean Circ J
50122020 Dec10.4070/kcj.2020.0337Received: Jul 31, 2020 Revised: Aug 20, 2020 Accepted: Sep 16, 20201129 https://e-kcj.org Correspondence to
A 76-year-old woman was admitted for electrophysiologic study. The patient was obese with a body mass index of 32.9 kg/m 2 , but did not have any risk factors of deep vein thrombosis. Both femoral veins were catheterized. During the study, atrioventricular (AV) nodal reentrant tachycardia was induced, and AV nodal slow pathway was ablated. Hemostasis was achieved through use of manual compression at puncture sites and absolute bed rest. One day after the procedure some ecchymosis was observed at the right inguinal area, however the left inguinal area was clear and the patient was subsequently discharged. One week later the patient reported discomfort in the left inguinal area during sleep, and progressive swelling of the left lower extremity. Upon examination, the patient's left lower extremity was swollen, red, and cooler than the right extremity (Figure 1A). Computed tomography (CT) of the patient's left A B C D E Figure 1. Photograph of patient's lower extremity and CT images. (A) Ecchymosis at right inguinal and thigh areas and swollen entire left lower extremity. (B) Coronal view of CT image showed deep vein thrombosis (arrow) at left iliac and femoral veins. (C-D) Horizontal views of CT showed extensive deep vein thrombosis (arrow) in the entire vein of the left lower extremity (C, inguinal level, D, thigh level, and E, calf level). CT = computed tomography.
lower extremity revealed extensive thrombosis in the left iliac and femoral veins in coronal sections and extensive swelling and deep vein thrombosis in horizontal views . There was no extrinsic venous compression on CT images, thus, May-Thurner syndrome was ruled out. The patient was placed on Apixaban 10 mg twice a day (b.i.d.) for 10 days, then reduced 5 mg b.i.d. orally for 3 months. Swelling in the left lower extremity was reduced after the patient began Apixaban and was normalized after 2 weeks of treatment. This case displayed an unusual delayed complication of electrophysiologic study and serves as an instructive case for electrophysiologists to be aware of possible delayed complications.
The informed consent that allows the publication of clinical data was obtained from the patient. |
The association of apolipoproteins with later-life all-cause and cardiovascular mortality: a population-based study stratified by age
Fig. S1. The sequence of mergers in the dendrogram from hierarchical cluster analysis (using Ward's methods) performed on TC, triglycerides, ApoB, ApoA-I, and ApoB/ApoA-I ratio in z-scores. . The sequence of mergers in the dendrogram from hierarchical cluster analysis (using Ward's methods) performed on TC, triglycerides, ApoB, ApoA-I, and ApoB/ApoA-I ratio in z-scores. . Boxplots of total cholesterol, triglycerides, ApoB, ApoA-I, and ApoB/ApoA-I ratio in zscores in the 3 clusters among people aged 39-59, 60-79, and ≥80 years, respectively. Cluster analyses were performed separately in the three age groups. Hazard ratios (solid line for all-cause mortality and dash line for cardiovascular mortality) and 95% confidence intervals (gray area) are retrieved from Cox regression models with restricted cubic splines, adjusted for age, sex, and history of coronary heart disease, heart failure, atrial fibrillation, hypertension, diabetes, ischemic stroke, and transient ischemic attack. Solid gray line indicates the distribution of lipid biomarkers in the study population. . Hazard ratios and 95% confidence interval for all-cause and cardiovascular mortality associated with LDL and HDL cholesterol in categories among people aged 39-59 years (left), 60-79 years (middle), and ≥80 years (right), respectively, adjusted for age, sex, and history of coronary heart disease, heart failure, atrial fibrillation, hypertension, diabetes, ischemic stroke, and transient ischemic attack.
[fig] Figure: S2. Boxplots of total cholesterol, triglycerides, ApoB, ApoA-I, and ApoB/ApoA-I ratio in zscores in the 3 clusters among people aged 39-59, 60-79, and ≥80 years, respectively. Cluster analyses were performed separately in the three age groups. [/fig]
[table] Table S1: Biomarker concentration levels for the 3 lipid biomarker clusters. Statistically different (p<0.001) from cluster 1 for all lipid biomarkers. b Statistically different (p<0.001) from cluster 2 for all lipid biomarkers. [/table]
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PHF20 is crucial for epigenetic control of starvation-induced autophagy through enhancer activation
Autophagy is a catabolic pathway that maintains cellular homeostasis under various stress conditions, including conditions of nutrient deprivation.To elevate autophagic flux to a sufficient level under stress conditions, transcriptional activation of autophagy genes occurs to replenish autophagy components. Thus, the transcriptional and epigenetic control of the genes regulating autophagy is essential for cellular homeostasis. Here, we applied integrated transcriptomic and epigenomic profiling to reveal the roles of plant homeodomain finger protein 20 (PHF20), which is an epigenetic reader possessing methyl binding activity, in controlling the expression of autophagy genes. Phf20 deficiency led to impaired autophagic flux and autophagy gene expression under glucose starvation. Interestingly, the genome-wide characterization of chromatin states by Assay for Transposase-Accessible Chromatin (ATAC)-sequencing revealed that the PHF20-dependent chromatin remodelling occurs in enhancers that are co-occupied by dimethylated lysine 36 on histone H3 (H3K36me2). Importantly, the recognition of H3K36me2 by PHF20 was found to be highly correlated with increased levels of H3K4me1/2 at the enhancer regions. Collectively, these results indicate that PHF20 regulates autophagy genes through enhancer activation via H3K36me2 recognition as an epigenetic reader. Our findings emphasize the importance of nuclear events in the regulation of autophagy.
# Introduction
Autophagy is a highly conserved process that maintains cellular homeostasis by eliminating unnecessary proteins and damaged organelles [bib_ref] Autophagy as a regulated pathway of cellular degradation, Klionsky [/bib_ref]. Under stress conditions such as nutrient starvation, autophagy is highly induced to perform a cytoprotective function [bib_ref] Autophagy in metazoans: cell survival in the land of plenty, Lum [/bib_ref]. Since autophagy is essential for both cell survival and protection against various types of environmental damage, dysregulated autophagy can cause serious human diseases, including diabetes, neurodegenerative diseases, and cancer [bib_ref] Autophagy in human health and disease, Choi [/bib_ref]. As autophagy proceeds, the protein components of autophagosomes, along with their autophagy cargoes, are rapidly degraded by lysosomes [bib_ref] Autophagy: process and function, Mizushima [/bib_ref]. Thus, the transcription of autophagy components should be increased to avoid the depletion of the autophagosome and to maintain an optimal autophagic flux under cellular stress conditions [bib_ref] The return of the nucleus: transcriptional and epigenetic control of autophagy, Fullgrabe [/bib_ref]. Previous studies have mainly reported the functions of transcription factors, including transcription factor EB (TFEB) and the forkhead box O (FOXO) protein family, to be involved in the regulation of autophagy [bib_ref] FoxO3 controls autophagy in skeletal muscle in vivo, Mammucari [/bib_ref] [bib_ref] FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway, Zhou [/bib_ref]. TFEB recognizes the CACGTG sequence in DNA (the 'CLEAR' motif), and activates the transcription of its specific target genes, including autophagy and lysosomal genes.
Gene expression is tightly regulated by not only transcription factors but also the chromatin structures that are modulated by chromatin remodelling factors [bib_ref] Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the..., Heintzman [/bib_ref]. Posttranslational modifications of histone tails influence the chromatin structures associated with transcriptional activation or repression. Active promoter regions are marked by H3K4me3, active enhancer regions are closely associated with H3K4me1 and H3K27ac, and heterochromatin regions are marked by H3K9me3 [bib_ref] Histone methylation: a dynamic mark in health, disease and inheritance, Greer [/bib_ref] [bib_ref] Histone H3K27ac separates active from poised enhancers and predicts developmental state, Creyghton [/bib_ref] [bib_ref] Histone methylation modifiers in cellular signaling pathways, Alam [/bib_ref] [bib_ref] Recognition of enhancer element-specific histone methylation by TIP60 in transcriptional activation, Jeong [/bib_ref] [bib_ref] A distinct mechanism for coactivator versus corepressor function by histone methyltransferase G9a..., Purcell [/bib_ref] [bib_ref] The selection and function of cell type-specific enhancers, Heinz [/bib_ref] [bib_ref] Histone H3 variants and modifications on transcribed genes, Workman [/bib_ref]. The enzymes that induce or remove histone modifications (called writers and erasers, respectively) are orchestrated to establish a specific repertoire of histone modifications under various cellular states [bib_ref] Epigenetic tools (The writers, the readers and the erasers) and their implications..., Biswas [/bib_ref] [bib_ref] Writing, erasing and reading histone lysine methylations, Hyun [/bib_ref] [bib_ref] Crosstalk among histone modifications, Suganuma [/bib_ref]. Most studies investigating the regulation of autophagy genes have focused on elucidating the mechanisms of writers and erasers [bib_ref] Epigenetic regulation of autophagy by the methyltransferase G9a, Artal-Martinez De Narvajas [/bib_ref] [bib_ref] The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy, Fullgrabe [/bib_ref] [bib_ref] Epigenetic regulation of autophagy by the methyltransferase EZH2 through an MTOR-dependent pathway, Wei [/bib_ref] [bib_ref] Function and molecular mechanism of acetylation in autophagy regulation, Yi [/bib_ref]. For example, H3R17me2 levels are increased by coactivatorassociated arginine methyltransferase 1 (CARM1) under glucose starvation conditions, thereby leading to the activation of TFEB target genes [bib_ref] AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy, Shin [/bib_ref]. In contrast, H4K16ac levels are reduced by decreased Males absent on the first (MOF) histone acetyltransferase activity and sirtuin 1 activation upon autophagic stimulation. Given that epigenetic readers recognize specific histone modifications and exert their functions by bringing another effector complex to that site or by serving as an effector itself, elucidating the molecular functions of epigenetic readers is crucial for a comprehensive understanding of the regulation of autophagy genes [bib_ref] Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene..., Lan [/bib_ref] [bib_ref] Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2, Pena [/bib_ref] [bib_ref] ING4 mediates crosstalk between histone H3 K4 trimethylation and H3 acetylation to..., Hung [/bib_ref] [bib_ref] ING2 PHD domain links histone H3 lysine 4 methylation to active gene..., Shi [/bib_ref] [bib_ref] Opposite role of yeast ING family members in p53-dependent transcriptional activation, Nourani [/bib_ref] [bib_ref] Readers of histone modifications, Yun [/bib_ref].
PHF20, a member of the PHF family, contains two conserved Tudor domains and one plant homeodomain (PHD) [bib_ref] PHF20 is an effector protein of p53 double lysine methylation that stabilizes..., Cui [/bib_ref]. As a core component of MOF-nonspecific lethal (NSL) protein complex, PHF20 recognizes methylation of histone or non-histone targets and recruits NSL complex to target promoters, thereby enhancing histone H4 acetylation [bib_ref] Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from..., Cai [/bib_ref] [bib_ref] Two mammalian MOF complexes regulate transcription activation by distinct mechanisms, Li [/bib_ref] [bib_ref] hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in..., Taipale [/bib_ref] [bib_ref] Histone H4 lysine 16 acetylation breaks the genome's silence, Shia [/bib_ref]. PHF20 recognizes H3K4me2 through PHD and interacts with methyl residues on non-histone proteins, including estrogen receptor ␣, p53, and p65 through Tudor domains [bib_ref] PHF20 readers link methylation of histone H3K4 and p53 with H4K16 acetylation, Klein [/bib_ref] [bib_ref] G9a-mediated methylation of ER␣ links the PHF20/MOF histone acetyltransferase complex to hormonal..., Zhang [/bib_ref] [bib_ref] regulates NF-B signalling by disrupting recruitment of PP2A to p65, Zhang [/bib_ref]. Phf20-deficient (Phf20 −/− ) mice show a high rate of perinatal lethality, with the surviving adults having a smaller body size than the wild-type (WT) mice, which is a well-known characteristic of autophagy-defective mice [bib_ref] Loss of the methyl lysine effector protein PHF20 impacts the expression of..., Badeaux [/bib_ref].
In this study, we found that Phf20 deficiency leads to the failure to maintain autophagic flux under glucose starvation condition using genome-wide analyses, providing insights into the previously unrecognized epigenetic regulatory mechanism of PHF20 during autophagy.
# Materials and methods
## Reagents
The following antibodies were used: anti-Flag (F3165) and anti--actin (A1978) (Sigma-Aldrich, St. Louis, MO, USA); anti-GFP (sc-9996) and anti-Lamin A/C (sc-6215) (Santa Cruz biotechnology, Dallas, TX, USA); anti-Tubulin (LF-PA0146A) (AbFrontier, Seoul, Korea); anti-PHF20 (#3934), anti-WDR5 (#13105), and anti-LC3 (#2775) (Cell Signaling Technology, Danvers, MA, USA); anti-HA (#MMS-101R) (Covance, Princeton, NJ, USA); Alexa Fluor 488 donkey anti-rabbit IgG (A21206) and Alexa Fluor 594 donkey anti-mouse IgG (A21203) (Invitrogen, Waltham, MA, USA). The following chemicals were used: hygromycin (H3274), puromycin (P8833), and CQ (C6628) (Sigma-Aldrich, St. Louis, MO, USA); Bafilomycin A1 (#11038) (Cayman, Ann Arbor, MI, USA); and rapamycin (R-5000) (LC laboratories, Woburn, MA, USA).
## Cell culture and transfection
We generated Phf20 −/− immortalized mouse embryonic fibroblasts (MEFs) by using 3T3 protocol. WT and Phf20 −/− MEFs were cultured at 37 - C in Dulbecco's modified Eagle's medium (DMEM) (SH30243.01, HyClone, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) (SH30084.03, HyClone) and ZellShield ® (13-0050, Minerva biolabs, Hillsborough, NJ, USA) in a humidified incubator with 5% CO 2 . For glucose or amino acid starvation, cells were washed with pre-warmed Dulbecco's phosphate-buffered saline (DPBS) (SH30028.02, HyClone) and then exchanged media with glucose-free DMEM (LM001-56, Welgene, Gyeongsan-si, Korea) or amino acid-free DMEM (LM001-90, Welgene) supplemented with 10% dialyzed FBS (26400044, Gibco, Amarillo, TX, USA) and ZellShield ® . Following reagents were used for cellular transfection: TurboFect (#R0531, Ther-moFisher, Waltham, MA, USA) and Lipofectamine 3000 (L3000-001, Invitrogen). All cell lines were maintained without mycoplasma contamination.
## Lentivirus construction and production
3X Flag-PHF20 WT, Tudor and W97A mutants were cloned in pLVX vector, lentiviral shRNA constructs were cloned in pLKO.1 vector and guide RNA constructs for CRISPRi were cloned in lentiGuide-Puro (#52963, Addgene, Wartertown, MA, USA) vector. To generate lentivirus, constructs were co-transfected with virus packaging vectors (psPAX2 and VSV-G) in HEK293T cells. 48 h after transfection, virus containing media were collected and filtered through a 0.45 m-membrane filter. Lenti-X concentrator was added to filtered media according to the manufacturer's instructions (631231, Clontech, Mountain View, CA, USA). Collected virus was resuspended in DPBS and infected to cells with polybrene. Hygromycin selection was performed 10 days post-infection for pLVX vector and puromycin selection was performed 36 h post-infection for pLKO.1 vector. Following sequences were targeted by gRNA: Atg13: 5'-TGAGATGGTGTGTATAAATG-3' and 5'-CATTTATACACACCATCTCA-3'; Ulk1: 5'-ACTGACCCACTTAACTCATG-3' and 5'-CATGAGTTAAGTGGGTCAGT-3'.
## Preparation for obtaining whole-cell lysates
To harvest cells, cells were briefly rinsed with cold PBS and collected from the plate with scrapper. Then, cells were resuspended in EBC200 buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl [pH 7.5], and 2 mM ethylenediaminetetraacetic acid (EDTA)) supplemented with protease inhibitors and sonicated using a Branson Sonifier 450 (Branson, Brookfield, CT, USA) at output 3 and a duty cycle of 30 for 10 pulses. Protein concentration in each lysate was quantified with the Bradford method and normalized with same concentration.
## Autophagic vacuole staining
Autophagic vacuoles were stained using the CYTO-ID ® autophagy detection kit (ENZ-51031, Enzo Life Sciences, Farmingdale, NY, USA) and observed through fluorescence microscopy. Cells grown on coverslips at a density of 2 × 10 4 cells were incubated with DMEM containing CYTO-ID ® green detection reagent (1:500) and Hoechst 33342 (1:1000) at 37 - C for 30 min. After staining, the cells were washed with PBS and then fixed with 2% paraformaldehyde in PBS at 20-22 - C for 10 min. Cells were then mounted and visualized under a confocal microscope (LSM700, Zeiss, Oberkochen, Germany).
## Immunofluorescence
Cells grown on coverslips at a density of 5 × 10 4 cells were washed with PBS and then fixed with 2% paraformaldehyde in PBS at 20-22 - C for 10 min. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS (PBS-T) and incubated at 20-22 - C for 10 min. Blocking was performed with 3% bovine serum in PBS-T for 1 h. For staining, the cells were incubated with antibodies at 20-22 - C for 2 h, followed by incubation with fluorescent-labeled secondary antibodies for 1 h. Cells were then mounted and visualized under a confocal microscope (LSM700, Zeiss). For autophagy studies, MEFs were transfected with GFP-LC3 or mCherry-GFP-LC3 and sub-cultured onto coverslips. The following day, cells were incubated with complete media or glucosestarved media for 18 h.
## Rna purification and quantitative real-time pcr (qrt-pcr)
Total RNAs were purified with Trizol (15596026, Invitrogen). Purified RNAs were reverse-transcribed using SuPrimeScript cDNA Synthesis Kit (SRK-1000, GeNet-Bio, Daejeon, Korea). The reaction was performed with 2.5 g of purified RNAs as template, Oligo dT and random hexamer for primer. Quantitative RT-PCR was reacted with SYBR TOPreal qPCR 2X PreMix (RT501, Enzynomics, Daejeon, Korea) following manufacturer's protocol. SYBR green signal was detected by an ABI prism 7500 system (Applied Biosystems, Waltham, MA, USA). Abundance of mRNA was quantified by the ddCt method using expression of HPRT, -actin as control. All reactions were performed as triplicates. The primers used for qRT-PCR are listed in .
# Rna-sequencing analysis
Total RNAs were extracted from WT and Phf20 -/-MEFs with or without glucose starvation, respectively. Then, RNA-seq libraries were produced using Illumina's TruSeq Stranded mRNA LT Sample Prep Kit. After pairedend sequencing of the RNA-seq libraries, adapters and reads with low quality were filtered out by Trimmomatic (v0.36) [bib_ref] Trimmomatic: a flexible trimmer for illumina sequence data, Bolger [/bib_ref]. Then, the trimmed reads were aligned onto the mm10 genome reference using STAR (v2.5.3a) [bib_ref] STAR: ultrafast universal RNA-seq aligner, Dobin [/bib_ref] , and Transcripts Per Million (TPM) per gene was calculated by RSEM (v1.3.0) [bib_ref] RSEM: accurate transcript quantification from RNA-Seq data with or without a reference..., Li [/bib_ref]. The TPM values were log 2transformed for downstream analyses such as hierarchical clustering, k-means clustering and functional analysis. kmeans clustering was performed to identify the genes regulated by PHF20, and DAVID (v6.8) (48) was utilized for gene ontology. For Gene Set Enrichment Analysis (GSEA) (v4.0.3) (49), mm10 annotated protein coding genes were mapped to human protein coding genes using biomaRt (v2.40.5) (50) in R. Phenotype label was assigned as 1:3:1:1 for WT MEFs normal condition: WT MEFs glucose starvation: Phf20 -/-MEFs normal condition: Phf20 -/-MEFs glucose starvation, and genes were ranked by the Pearson correlation coefficient. Finally, C2 and C5 gene sets in MSigDB (molecular signatures database) (v7.0) [bib_ref] The molecular signatures database (MSigDB) hallmark gene set collection, Liberzon [/bib_ref] of the Broad Institute were used for the enrichment score.
Assay for transposase-accessible chromatin using sequencing (ATAC)-sequencing analysis ATAC-seq libraries were prepared for sequencing using Illumina Tagment DNA TDE1 Enzyme and Buffer Kits (#20034197, Illumina, San Diego, CA, USA) and pairedend sequencing was performed by Illumina platform. Then, paired-end reads were aligned onto mm10 using Burrows-Wheeler Alignment tool (BWA) (v0.7.12) (53) and peak calling was conducted by Model-based Analysis for ChIP-Seq (MACS) (v2.1.2) [bib_ref] Model-based analysis of chip-Seq (MACS), Zhang [/bib_ref]. For hierarchical clustering based on the peak intensities, the significant peaks were selected with a cut-off false discovery rate (FDR) 0.01 for each sample then merged across the samples. Reads per peak, as an intensity, was counted using BEDTools (v2.25.0) [bib_ref] BEDTools: a flexible suite of utilities for comparing genomic features, Quinlan [/bib_ref]. To identify differentially opening peaks (DOPs) between samples, DESeq2 (v1.26.0) (56) was applied for the intensities. For average profile of DOPs, normalized read counts centered on peak summits were calculated and plotted by deep-Tools2 (v3.1.1) (57)
## Purification of gst-fusion proteins and in vitro peptide binding assay
GST-tagged PHF20 Tudor 1 and 2 domains (Tudor 1&2, 1-147 a.a) of WT or W97A mutant constructs were cloned in pGEX-4T-1 vector and were transformed in Rosetta Escherichia coli strain. The protein was purified with glutathione beads (GE17-0756-01, GE Healthcare, Chicago, IL, USA) and eluted with elution buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM reduced glutathione, and 1 mM DTT supplemented with 1X complete protease inhibitor). For in vitro peptide binding assay, 2 g of WT and W97A purified proteins were incubated overnight with 1 g of biotin-labeled protein peptides in the 300 l of binding buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, and 0.05% NP-40). Then, 30 l of streptavidin sepharose beads were added to each tube and incubated for 1 hr. After binding, beads were washed 4 times with binding buffer and samples were boiled with 30 l of SDS sampling buffer.
## In vitro histone peptide binding array
In vitro histone peptide binding array was performed with MODified™ Histone Peptide Array Kit (13001, Active Motif, Carlsbad, CA, USA) following manufacturer's protocol. In brief, array kit was blocked with 5% milk in TTBS (10 mM Tris-HCl [pH 7.5], 150 mM NaCl and 0.05% Tween-20) at 4 - C overnight. After that, the kit was incubated with Nucleic Acids purified GST-PHF20 Tudor 1&2 WT protein in binding buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 1 mM EDTA, 10% glycerol, and 0.1 mM DTT) for 1 h. Then, primary GST antibody and secondary antibody were treated and bound GST proteins were detected with ECL solution.
## Chromatin immunoprecipitation (chip) assay
Before crosslinking, cells were washed three times with cold PBS to remove amine-containing proteins from cells and media. Then, ethylene glycolbis(succinimidylsuccinate) (EGS) was treated to the cell for final concentration of 2 mM at 20-22 - C. After 20 min, 1% formaldehyde was added and cells were incubated for 10 min. After glycine quenching, cells were harvested and resuspended with ChIP lysis buffer containing 50 mM Tris-HCl [pH 8.1], 10 mM EDTA, 1% SDS and protease inhibitor cocktail. DNA was fragmented with sonication until average size reaches 250 bp. Dilution buffer containing 20 mM Tris-HCl [pH 8.1], 150 mM NaCl, 2 mM EDTA and 1% Triton X-100 were added to chromatin extract with a volume of ten times. Diluted samples were subjected to immunoprecipitation with assigned antibodies overnight at 4 - C. Then, 40 l of Protein A/G sepharose beads were added to capture immunocomplexes. .
## Chromatin immunoprecipitation (chip)-sequencing analysis
Paired-end reads were aligned onto the mm10 reference genome using BWA (v0.7.12), and MACS (v2.1.2) was utilized to identify significant peaks with cut-offs of false discovery rate (FDR) 0.01 and signal value 5.
## Cut&run assay
CUT&RUN assay kit (#86652, Cell Signaling) was used following manufacturer's instruction. In brief, collected cells were washed with wash buffer and permeabilized with Digitonin solution. Then, cells were bound to Concanavalin A magnetic beads and incubated with antibody overnight at 4 - C. Next, pAG-MNase was treated to fragment DNA where target protein is attached. DNA extract was purified with DNA Purification Buffers and Spin Columns (#14209, Cell signaling) and subjected to qRT-PCR. The primers used for qRT-PCR are listed in .
## Chromosome conformation capture (3c) assay
The 3C assays were performed as modified version of previous methods [bib_ref] Quantitative analysis of chromosome conformation capture assays (3C-qPCR), Hagege [/bib_ref] [bib_ref] Analysis of long-range chromatin interactions using chromosome conformation capture, Naumova [/bib_ref]. In brief, 1 × 10 7 cells were crosslinked in 1% formaldehyde/media for 10 min at 20-22 - C. After crosslinking step, glycine was added to a final concentration of 0.125 M and incubated for 10 min. After washing with DPBS for 2 times, cells were harvested and lysed with Buffer I (10 mM HEPES [pH 6.5], 0.25% Triton X-100, 10 mM EDTA and 0.5 mM EGTA) and Buffer II (10 mM HEPES [pH 6.5], 200 mM NaCl, 1 mM EDTA, and 0.5 EGTA) at 4 - C for 5 min, respectively. Cells were additionally lysed with lysis buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl, 0.2% NP-40 and 1× complete protease inhibitor). After centrifugation, the pelleted nuclei were resuspended with 1.2× restriction enzyme buffer M (Takara, Kusatsu, Japan) with 0.3% (w/v) SDS and incubated at 37 - C while shaking at 900 r.p.m. Triton X-100 was added to a final concentration of 2% (v/v) and incubated at 37 - C while shaking at 900 r.p.m. 400 U of restriction enzyme HindIII (1060BH, Takara) was added and incubated overnight at 37 - C while shaking at 900 r.p.m. for chromatin digestion. For restriction enzyme inactivation, SDS was added to a final concentration of 1.6% and samples were incubated for 20 min at 65 - C while shaking at 900 r.p.m. Before ligation, 1.15× filtered ligation buffer (66 mM Tris-HCl [pH 7.5], 5 mM DTT, 5 mM MgCl 2 and 1 mM ATP) and 1% Triton X-100 was added and were incubated for 1 h at 37 - C while shaking gently. The DNA was ligated by 100U T4 DNA ligase (M053L, Enzynomics) at 16 - C for 4 h followed by 30 min incubation at 20-22 - C. 300 g of Protease K (P2308, Sigma-aldrich) was added and the DNA was de-crosslinked at 65 - C overnight. DNA purification was performed by phenol-chloroform extraction and ethanol precipitation. The following PCR program was used: 95 - C for 10 min, followed by 38 cycles of 95 - C for 10 s, 60 - C for 10 s, and 72 - C for 10 s. The following primers were used.
Atg13 promoter fwd (P) 5'-ACTGTTTTGAAAGCGGGTTG-3'; Atg13 enhancer fwd (E) 5'-CGGTTGGTTCCTTGTGAATC-3'; Ulk1 promoter rev (P) 5'-TCCCCACAGTTTTTGGTTTC-3'; Ulk1 enhancer rev (E) 5'-TACCCACAGGGCCATCTTTA-3'; Supt5 promoter rev (P) 5'-GAGCAGGCCCCTAAAGTCTC-3'; Supt5 enhancer rev (E) 5'-TGGCTTTTTAAACCGTGAGG-3';
# Statistical analysis
All experiments were performed independently at least three times. Prism v5 software (GraphPad) was used for statistical analysis. Student's t-test was used for comparison between two groups. Analysis of variance (ANOVA) with post hoc tests was used for comparison of multiple samples and discrimination of significant relationships. P-values <0.05 were considered statistically significant.
# Results
## Phf20 is crucial for starvation-induced autophagy
As the phenotypes of Phf20 −/− mice showing perinatal lethality are often observed for autophagy-defective mice, we tested the possibility that PHF20 is involved in autophagy. To detect autophagic activity, we analyzed the conversion of non-lipidated light chain 3 (LC3)-I form to lipidated LC3-II form, which is a common marker of autophagic occurrence. We induced autophagy in WT and Phf20 −/− mouse embryonic fibroblasts (MEFs) by glucose and amino acid starvation, and found that LC3-II conversion in Phf20 −/− MEFs was attenuated as compared to that in WT MEFs under both glucose and amino acid starvation conditions [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref] and 1B). The same results were observed upon rapamycin treatment, which induces autophagy by inhibiting mammalian target of rapamycin (mTOR) [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref]. The formation of green fluorescent protein (GFP)-tagged LC3-positive autophagosomes was examined to evaluate the role of PHF20 in autophagy. Upon glucose starvation, the number of GFP-LC3 punctate cells increased in WT MEFs, however, this increase in number of puncta was attenuated in Phf20 −/− MEFs [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref]. Next, we compared the autophagic flux between WT and Phf20 −/− MEFs by treating the cells with lysosomal inhibitors that prevent the degradation of the mature autophagosome. We treated WT and Phf20 −/− MEFs with chloroquine (CQ) and compared the induction of autophagic flux in the cells under glucose starvation [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref]. While the autophagic flux in WT MEFs was greatly increased by glucose starvation, Phf20 −/− MEFs failed to show significant increase in autophagic flux [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref]. Similar results were observed in the absence or presence of Bafilomycin A treatment with Cyto-ID staining, which is an autophagosome-specific fluorescent reporter [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref]. The number of autophagosomes was increased to a much higher extent by glucose starvation in WT MEFs than in Phf20 −/− MEFs. Thereafter, we used the mCherry-GFP-LC3 reporter to examine the total number of autophagosomes induced and the extent of autophagic flux at the same time. During the formation of an autophagosome, mCherry-GFP-LC3 conjugates with the autophagosome membrane and stains the vesicle yellow, which results from the fluorescence of both mCherry and GFP. After a lysosome fuses with an autophagosome to form an autolysosome, only red fluorescence is observed, because the fluorescent activity of GFP is vulnerable to the acidic environment. Consecutively, WT MEFs showed increased number of both yellow and red puncta under glucose starvation, whereas Phf20 −/− MEFs had significantly attenuated number of both puncta [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref]. Therefore, these results indicate that Phf20 deficiency impairs the induction of autophagic flux under glucose starvation.
## Phf20 functions as a transcriptional coactivator of autophagy genes
To examine the role of PHF20 in autophagy at the transcriptional level, we carried out RNA-sequencing (RNAseq) of WT and Phf20 −/− MEFs with or without glucose starvation [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref]. In unsupervised hierarchical clustering, the Phf20 −/− MEFs were closely clustered independent of the starvation conditions, thereby suggesting that Phf20 deletion eliminates the transcriptional responses to glucose starvation [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref]. We then performed k-means clustering (k = 8) to figure out the functional role of PHF20 in regulating gene expression [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref] and Supplementary . The genes in cluster 1 were expressed in a PHF20-dependent manner, as the deletion of Phf20 led to the failure of activation of the genes in WT MEFs upon glucose starvation [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref] and Supplementary [fig_ref] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation [/fig_ref]. Interestingly, gene ontology (GO) analysis revealed that autophagy genes were significantly represented in cluster 1, indicating that PHF20 is involved in transcriptional activation of autophagy genes [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref] and .
Next, we conducted gene set enrichment analysis (GSEA) by ranking the genes based on Pearson correlation coefficient in a PHF20-dependent manner. The results confirmed that the gene sets of the autophagic process were significantly enriched in the PHF20-dependent cluster [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref]. Furthermore, we observed that the activation of genes related to autophagy initiation, phagophore expansion, and cargo recruitment and trafficking [bib_ref] Pontin arginine methylation by CARM1 is crucial for epigenetic regulation of autophagy, Yu [/bib_ref] in WT MEFs upon glucose starvation was repressed by Phf20 deletion, thereby showing the transcriptional dependency of autophagy on PHF20 [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref]. We further validated the function of PHF20 in transcriptional regulation using quantitative realtime polymerase chain reaction (qRT-PCR) for the genes associated with autophagy such as those encoding the autophagy (Atg) family proteins and the autophagy receptor sequestosome 1 (Sqstm1), also known as p62 [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref]. Taken together, the results of gene expression profiling revealed that PHF20 acts as a transcriptional coactivator during autophagy on a transcriptome-wide scale.
## Phf20 modulates autophagy genes through enhancer activation
As PHF20 is a chromatin-binding protein, we carried out ATAC-seq to elucidate the role of PHF20 in the alteration of chromatin structures during autophagy [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref]. After peak calling for open chromatin regions in ATAC-seq, hierarchical clustering using the peak intensities showed that clusters were segregated between WT and Phf20 −/− [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref]. The dendrogram in [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref] indicates that open chromatin structures were globally altered by Phf20 deletion, whereas the effect of glucose starvation on chromatin structures was relatively minimal. We then identified differentially opening peaks (DOPs) between WT and Phf20 −/− under each condition. We observed that 16,976 and 20,906 peaks were significantly changed by Phf20 deletion under normal and glucose starvation condition, respectively [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref]. Next, we conducted GO term analysis on the genes which show starvation-induced chromatin opening peaks [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref] and Supplementary . The results showed that autophagy-related GO terms were significantly represented in WT MEFs, indicating a PHF20dependent chromatin opening of autophagy-related genes under glucose starvation.
Nucleic Acids Research, 2022, Vol. 50, No. 14 7861 Bars, mean ± SEM; ** P < 0.01, *** P < 0.001. Statistical analysis using two-tailed t-test. (G) Representative confocal images of mCherry-GFP-LC3 assays in WT or Phf20 −/− MEFs. Colocalization of mCherry and GFP signal (yellow puncta) represents autophagosomal vesicles that have not fused with a lysosomal compartment (phagophores or autophagosomes). mCherry signal without GFP signal (red puncta) represents acidic autophagosomal vesicles (acidic amphisomes or autolysosomes). Nucleus are stained with DAPI (Blue). Scale bar, 50 m. The graph indicates the number of puncta per cell. Bars, mean ± SEM; ** P < 0.01, *** P < 0.001. Statistical analysis using two-tailed t-test. To investigate precisely which chromatin states were regulated by PHF20 on a genome-wide scale, we utilized chromHMM, which is a software for discovering chromatin states by learning chromatin signatures based on the multivariate Hidden Markov Model [bib_ref] ChromHMM: automating chromatin-state discovery and characterization, Ernst [/bib_ref] [bib_ref] Chromatin-state discovery and genome annotation with ChromHMM, Ernst [/bib_ref]. We collected 12 different publicly available ChIP-seq datasets, such as RNA polymerase II and CTCF ChIP-seq datasets, and various histone ChIP-seq datasets derived from studies using MEFs [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref]. After learning the diverse chromatin signatures, we were able to generate genome-wide chromatin annotations consisting of 16 states . To specify the chromatin states regulated by PHF20, we calculated the average profile of the DOPs for each state [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref] and Supplementary [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref]. We found that states 6, 7 and 8 show relatively strong dependency on PHF20 for starvation-induced chromatin opening, defining PHF20 dependency as states where chromatin became less accessible by Phf20 depletion under glucose starvation. The state 3 was excluded because it was a repetitive region and had no significant signal of all histone marks. Regarding the states 4, 5 and 9, they exhibited relatively weak PHF20 dependency, meaning that the chromatin accessibility difference (WT Glc starv -KO Glc starv) was smaller. As a result, we finally defined the states 6, 7 and 8 as PHF20-dependent states [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref] , Supplementary [fig_ref] Figure 2: PHF20 induces autophagy genes under glucose starvation [/fig_ref] and S3). Based on the opened patterns of the DOPs along with the chromHMM states, we hypothesized that DOPs in states 6, 7 and 8 lead to the PHF20-dependent gene expression under glucose starvation. To confirm this, we performed an integrative analysis of ATAC-seq with the gene clusters from RNA-seq, which were distinguished by PHF20-dependent gene expression patterns. First, we counted the number of DOPs within 50 kb from the transcriptional start site (TSS) of each gene. Next, we compared the proportions of DOPs between RNA-seq clusters. Interestingly, states 6 and 8 had greater proportion of DOPs in the RNA-seq cluster with PHF20-dependent expression (cluster 1) [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref] , indicating that PHF20-dependent expression is regulated by DOPs in states 6 and 8 near their TSSs (<50 kb). Further, we investigated the genomic regional distribution of the DOPs in states 6 and 8. Interestingly, DOPs belonging to PHF20-dependent states 6 and 8 are more condensed in intragenic regions, suggesting that PHF20 is mainly associated with chromatin opening of intragenic regions [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref]. Collectively, these results indicate that PHF20 activates its target DOPs under glucose starvation, and activation of these DOPs is closely related to the PHF20-dependent expression of autophagy genes.
## Phf20 deletion reduces active enhancer markers on its target dops
Since the chromHMM showed that PHF20-dependent DOPs are localized in non-promoter regions, including an H3K4me-enriched enhancer state (state 8: Enhancer region as shown in [fig_ref] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions [/fig_ref] , we hypothesized that PHF20 is required for the activation of cis-regulatory elements to upregulate autophagy-related genes upon glucose starvation. To test this hypothesis, we performed ChIP-seq for H3K4me1 and H3K4me2, which are known to be closely linked to active cis-regulatory elements. Average profiling and read density heatmaps around the peak center revealed that the levels of both H3K4me1 and H3K4me2 increased on PHF20 dependently opened chromatin regions in WT MEFs, but not in Phf20 -/-MEFs upon glucose starvation [fig_ref] Figure 4: Phf20 deletion reduces active enhancer markers on its target DOPs [/fig_ref]. In particular, the DOPs near the Supt5, Ulk1, and Atg13 loci showed marked chromatin opening during autophagy, along with H3K4me1 and H3K4me2 enrichment in WT, but not in Phf20 -/-MEFs [fig_ref] Figure 4: Phf20 deletion reduces active enhancer markers on its target DOPs [/fig_ref]. Moreover, these three DOPs are closely located to the state 8 enhancer region of chromHMM which shows a PHF20dependent opening pattern.
## Phf20 activates enhancers via the recognition of h3k36me2 and the recruitment of mixed lineage leukemia 3/4 (mll3/4) complex
Since the PHF20-dependent chromatin states show a high level of H3K36 methylation as determined by the chromHMM analysis [fig_ref] Figure 5: PHF20 recognizes H3K36me2 via its Tudor 1 and 2 domains [/fig_ref] , we tested the possibility that PHF20 is responsible for chromatin opening at the H3K36me-enriched regions during autophagy. For this, we first examined whether PHF20 recognizes H3K36 methylation directly. Crystal structure of PHF20 predicted that the Tudor domain of PHF20 has a potential for binding di-methylated histone substrates including H3K36me2 [bib_ref] Crystal structures of the tudor domains of human PHF20 reveal novel structural..., Adams-Cioaba [/bib_ref]. Moreover, the comparison of the 3D structure of the PHF20 Tudor domain with that of H3K36me2-bound PHF1 Tudor domain from structural modeling allowed us to predict that the PHF20 Tudor domain possesses an aromatic cage structure to be able to accommodate H3K36me2 binding as in the case of PHF1 Tudor domain [bib_ref] An H3K36 methylation-engaging tudor motif of polycomb-like proteins mediates PRC2 complex targeting, Cai [/bib_ref]. Therefore, we tested the binding affinity of GST-PHF20 Tudor 1 and 2 domains (Tudor 1&2) to various histone modifications using an in vitro histone peptide binding array [fig_ref] Figure 5: PHF20 recognizes H3K36me2 via its Tudor 1 and 2 domains [/fig_ref]. The peptide binding array revealed specific binding of PHF20 Tudor 1&2 to H3K36me2 as well as other di-methylated lysine peptides [fig_ref] Figure 5: PHF20 recognizes H3K36me2 via its Tudor 1 and 2 domains [/fig_ref]. Next, we performed an in vitro peptide binding assay to examine the binding affinity of GST-PHF20 Tudor 1&2 using WT and mutant W97A; the mutant W97A carries a mutation corresponding to a core aromatic residue to block substrate binding to H3K36me2. GST-PHF20 Tudor 1&2 of WT protein, but not W97A mutant protein, selectively bound the H3K36me2 peptide [fig_ref] Figure 5: PHF20 recognizes H3K36me2 via its Tudor 1 and 2 domains [/fig_ref]. To test the effect of H3K36me2 binding affinity on the recruitment of PHF20, we conducted the CUT & RUN assay, a chromatin immunocleavage assay with a primary antibody and micrococcal nuclease conjugated with protein A (pA-MN) [bib_ref] High-Resolution chromatin profiling using CUT&RUN, Hainer [/bib_ref] , with Flag-PHF20 WT and Tudor mutant [fig_ref] Figure 5: PHF20 recognizes H3K36me2 via its Tudor 1 and 2 domains [/fig_ref]. While the binding of PHF20 WT on the target site increased upon glucose starvation, the Tudor mutant which cannot bind H3K36me2 failed to show increased recruitment upon glucose starvation [fig_ref] Figure 5: PHF20 recognizes H3K36me2 via its Tudor 1 and 2 domains [/fig_ref].
Next, we tried to find out the effector molecules that directly activate the H3K36me2-enriched enhancer regions, given that PHF20 has no enzymatic activity. Since the MLL complexes are well-known methyltransferase complexes for both H3K4me1 and H3K4me2 (66-70), we examined the interaction between PHF20 and MLL components including WDR5 and RbBP5. Co-immunoprecipitation assay revealed that WDR5 and RbBP5 showed comparable binding to PHF20 under glucose starvation . Moreover, PHF20 interacted with KDM6A/UTX, a specific component of MLL3/4 complex, but not with Menin, a specific component of MLL1/2 complex, indicating that PHF20 has a binding preference for MLL3/4 complex . Because MLL3/4 complex are known to play an important role in establishing H3K4me1 on enhancer, this result supports that PHF20 is mainly responsible for enhancer activation upon glucose starvation. To test whether PHF20 recruits the MLL3/4 complex to H3K36me2-enriched target sites under glucose starvation, we performed ChIP assay and checked the recruitment of WDR5 to PHF20dependent DOPs . WDR5 was recruited to the target DOPs under glucose starvation in WT MEFs, but not in Phf20-/-MEFs. Moreover, the transcription of eRNA, which is closely correlated with enhancer activity [bib_ref] Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA, Wang [/bib_ref] [bib_ref] Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation, Li [/bib_ref] , increased under glucose starvation in WT MEFs, but not in Phf20 -/-MEFs . Next, we tested the effect of PHF20-dependent enhancer activity on its target gene expression using CRISPRi. We used a fusion protein of the enzymatically inactive dCas9 and Krüppel-associated box (KRAB) repressor (dCas9-KRAB) to repress the target enhancer regions [bib_ref] Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory..., Thakore [/bib_ref]. Inhibition of the enhancer region by CRISPRi led to a decrease in target gene expression exemplified by Atg13 and Ulk1 without affecting promoter activity and F). At last, we confirmed the PHF20 dependent promoter-enhancer looping with chromosome conformation capture (3C) assay . The looping between the PHF20 dependent target gene promoters and DOP regions increased under glucose starvation in WT MEFs, but not in Phf20 -/-MEFs. Taken together, our data indicate that PHF20 increases the expression of autophagy genes via enhancer activation by recruiting the MLL3/4 complex to the target DOPs under glucose starvation [fig_ref] Figure 7: PHF20 is crucial for epigenetic regulation of autophagy via H3K36me2-dependent enhancer activation [/fig_ref].
# Discussion
Replenishment of autophagy proteins by transcriptional activation is an essential process for prolonged autophagy. This process is achieved by maintaining adequate levels of autophagic flux. To precisely control the expression of specific target genes, epigenetic regulation is crucial. Here, we defined the integrated signaling pathway that connects the upstream inducing signal of autophagy to the downstream target gene expression through epigenetic regulation. Genome-wide analyses and molecular mechanistic studies revealed that PHF20 functions as a versatile platform for recruiting MLL3/4 methyltransferase complexes with increased histone H3K4 methylation and the subsequent activation of autophagy genes. Integrative analysis of RNA-seq and ATAC-seq results showed that PHF20 altered the chromatin structure to activate the transcription of autophagy-related genes on a genome-wide scale and the global chromatin opening by PHF20 was more prominent under glucose starvation. With respect to its region of activity on the genome, enhancers and gene bodies were the chromatin states where PHF20 evidently worked, suggesting that PHF20 is likely associated with long range interactions in the 3D genome structure [fig_ref] Figure 7: PHF20 is crucial for epigenetic regulation of autophagy via H3K36me2-dependent enhancer activation [/fig_ref]. These specificities of PHF20 for chromatin states were found to be achieved by interaction with the modified histone marks. Therefore, our genome-wide approaches indicate that epigenetic regulation of chromatin is crucial for the response to autophagy. Intriguingly, glucose starvation-induced PHF20-MLL3/4 complex can work in the distal regions and activate autophagy genes through enhancer activation. Histone H3K36me2 in enhancer regions is recognized by PHF20 through the Tudor 1 and 2 domains and this recognition is required for the activation of the transcription of autophagy genes, leading to the continued autophagic flux. Given that PHF20 plays an important role in stress-induced autophagy, connections between H3K36me2-enriched regions and enhancer activation by PHF20 reveal a new way in which cells cope with various harmful conditions.
As neither PHF20 protein level nor H3K36me2 level is increased by glucose starvation, we speculate that certain signal-induced post-translational modifications of PHF20 may contribute to the increased recruitment of PHF20 to H3K36me2-enriched chromatin regions during starvationinduced autophagy. Another possibility is that certain transcription factors and coregulators function to facilitate the enhanced binding of PHF20 to H3K36me2-enriched target sites. Since deletion of the Tudor domain of PHF20 led to the failure of PHF20 recruitment to the H3K36me2enriched region, certain post-translational modifications of PHF20 may occur on the Tudor domain or nearby regions to make it effective for accommodating H3K36me2 binding. Moreover, we found that PHF20 Tudor domain also showed comparable binding to H4K20me2 and H3K27me2 from in vitro peptide binding array. In this study, we only focused on H3K36me2 to further studies, although it is possible that H4K20me2 or H3K27me2 might have functions to induce the binding of PHF20 to the target sites. SET1 family methyltransferases including the MLL family proteins should be associated with WRAD components--which comprise WDR5, RbBP5, Ash2L and DPY-30--for their complete activation [bib_ref] COMPASS: a complex of proteins associated with a trithorax-related SET domain protein, Miller [/bib_ref] [bib_ref] WRAD: enabler of the SET1-family of H3K4 methyltransferases, Ernst [/bib_ref]. WRAD induces the allosteric activation of methyltransferases or recruits methyltransferases to the appropriate target sites [bib_ref] On the mechanism of multiple lysine methylation by the human mixed lineage..., Patel [/bib_ref] [bib_ref] A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3..., Patel [/bib_ref] [bib_ref] Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL..., Steward [/bib_ref] [bib_ref] An Ash2L/rbbp5 heterodimer stimulates the MLL1 methyltransferase activity through coordinated substrate interactions..., Bryk [/bib_ref]. Since the MLL complex is responsible for all three types of H3K4 methylations, each subtype of MLL complex possesses distinct enzymatic activity toward its substrate; MLL1/2 is a major methyltransferase for H3K4me3 on promoters [bib_ref] The mll2 branch of the COMPASS family regulates bivalent promoters in mouse..., Hu [/bib_ref] [bib_ref] Global analysis of H3K4 methylation defines MLL family member targets and points..., Wang [/bib_ref] , while MLL3/4 is responsible for the accumulation of H3K4me1 on active enhancers [bib_ref] Enhancer-associated H3K4 monomethylation by Trithorax-related, the drosophila homolog of mammalian mll3/mll4, Herz [/bib_ref] [bib_ref] Structural insights into trans-histone regulation of H3K4 methylation by unique histone H4..., Liu [/bib_ref]. Therefore, the genomic site where each subtype of the MLL complex is recruited under specific conditions should be tightly regulated. Our finding regarding the recruitment of WDR5 and RbBP5 by PHF20 to the target enhancer site suggests that PHF20 plays an important role in inducing H3K4 methylation via the MLL complex. Especially, our immunoprecipitation data shows that PHF20 has a binding preference to MLL3/4 complex compared to MLL1/2 complex . Therefore, our data suggest that PHF20 with H3K36me2 binding activity might contribute to the regulation of the subtype-specific target decision between the MLL complexes, depending on the upstream signals.
The epigenetic and transcriptional control of autophagy is mainly triggered by upstream signaling cascades, and then regulated by epigenetic enzymes in the nucleus. Hi- . PHF20 activates enhancers by recruiting MLL3/4 complex. (A and B) Co-immunoprecipitation assay of endogenous PHF20 with WRAD components including WDR5 and RbBP5 (A) or MLL subtype specific components exemplified by Menin (MLL1/2 complex-specific) and KDM6A/UTX (MLL3/4 complex-specific) (B) under glucose starvation. (C) Chromatin immunoprecipitation (ChIP) assay was detected by individual qRT-PCR with primers for Atg13, Ulk1 and Supt5 DOP regions. Bars, mean ± SEM; *** P < 0.001, ** P < 0.01. Statistical analysis using two-tailed t-test. (D) qRT-PCR for expression of enhancer RNA (eRNA) on Atg13, Ulk1 and Supt5 DOP regions. Bars, mean ± SEM; *** P < 0.001. Statistical analysis using twotailed t-test. (E) qRT-PCR for ChIP assay with histone H3K4me1 and H3K4me2 antibodies on Atg13 DOP region (left panel) and qRT-PCR of Atg13 mRNA (right panel). sgAtg13 cell line was generated by CRISPRi system with sgRNA targeting Atg13 DOP region. Bars, mean ± SEM; *** P < 0.001, ** P < 0.01. Statistical analysis using two-tailed t-test. (F) qRT-PCR analysis after ChIP assay with H3K4me1 and H3K4me2 antibodies on Ulk1 DOP region (left panel) and qRT-PCR of Ulk1 mRNA (right panel). sgUlk1 cell line was generated by CRISPRi system with sgRNA targeting Ulk1 DOP region. Bars, mean ± SEM; *** P < 0.001. Statistical analysis using two-tailed t-test. (G-I) Chromosome conformation capture (3C) assay on promoter-enhancer region of PHF20 target genes including Atg13 (G), Ulk1 (H) and Supt5 (I). PCR products were detected by DNA gel electrophoresis. DNA sequencing results were indicated (middle box). The models describe promoters (green blocks) with possible enhancer elements (blue blocks). Black lines represent HindIII restriction sites. Red arrows represent the site and the direction of primers used in PCR (bottom box). stone modifications and epigenetic enzymes are linked to the transcriptional regulation of autophagy depending upstream signals. We have previously identified CARM1 arginine methyltransferase as an essential regulator of both TFEB and FOXO transcription factors. CARM1 directly functions as a coactivator for TFEB with increased H3R17 methylation. CARM1 also has a nonhistone substrate, Pontin, as well as a histone substrate, H3R17me2; methylated Pontin functions as a coactivator of FOXO, with increased H4 acetylation by the Tip60 coactivator [bib_ref] Pontin arginine methylation by CARM1 is crucial for epigenetic regulation of autophagy, Yu [/bib_ref]. Compared to the specific PHF20 binding to an enhancer region, methylated Pontin can bind both the distal DNA region and the promoter region via FOXO3a binding. Although further studies are needed to understand how PHF20-dependent enhancers and methylated Pontin-FOXO3a-dependent enhancers are orchestrated to work with promoters to regulate autophagy genes upon glucose starvation, our study suggests the possibility that there exist various ways of enhancer activation via a distinct signaling axis. We speculate that pharmacological manipulation would be helpful in controlling autophagy as well as autophagy-related diseases by selectively blocking transcription factors, coregulators and various signaling axes.
Various histone marks with their corresponding epigenetic writers, such as H3R17me2 and CARM1, H4K16ac and hMOF, H3K9me2 and G9a, and H3K27me3 and EZH2, which are involved in the epigenetic regulation of autophagy have been reported. Given that PHF20 is an epi-genetic reader without possessing enzymatic activity, our studies extend the nuclear events of autophagy to highlight the role of epigenetic readers in recruiting epigenetic writers/erasers together to the target sites. Therefore, identification of autophagy-specific epigenetic writers/erasers and the corresponding epigenetic readers can both provide a basis for understanding the transcriptional outcome eliciting the autophagic process and be applicable to the development of therapeutic approaches for the dysregulated autophagic processes which lead to human diseases.
Our findings provide a novel insight into the function of PHF20 in regulating the expression of autophagy genes via the recognition of H3K36me2 and highlight the importance of enhancers in the regulation of autophagy genes. Furthermore, our findings will have application in future drug development research, such as in determining the therapeutic targets for autophagy-related diseases.
[fig] Figure 1: PHF20 is crucial for inducing autophagic flux under glucose starvation. (A-C) Immunoblot analysis of light chain 3 (LC3) levels in cell lysates of WT or Phf20 −/− MEFs after glucose starvation (A), amino acid starvation (B), and rapamycin (150 nM) treatment (C). The number below indicates LC3-II/-actin ratio. (D) Representative confocal images of GFP-LC3 puncta formed under control or glucose starvation conditions. Scale bar, 50 m. The graph indicates the number of LC3-positive cells. Bars, mean ± standard error of the mean (SEM); *** P < 0.001. Statistical analysis using twotailed t-test. (E) Autophagic flux was analyzed in WT or Phf20 −/− MEFs in the presence or absence chloroquine (10 M; 4 h) under glucose starvation conditions. The LC3-II/-actin ratio is indicated. (F) Representative confocal images of autophagic vacuoles in WT or Phf20 −/− MEFs in the presence or absence of bafilomycin A1 (200 nM; 2 h). Autophagic vacuoles were detected using the CYTO-ID staining. Nucleus are stained with Hoechst (Blue). Scale bar, 50 m. The graph indicates the number of autophagic vacuoles per cells. [/fig]
[fig] Figure 2: PHF20 induces autophagy genes under glucose starvation. (A) Workflow of RNA-sequencing and downstream analysis. (B) Unsupervised hierarchical clustering using top 10% variably expressed genes. The y-axis shows distance in Spearman correlation coefficient. (C) Heat map of k-means clustering of total protein coding genes in WT and Phf20 −/− with or without glucose starvation (n = 12208, k = 8). The genes are clustered in 8 different groups based on relative gene expression across the samples. Cluster 1 which shows PHF20 dependent gene activation pattern is highlighted in red. (D) z-score centroids of Cluster 1. The black line and grey lines indicate the centroid and genes, respectively. (E) Functional Gene Ontology (GO) analysis on the genes in Cluster 1. Autophagy related terms are shown significantly in Cluster 1 but not in other clusters. (F) Gene Set Enrichment Analysis (GSEA) for the genes correlated with the gene expression in Cluster 1. FDR, false discovery rate; NES, normalized enrichment score. (G) Expression levels of genes involved in autophagy initiation, phagophore expansion, and cargo recruitment and trafficking. (H) mRNA expression of autophagy-related genes with quantitative real time-PCR (qRT-PCR).Bars, mean ± SEM; *** P < 0.001. Statistical analysis using two-tailed t-test. [/fig]
[fig] Figure 3: PHF20 affects chromatin opening of intragenic enhancer regions. (A) Workflow of ATAC-sequencing analysis. (B) Unsupervised hierarchical clustering using top 10% variably opened peaks (Spearman distance). The height implies similarity of opening peaks by each sample. (C) Differentially opening peaks (DOPs) between WT and Phf20 -/under each condition. Red dots represent statistically significant DOPs that are less than adjusted Pvalue 0.05. N, the number of DOPs that are greater than fold change 2 and less than adjusted P-value 0.05. (D) Functional analysis for the DOPs between conditions. GO results are shown for the genes whose TSS are within 10 kb from the DOPs. Autophagy-related terms are significantly found in WT but not Phf20 -/-. (E) Chromatin state using chromHMM software. Each row represents one chromatin state. From left to right: Histone mark and probability used to define the states (State emission). Chromatin state enrichment in genomic features (genomic annotation). Description of 16 states (Descriptions). (F) Average plots of DOPs in state 6, 7 and 8 by each sample. (G) Comparing the chromHMM state ratio of DOPs in each RNA-sequencing cluster to the ratio of DOPs (n = 33 443) in total protein-coding genes. DOPs distributed under 50 kb from TSS are counted. Statistical analysis using chi-square test. Total protein-coding genes have 4.43% (n = 1481 from 33 443 total DOPs, <50 kb) of state 6 DOPs, and 7.54% (n = 2521 from 33 443 total DOPs, <50 kb) of state 8 DOPs. (H) Proportion of DOPs at genomic location. DOPs in states 6 and 8 are enriched in the intragenic region. [/fig]
[fig] Figure 4: Phf20 deletion reduces active enhancer markers on its target DOPs. (A) Average profiles of H3K4me1 and H3K4me2 signals in WT Glc starv. > Phf20 -/-Glc starv. DOPs for each condition. (B) Read density heatmaps around peak center of H3K4me1 and H3K4me2 signals in WT Glc starv. >Phf20 -/-Glc starv. DOPs for each condition. (C) UCSC Genome Browser (GB) tracks of ATAC-seq signal (green), ChIP-seq signals for H3K4me1 (blue) and H3K4me2 (darkbrown), and chromHMM chromatin states around the DOPs of Supt5, Ulk1 and Atg13. Y-axis represents normalized read counts. [/fig]
[fig] Figure 5: PHF20 recognizes H3K36me2 via its Tudor 1 and 2 domains. (A) Relative H3K36me2 ChIP-seq peak intensity of PHF20-dependent (states 6 and 8) and PHF20-independent states in chromHMM. (B) Screening for histone peptide binding of PHF20 Tudor 1 and 2 domains (Tudor 1&2) with MODi-fied™ Histone Peptide Array. GST-PHF20 Tudor 1&2 construct was detected with GST antibody. Histone peptides with significant binding intensity are indicated with red, yellow, and blue circles. Each dot indicated with the color contains the following histone peptides. red:H3K27me2, yellow:H3K36me2, and blue:H4K20me2. (C) Top five histone peptides with the highest binding intensity. Binding intensity was calculated with MODified™ Histone Peptide Array analysis program. (D) In vitro peptide binding assay using GST-PHF20 Tudor 1&2 of WT and W97A constructs was performed, followed by immunoblot analysis with anti-GST antibody. (E) CUT&RUN assay of Flag-tagged PHF20 constructs on Atg13 DOP region. (F) Schematic model for H3K36me2 recognition of PHF20 WT and Tudor domain deletion mutant. [/fig]
[fig] Figure 7: PHF20 is crucial for epigenetic regulation of autophagy via H3K36me2-dependent enhancer activation. Model of working mechanism of PHF20 in autophagy gene regulation via activation of enhancers that are co-occupied by H3K36me2. [/fig]
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Challenges in fibromyalgia diagnosis: from meaning of symptoms to fibromyalgia labeling
Fibromyalgia (FM) is a contested illness with ill-defined boundaries. There is no clearly defined cut-point that separates FM from non-FM. Diagnosis of FM has been faced with several challenges that occur, including patients' health care-seeking behavior, symptoms recognition, and FM labeling by physicians. This review focuses on important but less visible factors that have a profound influence on under-or over-diagnosis of FM. FM shows different phenotypes and disease expression in patients and even in one patient over time. Psychosocial and cultural factors seem to be a contemporary ferment in FM which play a major role in physician diagnosis even more than having severe symptom levels in FM patients. Although the FM criteria are the only current methods which can be used for classification of FM patients in surveys, research, and clinical settings, there are several key pieces missing in the fibromyalgia diagnostic puzzle, such as invalidation, psychosocial factors, and heterogeneous disease expression. Regarding the complex nature of FM, as well as the arbitrary and illusory constructs of the existing FM criteria, FM diagnosis frequently fails to provide a clinical diagnosis fit to reality. A physicians' judgment, obtained in real communicative environments with patients, beyond the existing constructional scores, seems the only reliable way for more valid diagnoses. It plays a pivotal role in the meaning and conceptualization of symptoms and psychosocial factors, making diagnoses and labeling of FM. It is better to see FM as a whole, not as a medical specialty or constructional scores. (Korean J Pain 2018; 31: 147-54)
# Introduction
Fibromyalgia (FM) is a contested illness where there is a considerable controversy in its nature, existence, assessment, and diagnosis [bib_ref] Culture, science and the changing nature of fibromyalgia, Wolfe [/bib_ref] [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref] [bib_ref] Fibromyalgia: from pathophysiology to therapy, Schmidt-Wilcke [/bib_ref]. There are several challenges in factors that influence profoundly on under-or over-diagnosis of FM by physicians. We hope this review creates a new gate to a holistic and real understanding of FM diagnosis beyond existing arbitrary and constructional scores.
## Main body
## Fuzzy boundaries of fibromyalgia with broad and time-changing phenotypes
Chronic widespread pain, sleep problems or unrefreshing sleep, physical exhaustion, and cognitive difficulties are the core symptoms of FM [bib_ref] Identifying the clinical domains of fibromyalgia: contributions from clinician and patient Delphi..., Mease [/bib_ref] [bib_ref] OMERACT-based fibromyalgia symptom subgroups: an exploratory cluster analysis, Vincent [/bib_ref]. However, most patients diagnosed with FM report a wide range of additional somatic and psychological symptoms [bib_ref] Identifying the clinical domains of fibromyalgia: contributions from clinician and patient Delphi..., Mease [/bib_ref]. It has fuzzy boundaries that are cross-linked to other illness (such as psychological disorders) as subsets, mistaken diagnosis and comorbid conditions [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref]. There are more confusing issues when we review our knowledge about FM neurobiology, illness definition, and accepted diagnostic criteria.
Although not yet fully understood, the new concept suggests that FM is a heterogeneous condition which likely has multiple potential etiologies [bib_ref] Neurobiology of fibromyalgia and chronic widespread pain, Sluka [/bib_ref]. It is important to not consider FM as 'yes' or 'no' but rather as the end of a continuum [bib_ref] Neurobiology of fibromyalgia and chronic widespread pain, Sluka [/bib_ref] [bib_ref] Fibromyalgia criteria and severity scales for clinical and epidemiological studies: a modification..., Wolfe [/bib_ref] [bib_ref] Fibromyalgia prevalence, somatic symptom reporting, and the dimensionality of polysymptomatic distress: results..., Wolfe [/bib_ref]. On the one end of this continuum is a purely peripherally driven painful condition and the other end of the continuum is when pain is nearly completely the result of altered central augmentation [bib_ref] Neurobiology of fibromyalgia and chronic widespread pain, Sluka [/bib_ref].
In fact, many clinical researches suggest that various individuals with chronic pain, including FM, are at various points in this continuum [bib_ref] Lack of pressure pain modulation by heterotopic noxious conditioning stimulation in patients..., Kosek [/bib_ref] [bib_ref] Fibromyalgia: a clinical review, Clauw [/bib_ref]. Thus, some patients may have stronger peripheral than central components, some mixed components, and many have stronger central components. Consequently, FM Patients may present with different phenotypes according to their different underlying neurobiology [bib_ref] Neurobiology of fibromyalgia and chronic widespread pain, Sluka [/bib_ref].
Furthermore, it is also debated whether patients maintain their characteristic phenotype and disease expression with time, or whether disease expression changes [bib_ref] Treat-to-target strategy for fibromyalgia: opening the dialogue, Häuser [/bib_ref]. It is not surprising that an individual FM core symptom may emerge or decline as a predominant symptom over time [bib_ref] Reliability of ACR criteria over time to differentiate classic fibromyalgia from nonspecific..., Bidari [/bib_ref].
Due to diversity of symptoms presentation and severity over time, it is possible that an individual with FM seeks medical help and is diagnosed with the FM label at one time, and the same patient, at another time, is given a diagnostic label that merely connotes more local complaints, such as chronic low back pain, headache, or temporomandibular joint disorder, irritable bowel syndrome (IBS), and so on [bib_ref] Neurobiology of fibromyalgia and chronic widespread pain, Sluka [/bib_ref] [bib_ref] Implications of proposed fibromyalgia criteria across other functional pain syndromes, Egloff [/bib_ref]. It seems that some FM core symptoms diminish or fade over time [bib_ref] The longitudinal outcome of fibromyalgia: a study of 1555 patients, Walitt [/bib_ref] , or patients do not mention actively in their core symptoms which may be non-significant or may be missed by the clinician.
Core symptom variation both within a patient over time as well as between patients in the setting of etiologic and symptom heterogeneity produces considerable perplexity in FM diagnosis, even in hands of experts. The clinicians who are familiar with FM contend that "they can recognize FM", but experts still debate practical clinical diagnostic criteria [bib_ref] Treat-to-target strategy for fibromyalgia: opening the dialogue, Häuser [/bib_ref] [bib_ref] Survey of physician experiences and perceptions about the diagnosis and treatment of..., Perrot [/bib_ref]. So, the precise diagnosis in an individual patient may be elusive, with symptoms present for years leading to many health care encounters and diagnostic delay [bib_ref] The iceberg nature of fibromyalgia burden: the clinical and economic aspects, Ghavidel-Parsa [/bib_ref].
## Patients and physicians' confounding factors in fm diagnosis
The substantial role for psychosocial and environmental determinants in FM patients capturing and diagnosis cannot be denied [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref]. It was demonstrated that having the severe core symptoms that define FM is not essential to receiving an FM diagnosis. Rather, demographic and social disadvantage appears to be more important than symptom severity in making FM clinical diagnosis by clinicians [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref]. For obtaining an FM diagnosis, symptomatic persons must seek medical care from clinicians and those clinicians must interpret and label the described symptoms as being FM. A patient cannot obtain an FM diagnosis unless they want to see a clinician who is willing to make that diagnosis. So, clinical diagnosis of FM patients is necessarily confounded by patient's health care seeking behavior and clinical selection [bib_ref] The prevalence and characteristics of fibromyalgia in the 2012 National Health Interview..., Walitt [/bib_ref]. Furthermore, in a clinical setting, physicians' beliefs and biases influence FM diagnosis [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref]. All FM assessments are subjective and there is no clear gold standard for FM diagnosis. The constellation of severe symptoms can be clinically interpreted and diagnosed in many different ways, perhaps influenced by clinicians [bib_ref] Editorial: the status of fibromyalgia criteria, Wolfe [/bib_ref]. This leads to a large number of apparently over-and under-diagnosed subjects in clinical settings [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref] [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref]. On one hand, a diagnosis of FM can legitimize vague and difficult symptoms, allowing entrée into official diagnosis or providing a way 149 www.epain.org toward official disability status. So, resultantly, millions of people may be given a FM diagnosis without satisfying the severity cutoff of FM diagnosis [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref] [bib_ref] Comparison of physician-based and patient-based criteria for the diagnosis of fibromyalgia, Wolfe [/bib_ref].
On the other hand, it appears that many clinicians, when faced with the opportunity to diagnose FM may miss making such a diagnosis. This action may be related to the complexity and diversity of FM symptoms, the presence of other medical diagnoses, a lack of knowledge; or a disagreement about the nature and meaning of symptoms and how they should be interpreted [bib_ref] The prevalence and characteristics of fibromyalgia in the 2012 National Health Interview..., Walitt [/bib_ref] [bib_ref] When do symptoms become a disease?, Aronowitz [/bib_ref]. So, it is not so surprising to see patients who have gone from doctor to doctor and who underwent multiple diagnostic tests and received alternative diagnoses such as lupus erythematous, rheumatoid arthritis, non-specific "arthritis" and malingering [bib_ref] The iceberg nature of fibromyalgia burden: the clinical and economic aspects, Ghavidel-Parsa [/bib_ref] [bib_ref] The problem in differentiation between psoriatic-related polyenthesitis and fibromyalgia, Marchesoni [/bib_ref].
FM patients may experience and mention some level of lack of understanding or discounting by their clinicians, family, or other people that interact with them. The term "invalidation" is used to describe the patients' perception that their illness and symptoms is not recognized by their social environment [bib_ref] Understanding the lack of understanding: invalidation from the perspective of the patient..., Kool [/bib_ref]. Although invalidation is not exclusive to FM patients and also affects other rheumatic diseases, it most often perceived and expressed by FM patients [bib_ref] Invalidation in patients with rheumatic diseases: clinical and psychological framework, Santiago [/bib_ref] [bib_ref] Lack of understanding in fibromyalgia and rheumatoid arthritis: the Illness Invalidation Inventory..., Kool [/bib_ref]. Practicing clinicians are often confronted with a high prevalence of invalidation in FM patients. In recent years, some researchers have suggested that patients' perception of invalidation by clinicians could be a clue for the diagnosis of FM. In fact, it would certainly be helpful to be clear whether invalidation assessment may be useful for diagnosis or classification of FM [bib_ref] Correlation of invalidation with symptom severity and health status in fibromyalgia, Ghavidel-Parsa [/bib_ref].
Resultantly, it seems that both patients' behavior and clinical symptoms as well as physicians' symptom interpretation, beliefs, invalidation, or intimacy with patients can clearly influence whether or not an FM diagnosis is given.
## Application of the fm classification criteria in our diagnostic lexicon
The existing FM classification criteria has clearly altered and introduced a more precise case definition of FM in the recent decades. The recent criteria for the diagnosis of FM are clearly a steps towards a better understanding of this condition and are currently used for survey and clinical purposes. However, it seems much more effort must be made in this direction. The existing diagnostic criteria don't seem to cover the broad heterogeneity and variation in symptoms and severity.
Regarding the intuitive complexity of the nature of FM, as well as the presence of these broad confounding factors in diagnosis, using a more valid diagnostic instrument seems essential. It seems that FM is better conceptualized as a symptom and etiological continuum rather than as a discreet dichotomous entity inferred by existing criteria [bib_ref] Fibromyalgia prevalence, somatic symptom reporting, and the dimensionality of polysymptomatic distress: results..., Wolfe [/bib_ref] [bib_ref] The American College of Rheumatology 1990 criteria for the classification of fibromyalgia...., Wolfe [/bib_ref] [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref] [bib_ref] 2016 revisions to the 2010/2011 fibromyalgia diagnostic criteria, Wolfe [/bib_ref] [bib_ref] Criteria for the diagnosis of fibromyalgia: validation of the modified 2010 preliminary..., Bennett [/bib_ref].
No clear distinguishing point exists where FM stops being FM and becomes some other illness with more localized complaints, or no illness at all [bib_ref] The prevalence and characteristics of fibromyalgia in the 2012 National Health Interview..., Walitt [/bib_ref]. This raises a fundamental question about how diagnostic labeling should be applied according to the diagnostic criteria, because one patient may be labeled as FM on one occasion and non-FM on another. FM patients often move on this spectrum, occasionally toward a typical FM phenotype and at other times toward a normal phenotype [bib_ref] Reliability of ACR criteria over time to differentiate classic fibromyalgia from nonspecific..., Bidari [/bib_ref] , probably due to the effects of certain psychological, environmental, or other unknown factors. But, it is important to note that each patient is the same person in regards to genotype, neurobiology, and other risk factors.
Although, as mentioned before, psychological and environmental, or socio-cultural factors also play an important role in presentation, recognition and capturing, or even of labeling FM cases [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref] [bib_ref] The prevalence and characteristics of fibromyalgia in the 2012 National Health Interview..., Walitt [/bib_ref] , there is no place for these issues in the FM criteria. In other words, these factors undoubtedly influence diagnosis of FM and are even more important than symptom severity in the probability of receiving an FM diagnosis by medical healthcare providers [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref].
On the other hand, physicians will differ in their interpretation of patient complaints and also the severity of symptoms. A physician both records the complaint as presented by the patient, and also assesses the validity of the symptom report which the latter does not provide by the existing criteria. It is likely that some persons with FM may exaggerate subjective symptoms for reasons of secondary gain such as obtaining disability payments [bib_ref] Disability in fibromyalgia associates with symptom severity and occupation characteristics, Fitzcharles [/bib_ref].
So, full clinical evaluation such as assessment of dayto-day activities, information from all treating healthcare professionals, and careful observation during assessment for evidence of discrepancies in clinical findings should still remain the standard of patient evaluation and diagnosis, rather than relying on differently interpreted or perceived FM criteria items.
Regarding both the above-mentioned issues and some other points in criticizing the existing criteria (see "Arbitrary and illusory constructs of the existing FM criteria"), it seems these criteria have not met expectations. There is a critical question which deals with the existing criteria: to what extent are the symptom-based and arbitrary constructs of the existing FM criteria suitable as diagnostic instruments? Although these criteria have incorporated most core symptoms of FM, there appears to remain a long and hard path to achieve a holistic and intuitive instrument for FM diagnosis.
In Egolff view, "No specialist in internal medicine would be likely to confuse the New York Heart Association (NYHA) criteria for quantifying dyspnea in heart failure with the diagnosis of the underlying cardiomyopathy" [bib_ref] Implications of proposed fibromyalgia criteria across other functional pain syndromes, Egloff [/bib_ref]. In other words, use of symptom scores and cutoff points are not the type of instrument needed or suitable for the clinical diagnosis of diseases. It seems that the just symptom-based approach for diagnosis of disease was subject to criticism from the very beginning [bib_ref] Implications of proposed fibromyalgia criteria across other functional pain syndromes, Egloff [/bib_ref] [bib_ref] Unhelpful criteria sets for "diagnosis" and "assessment of severity" of fibromyalgia, Smythe [/bib_ref]. This approach results in a failure to provide a diagnostic instrument fit for clinical purposes [bib_ref] Fibromyalgia criteria and severity scales for clinical and epidemiological studies: a modification..., Wolfe [/bib_ref].
Diagnosis of FM is discretionary and patient symptoms level, psychosocial factors, and external societal factors influence that discretion [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref]. It is wise to quote George Ehrlich's sentence: "When one has tuberculosis, one has tuberculosis, whether or not it is diagnosed, but "no one has FM until it is diagnosed" [bib_ref] Pain is real; fibromyalgia isn't, Ehrlich [/bib_ref]. So, diagnostic labeling in FM must be used with caution and discretion [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref] [bib_ref] Pain is real; fibromyalgia isn't, Ehrlich [/bib_ref]. This double-edged tool can have both beneficial and harmful effects. On the one hand, delay in labeling can result in excessive testing, inappropriate treatment, and consequently economic burden on the healthcare system and frustration for patients and their families. On the other hand, extensive use of FM diagnosis as an explanation of mild-to-moderate levels of symptoms or illness impact has likely led to substantial harm to patient and societal costs [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref].
Finally, at the present time, no matter where we put FM criteria in our diagnostic lexicon, undoubtedly, these criteria are the only current methods which can be used for classification of FM patients in surveys, research, and clinical settings. However, there are several missing pieces in the diagnostic puzzle of FM. Until receiving more accurate data about diagnostic methods, it seems that there is no way except the discretionary policy of clinicians for the diagnosis and labeling of this disorder. Physicians' judgment obtained in a real communicative environment with patients, beyond the existing constructional scores, seems the only reliable way for more valid diagnoses. Much more effort is required to identify the many missing pieces of the FM puzzle, which seem to cause and perpetuate this medical problem.
## Substantial role of psychosocial factors in fm diagnosis
Data obtained from large population studies has provided new insight into FM diagnosis in the general population and clinical settings [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref] [bib_ref] The prevalence and characteristics of fibromyalgia in the 2012 National Health Interview..., Walitt [/bib_ref]. It revealed a fundamental role for psychosocial and cultural factors in the diagnostic acceptability of FM. These studies showed that having severe symptoms which define FM showed substantially less influence on how patients received FM diagnosis by physicians. Rather, health care-seeking behavior and clinical selection, as well as social and cultural factors are more important in physician diagnosis. So, 73% of the population who had enough severe symptoms to meet a criteria-based diagnosis did not receive a clinical diagnosis of FM [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref]. On the other hand, 75% of persons in the US population reporting a clinician diagnosis of fibromyalgia did not satisfy FM criteria [bib_ref] The prevalence and characteristics of fibromyalgia in the 2012 National Health Interview..., Walitt [/bib_ref]. This observation showed the large numbers of apparently over-and under-diagnosed subjects. In this regard, demographic factors and social disadvantage appears to be more important than symptom reporting in predicting FM diagnosis [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref]. It seems the under-and over-diagnosis of clinical FM may be at least partly related to the possible influence of social and cultural factors.
To have clinical diagnosis and to legitimize the illness, which does not have any visible sign of disease, a patient must receive an insurance diagnosis by physician. In developed countries, FM patients often take an active role in their diagnosis, recognizing their poly-symptomatic distress (PDS) as FM and seeking clinical care for confirmation and treatment [bib_ref] Comparison of physician-based and patient-based criteria for the diagnosis of fibromyalgia, Wolfe [/bib_ref].
As a result, the diagnosis of FM has been extended beyond expectations and consequently has lead to an over-diagnosis of this illness [bib_ref] The prevalence and characteristics of fibromyalgia in the 2012 National Health Interview..., Walitt [/bib_ref]. Self-reported clinical FM diagnosis appears to be increased by programs for clinical awareness, through educational activities for clinicians, academic research, patient advocacy, and directto-patient advertising, much of which has been financed 151 www.epain.org by the pharmaceutical industry [bib_ref] Culture, science and the changing nature of fibromyalgia, Wolfe [/bib_ref] [bib_ref] Comparison of physician-based and patient-based criteria for the diagnosis of fibromyalgia, Wolfe [/bib_ref] [bib_ref] Listening to Lyrica: contested illnesses and pharmaceutical determinism, Barker [/bib_ref].
In contrast, the substantial numbers of under-diagnosed FM cases showed high level of invalidation of patients by their medical care providers and social environment. Furthermore, many physicians express frustration directed not only at the FM issue, but also at patients. They will not see patients that are referred to them for suspicious FM and others will only see the patient for a one-time assessment to exclude other disorders [bib_ref] Pain is real; fibromyalgia isn't, Ehrlich [/bib_ref]. This misunderstanding can also be seen in family members or other people interacting with patients [bib_ref] Understanding the lack of understanding: invalidation from the perspective of the patient..., Kool [/bib_ref] [bib_ref] Correlation of invalidation with symptom severity and health status in fibromyalgia, Ghavidel-Parsa [/bib_ref]. It seems that this high level of invalidation may be related to the great psychological distress expressed by FM patients and also to inherently invisible FM symptoms and to sociocultural factors [bib_ref] Invalidation in patients with rheumatic diseases: clinical and psychological framework, Santiago [/bib_ref].
Furthermore, the surprising data about substantial discordance between physician-based and criteria-based diagnosis raise a simple but essential question concerning to what extent we are permitted to confine all of our discretion about symptoms' meaning and severity into the scores or criteria construction. Undoubtedly, the answer to this question can be an important step toward a more real diagnosis of FM.
## Arbitrary and illusory constructs of the existing fm criteria
In 1990, The ACR committee found that the presence of widespread pain combined with at least 11 out of 18 tender points best separated patients with fibromyalgia from controls that led to development of the 1990 ACR criteria [bib_ref] The American College of Rheumatology 1990 criteria for the classification of fibromyalgia...., Wolfe [/bib_ref]. However, tender point examination was hard for most non-rheumatologists to perform [bib_ref] Editorial: the status of fibromyalgia criteria, Wolfe [/bib_ref] [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref]. Numerous concerns were raised on the reliability and validity of the tender points in the clinical setting, which eventually led to stopping their use in clinics [bib_ref] Editorial: the status of fibromyalgia criteria, Wolfe [/bib_ref]. So, the 2010 ACR preliminary diagnostic criteria proposed to address numerous problems with the 1990 ACR criteria [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref]. The ACR 2010 criteria not only eliminated tender points, they also changed the case definition of FM to an illness characterized by self-reported, multiple painful regions (0-19 WPI) and additional key symptoms, such as problems with fatigue, sleep, cognition, and the extent of somatic symptom reporting [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref].
The change in definition from 1990 to 2010 gave an important role to non-pain symptoms in FM diagnosis.
Furthermore, there were a small proportion of patients whom clinicians diagnosed as having FM but who did not satisfy the 1990 criteria because of having either ＜ 11 tender points or slightly less than the full ACR 1990 definition of widespread pain [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref]. These cases were recognized simply by the 2010 ACR criteria. Moreover, another remarkable achievement of the 2010 criteria was introducing of the Poly-symptomatic distress (PSD) scale [bib_ref] Fibromyalgia criteria and severity scales for clinical and epidemiological studies: a modification..., Wolfe [/bib_ref]. With the PSD Scale, dichotomizing diagnosis and distress becomes less important [bib_ref] Fibromyalgia criteria and severity scales for clinical and epidemiological studies: a modification..., Wolfe [/bib_ref] [bib_ref] Editorial: the status of fibromyalgia criteria, Wolfe [/bib_ref].
Because it shows where the patient is on the continuum of distress or symptoms, as well as how far the patient is from the positive/negative dividing line [bib_ref] 2016 revisions to the 2010/2011 fibromyalgia diagnostic criteria, Wolfe [/bib_ref]. Although the 2010 ACR criteria mapped out FM as a dimensional or continuum disorder, this concept did not apply completely. There is still a cut point for FM diagnosis in the PSD or the 2010 ACR criteria where patients are classified as FM versus non-FM. Hence this fact raises a fundamental question about how much difference there truly is between patients on different side of the cut point. In fact, the boundaries of FM are not well defined and there is no clearly defined cut point that separates FM from non-FM [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref] [bib_ref] 2016 revisions to the 2010/2011 fibromyalgia diagnostic criteria, Wolfe [/bib_ref]. Moreover, all assessments are subjective, and both physicians and patients might differ in their assessment of severity [bib_ref] Editorial: the status of fibromyalgia criteria, Wolfe [/bib_ref]. So, this makes distinguishing cases and non-cases difficult and arbitrary [bib_ref] Culture, science and the changing nature of fibromyalgia, Wolfe [/bib_ref].
Additionally, it seems that dropping the tender point concept is not only the main advantage of the ACR 2010 criteria but also their greatest drawback [bib_ref] Implications of proposed fibromyalgia criteria across other functional pain syndromes, Egloff [/bib_ref]. The 2010 criteria study showed that muscle tenderness was one of the most important variables classifying cases and non-cases of FM, although tenderness was not used in the final formulation of the criteria [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref]. Numerous previous studies confirm that the feature of hyperalgesia is a crucial and intuitive characteristic of FM [bib_ref] Culture, science and the changing nature of fibromyalgia, Wolfe [/bib_ref] [bib_ref] Fibromyalgia: from pathophysiology to therapy, Schmidt-Wilcke [/bib_ref] [bib_ref] Neurophysiologic evidence for a central sensitization in patients with fibromyalgia, Desmeules [/bib_ref]. Although the majority of experts agree that tender points' examination and interpretation is difficult and may miss the intended target [bib_ref] Editorial: the status of fibromyalgia criteria, Wolfe [/bib_ref] [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref] , there are still some valid concerns that totally abandoning the tender point concept would omit examination of the distinctive 'hyperalgesia aspect' of FM [bib_ref] Implications of proposed fibromyalgia criteria across other functional pain syndromes, Egloff [/bib_ref].
Additionally, the 2010 criteria are inherently underrepresentative in regards to the nature of the pain. It only incorporates pain regions or distribution and does not consider pain quality and even quantity [bib_ref] Fibromyalgia: from pathophysiology to therapy, Schmidt-Wilcke [/bib_ref]. Evaluation of pain is multidimensional and fewer pain sites (fewer WPI) will not necessarily connote to lower pain significance or impact [bib_ref] A critical examination of the Polysymptomatic Distress Scale construct as a symptom..., Friend [/bib_ref]. So, reliance just on the pain site numbers for assessment of pain impact is not the right way to a valid diagnosis.
On the other hand, non-pain symptoms were selected from a larger set of symptoms, based on empirical-statistical criteria according to their importance in distinguishing patients with FM from those who are non-FM [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref]. This larger set did not include symptoms that had been found to be essential to FM recognition, for example, stiffness, balance, tenderness to touch, environmental sensitivity, and invalidation [bib_ref] Fibromyalgia: from pathophysiology to therapy, Schmidt-Wilcke [/bib_ref] [bib_ref] The American College of Rheumatology preliminary diagnostic criteria for fibromyalgia and measurement..., Wolfe [/bib_ref] [bib_ref] Criteria for the diagnosis of fibromyalgia: validation of the modified 2010 preliminary..., Bennett [/bib_ref].
Ultimately, no criteria can translate a real understanding of patient's symptoms. Comprehension of this fact is more important and perplexing since all the diagnostic methods of assessment are subjective.
The misclassification rate of the criteria was significant among different populations [bib_ref] The Japanese version of the modified ACR preliminary diagnostic criteria for fibromyalgia..., Usui [/bib_ref] [bib_ref] The prevalence of fibromyalgia in the general population: a comparison of the..., Jones [/bib_ref] [bib_ref] A questionnaire using the modified 2010 American College of Rheumatology criteria for..., Ferrari [/bib_ref] [bib_ref] Validation of the 2010 American College of Rheumatology preliminary diagnostic criteria for..., Bidari [/bib_ref] [bib_ref] Validation of the Fibromyalgia Survey Questionnaire within a cross-sectional survey, Häuser [/bib_ref] [bib_ref] Validation of the modified 2010 American College of Rheumatology diagnostic criteria for..., Segura-Jiménez [/bib_ref] , especially when it was applied to the patients with regional pain disorders in tertiary pain clinics [bib_ref] Implications of proposed fibromyalgia criteria across other functional pain syndromes, Egloff [/bib_ref]. These observations lead to the revision of the 2010 ACR criteria and to developing the 2016 modified criteria. The researchers attempted to impose the requirement of a meeting of widespread pain criterion, such as the 1990 criterion but with caution to take away the restrictions of the 1990 criterion [bib_ref] 2016 revisions to the 2010/2011 fibromyalgia diagnostic criteria, Wolfe [/bib_ref]. It seems that it is time to criticize changing the criteria. Has changing or revising the criteria over the past decades satisfied researchers or clinicians for making a valid diagnosis of FM? The answer is probably "no". Contrary to the declared claim of these criteria, they are not simple and practical for clinical use.
Most practitioners don't use any criteria for FM diagnosis in the clinical settings [bib_ref] Fibromyalgia: a short commentary, Wolfe [/bib_ref] [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref]. This may be related to the arbitrary nature of these criteria or the criteria's limitations in coverage of all FM characteristics. It seems they are not representative of the multidimensionality and complexity of the nature of FM. So, although the existing criteria can be useful in the recognition of FM, especially for research purposes, they are not able to replace the discretionary and holistic eye of expert practitioners.
## Labeling of fm diagnosis
FM is a real but untenable diagnosis [bib_ref] Pain is real; fibromyalgia isn't, Ehrlich [/bib_ref]. Diversity of symptoms and phenotypes even in one patient may lead to numerous labels over time by medical care providers [bib_ref] Neurobiology of fibromyalgia and chronic widespread pain, Sluka [/bib_ref] [bib_ref] The iceberg nature of fibromyalgia burden: the clinical and economic aspects, Ghavidel-Parsa [/bib_ref]. Furthermore, medicalization and a diagnostic expansion in a population with mild symptoms raise serious concerns about FM labeling. In developed countries, FM patients often take an active role in their diagnosis [bib_ref] Three-quarters of persons in the US population reporting a clinical diagnosis of..., Walitt [/bib_ref]. It is not uncommon to see self-diagnosis of FM [bib_ref] Comparison of physician-based and patient-based criteria for the diagnosis of fibromyalgia, Wolfe [/bib_ref]. The agency of social and electronic media and support groups extend this concept [bib_ref] Listening to Lyrica: contested illnesses and pharmaceutical determinism, Barker [/bib_ref]. The existing data make clear that both under-and over-labeling can lead to personal and societal burdens with respect to economic and clinical aspects [bib_ref] The iceberg nature of fibromyalgia burden: the clinical and economic aspects, Ghavidel-Parsa [/bib_ref]. So, to return to the statement that "no one has FM until it is diagnosed and labeled", it is clear that a cautious and holistic approach must be emphasized in regard to patients with suspicious FM.
Modern medicine has empowered societies, physicians, and patients with vast increases in access to medical information, investigations, and with the ability to have more options about diagnoses and treatments [bib_ref] The end of professional dominance, Furedi [/bib_ref]. However, it might be thought that little has changed after many years of research on FM diagnosis or treatment. Here, there is a quote to Frederick Wolfe: "A kind, conscientious physician treating a FM patient in 1980 or 1990 will have done as well as the 2016 health workers with access to all of the new publications and expensive if not very efficacious medications".
# Conclusions
Precise fibromyalgia diagnosis may be elusive. It seems that the meaning of FM symptoms and the conceptualization of patients' complaints in a logical manner needs to challenge all of experience and discretion of practicing physicians. This means that such logical and complex diagnostic challenges cannot be placed into the limited capacity of FM criteria.
Although these criteria are the only current methods which can be used for classification of FM patients in surveys, research, and clinical settings, there are several key pieces missing in the fibromyalgia diagnostic puzzle, such as invalidation, psychosocial factors, and heterogeneous disease expression.
Deciding if the patient labeled as having FM or not, and also evaluation of the FM patient for disease impact require a meticulous and discretionary approach to FM. It is better to see FM as a whole, and not as a medical specialty or constructional scores.
Korean J Pain Vol.31, No. 3, 2018 www.epain.org
ACKNOWLEDGEMENTSThis work was supported by the Deputy for Research of Guilan University of Medical Sciences, Rasht, Iran. |
Mesoscopic 3D imaging of pancreatic cancer and Langerhans islets based on tissue autofluorescence
The possibility to assess pancreatic anatomy with microscopic resolution in three dimensions (3D) would significantly add to pathological analyses of disease processes. Pancreatic ductal adenocarcinoma (PDAC) has a bleak prognosis with over 90% of the patients dying within 5 years after diagnosis. Cure can be achieved by surgical resection, but the efficiency remains drearily low. Here we demonstrate a method that without prior immunohistochemical labelling provides insight into the 3D microenvironment and spread of PDAC and premalignant cysts in intact surgical biopsies. The method is based solely on the autofluorescent properties of the investigated tissues using optical projection tomography and/or light-sheet fluorescence microscopy. It does not interfere with subsequent histopathological analysis and may facilitate identification of tumor-free resection margins within hours. We further demonstrate how the developed approach can be used to assess individual volumes and numbers of the islets of Langerhans in unprecedently large biopsies of human pancreatic tissue, thus providing a new means by which remaining islet mass may be assessed in settings of diabetes. Generally, the method may provide a fast approach to provide new anatomical insight into pancreatic pathophysiology.Modern optical imaging technologies are starting to show potential as tools for obtaining 3D insight into pancreatic disease based on whole mount immunolabelling of pancreatic tissues 1,2 . Still, there are several limitations for their use in clinical practice. Firstly, lengthy protocols are required to ensure reagent/antibody penetration into larger pancreatic tissue biopsies, with an upper limit in the lower mm rather than the cm range. Secondly, employing whole mount immunohistochemical protocols may hamper or prevent subsequent routine histopathological analyses of tissue sections.Although 3D imaging techniques, such as optical projection tomography (OPT) 3 that is based on the attenuation contrast of light 4 have been used for 3D imaging of mouse embryos without labels, few studies, if any, have used the autofluorescent (AF) properties of the pancreas for 3D assessments of mesoscopic sized (mm-cm scale) tissue specimens. OPT could be described as a CT scanner allowing for 3D imaging of biological specimen that employs light in the visible or near infrared part of the spectrum instead of using x-rays. Similarly, light sheet fluorescent microscopy (LSFM), aka selective plane illumination microscopy (SPIM) 5-7 has emerged as a powerful tool for high-resolution studies of optically cleared specimen (for a comprehensive review of OPT versus LSFM see Liu et al. 8 ). In contrast to OPT, LSFM acquires data by performing illumination and detection of the sample in two distinct optical paths that are organized orthogonally to each other. Hereby, a stack of optical sections is produced by either moving a laser generated light sheet through the specimen, alternatively by moving the specimen through the light sheet. Although benefitting from higher resolution scans, the latter platform has limitations in terms of maximum tissue-size and non-isotropic voxels for quantitative assessments. Scanning times are significantly longer for LSFM compared to OPT for similar sized specimen, depending on the applied resolution. Generalized, OPT may be more appropriate for larger samples at lower magnification, relating to scan times and OPEN
## Scientific reports
| (2020) 10:18246 | https://doi.org/10.1038/s41598-020-74616-6 www.nature.com/scientificreports/ data volumes, and subsequently for larger numbers of samples. LSFM in turn may be more appropriate for higher resolution studies (of smaller numbers of samples). Still, the techniques are to a high degree interchangeable and can often substitute each other. Previously, both techniques have been successfully implemented in studies of pancreatic islets in various models of rodent diabetes models based on specific antibody labelling. Autofluorescent (AF) properties of tissues have been used for decades as a source of contrast to demarcate specific cells, cellular constituents or cellular processes. As such, AF has been used to study various pathological features on tissue sections. The source of pancreatic AF is multifaceted and may originate from different compounds or molecules such as NADPH, laminins, collagens, elastin, porfyrin and lipofuscines (for review see e.g.. An important feature of naturally occurring AF is that different compounds have emission profiles that may enable channel separation of tissues and/or proteins. For example, collagens and elastins contribute to blood vessel structures and are strong sources of AF. Lipofuscin is a compound pigment formed as the result of lysosomal digestion that contain lipids, sugars and proteins. It has strong AF properties and is found in a variety of tissues including pancreatic islets. AF from lipofuscin bodies has moreover been used to study the longevity of islets in pancreatic tissue from diseased donors. Detection of specific AF properties has also been suggested as a means for cancer screening of various tissues, but not for assessing tumour spread in intact surgical biopsies of human pancreas.
Over the past decades, no improvement in survival has been achieved for PDAC and the incidence of this cancer form is increasing. Curative treatment can only be achieved through radical surgery combined with adjuvant chemotherapy. A key issue in this regard is obtaining tumour-free resection margins, which are normally determined by histopathological analyses on resected tissue sections. PDACs grow within a dense tumour stroma and cancer cells are spread at relatively large distances within this matrix. The extent of the tumour is therefore often underestimated leading to false negative resection margins. Using OPT and LSFM imaging, we here present protocols that facilitate assessments of tumor characteristics and extent without adversely affecting routine histopathological analyses, which instead could be guided by these protocols. Using endogenous tissue AF to obtain tissue contrast, the whole protocol including image display and analysis can be performed within hours. As such it holds potential to become a valuable addition to today´s toolbox of histopathological techniques for identification and characterization of pancreatic cancers and for determination of islet mass distributions.
# Materials and methods
The study was conducted in accordance with the Helsinki declaration of 1975 and approved by the ethical committee of Northern Sweden (2016/384-31, 2019-04593 and 09-175M/2009-1378-31). All patients signed informed consent for tissues to be collected. Pancreatic cancer tissue was collected during pancreatic cancer surgery from patients admitted to the Department of Surgery at Umeå University Hospital, Sweden or from diseased donors within the framework for the Nordic Network for Clinical Islet Transplantation (NNCIT).
## Samples.
Biopsies from the displayed tumour specimens were collected at surgery and fixed in formalin. The biopsy displayed inwas collected from a patient with histopathologically verified PDAC and the biopsy displayed inwas collected from a patient with intraductal papillary mucinous neoplasia (IPMN) without evidence of invasive cancer. Tissue biopsies for assessments of islet mass distribution were isolated from formalin fixed pancreata post-mortem ( Sample preparation for OPT or LSFM imaging. Approximate time schedules for the described "fast"
and "optimal quality" imaging protocols, are displayed in . We recommend using the fast track protocol for smaller (approx. 1 × 1 × 0.5 cm) samples, and samples that require rapid screening. For larger specimen and samples that bear a lot of membranes or adipose tissue, we recommend using the optimal quality protocol to ensure adequate contrast and transparency for post-imaging analysis.
FAST TRACK AF protocol. Specimen are fixed in 4% w/vol Paraformaldehyde (PFA, Sigma Aldrich 30525-89-4) at 4 degrees Celsius over a period of 2 h and 30 min. They are then transferred to 5 mL 30% Hydrogen Peroxide vol/vol H2O2 (Merck 7722-84-1) for 30 min at room temperature on rotation in the dark. Samples are afterwards dehydrated in 15 mL Methanol (MeOH, Fisher Chemical 67-56-1) for 30 min, changing solvent every 10 min, and quickly cleared in BABB (Benzyl Alcohol: Benzyl Benzoate = 1:2, CAS 100-51-6 Merck, 120-51-4 Acros Organics respectively) for 1 h and 30 min. During this period the solvent is changed every 30 min. Throughout these two steps samples are rotating at room temperature.
OPTIMAL QUALITY AF protocol. Specimen are fixed in 4% w/vol PFA at 4 degrees Celsius over a period of 2 h and 30 min. They are then dehydrated stepwise in Methanol/PBS1X 33-66-100%, with each step lasting 20 min at RT. Samples are then bleached in 10 ml fresh bleaching solution (H2O2 : MeOH : DMSO (Dimethyl sulfoxide 67-68-5, Merck) = 3:2:1) at room temperature in the dark, for 24 h, changing the bleaching solution to a fresh one overnight. Samples are then rehydrated in TBST/MeOH 33-66-100% at room temperature. During dehydration and dehydration samples are kept on rotation. Samples are then mounted in 1.5% w/vol Low melting point SeaPlaque Agarose (39346-81-1 Lonza) as previously described, dehydrated in MeOH for 30 min and cleared in BABB as described above. Samples are subsequently scanned by OPT or LSFM.
Pancreatic AF based optical projection tomography (OPT) imaging. OPT scanning was performed using an in house built Near Infrared-OPT setup as describedPancreatic AF based light sheet fluorescence microscopy (LSFM) imaging. Specimen that were OPT processed and imaged (see above), were reimaged by a LaVision biotech 2nd generation UltraMicroscope II (LaVision BioTec GmbH, Germany). The samples mounted in agarose were either trimmed in BABB (see sample preparation) to fit into the UltraMicroscope II sample holder or glued onto the sample holder before image acquisition. Samples displayed in. Tile scans with 20% overlap along the longitudinal y axis were obtained. In general, exposure time was 582-843 ms with a light sheet width of 40-50%, 3.87 µm thickness and 0.14 NA using a z-step of 4-5 µm at 0.80 X magnification.
## Post-image processing and image analysis. for post-opt processing (detailed information see sup-
plementary Dataset 1), ranges of pixel values of subsequent OPT image data were cut to increase signal-to-background ratio, a contrast limited adaptive histogram equalization (CLAHE)algorithm with a tile size of 32 × 32 and a post-acquisition misalignment detection and correction using Discrete Fourier Transform Alignment (DFTA)was applied. The processed frontal projections were then reconstructed to tomographic sections using the NRecon v1.6.9.18 software (Skyscan, Belgium). Tomographic OPT sections and resultant LSFM image data was converted, visualized and analyzed using Imaris File Converter and Imaris 9.5. (Bitplane, UK). Using the automatic surface algorithm in Imaris for the lowest wavelength of autofluorescence the anatomy was segmented, and the displayed texture was adjusted to make the segmented anatomy "transparent". The same automatic surface algorithm with different creation parameters was applied to segment out vessel/duct like tubular structures. For the signal intensity spot analysis (Video S2) the Imaris automatic spot algorithm was applied to the maximum intensity projection (MIP) data and statistically color coded based on the sum of intensities for the investigated channel, OPT; Ex: 425/60 nm, Em: 480 nm LP and for LSFM; Ex: 470/40 nm, Em: 525/50 nm. For OPT-base islet visualization and quantification (Figs. 3f and 5 and 6), the near infrared imaged OPT samples were baseline subtracted and automatically 3D surfaced with a voxel number filter. Artefacts and tiny hairs were removed for quantification and overall statistics were exported to excel file format. The average . Protocols for label free, AF based imaging of pancreatic tissue biopsies. Schematic outlining two alternative protocols for AF based imaging of pancreatic tissue biopsies (see methods for details). The fast track protocol (a) enables 3D analyses of AF features, including generation of full 3D and tomographic data, within in less than 7 h from that the sample is received. In the optimal quality track (b) we attempted to optimize every parameter of the protocol for imaging scenarios for which time is not a critical factor.
# Results
A protocol for AF based optical 3D assessments of pancreatic tissue. By scanning optically cleared pancreatic specimen in emission spectra ranging from 425 to 680 nm, we initially assessed which parts of the spectrum provide channel separation between recognisable features in PDAC and normal pancreatic tissue . When implementing either a "fast track" protocol that could be used in conjunction with clinical investigations, or a protocol in which every parameter of the tissue and image acquisition process was optimized (see "Materials and methods" section and AF based assessments of tumour microenvironment. As exemplified in, a PDAC tissue sample revealed noticeable tubular features as well as areas of higher and lower intensity, when subjected to tissue clearing and OPT and LSFM imaging (see also. Dual modality imaging of the same specimen with OPT and LSFM revealed similar features, although LSFM produces higher resolution images albeit at the expense of significantly longer scanning times and non iso-tropic voxels (see alsoand Videos S1, and S2). At the implemented wavelengths vascular structures appeared as prominent features in the tomographic data sets, allowing individual vessels and their trajectories to be chartered in interactive 3D data sets of the biopsiesand Video S3). PDACs are generally characterised as scarcely vascularized, a feature believed to cause high interstitial tissue pressure and poor systemic treatments effects. In contrast to this, our sample was surprisingly well vascularised on 3D examination. The normal pancreatic parenchyma displays a considerably higher AF signal intensity than the PDAC tissue. When applying a transparency filter on the iso-surfaced dataset in the Imaris software (see online methods and Supplementary Dataset 1), this low intensity AF region could be visualized in 3D within the entire volume of the biopsyand Video S4). The PDAC area is characterized by abundant stroma and extracellular matrix structures seen as a grey mesh in the transparent anatomy(compare. The approximate anatomical outline of the tissue could further be visualized in 3D signal intensity hot-spot histograms, based on the tissue AF signal intensity (Video S4). The outline based on AF closely resembled the outline of the cancer regions as determined by HTEX and Ck18 staining at each level (compare, whereas the endogenous tissue fluorescence from vascular/tubular structures partly overlapped with aSMA (compare. Based on the 2D histopathological information (e.g. HTEX), it is possible to localize the corresponding PDAC regions in the LSFM or OPT data sets. PDAC development is preceded by premalignant lesions that include pancreatic intraepithelial neoplasia (PanINs) and intraductal papillary mucinous neoplasms (IPMNs). By segmentation of the AF signal, PanIN regions within the tissue appears to exhibit 3D tubular structures with connecting branchesand Video S5).
When applying the proposed imaging schemes to an IPMN cyst, vessel trajectories could be followed similarly to the PDAC specimen. These surrounded the spherical cyst, which in itself did not produce an AF signal. Interestingly, when detecting AF in the near infrared (NIR) spectrum (Ex: 665/45 nm Em: 725/50 nm for OPT and Ex: 650/45 nm Em: 750/60 nm for LSFM), islets of Langerhans could easily be segmented,f, see also Figs. 5 and 6). In HTEX staining of the sectioned lesion, the vascular www.nature.com/scientificreports/ and endocrine components could be directly matched with tomographic slices. Whereas the core of the cyst did not display an AF signal in the investigated spectra, immunohistochemistry revealed a hollow structure with an epithelial lining and a relatively thick cyst wall, filled with mucin. Similar results were obtained by LSFM analyses (Video S7).
## Af based visualisation and quantification of the islets of langerhans. quantification of islets in
larger human pancreatic tissue volumes is challenging and most commonly involves extrapolation of 2D data resulting from labour-consuming stereological assessments. As noted, when analysing the surgical specimens, detection and segmentation of islets within pancreatic tissue is feasible based on their AF signal, which was confirmed by analyses of normal pancreas tissue obtained from deceased donors. Hence, by applying a NIR filter set in the OPT scanner, the endogenous fluorescence from the islets was clearly visualized (see-c,d-f, and Video S8). Similar results were obtained for LSFM imaging. The AF signal was determined to be islet specific in this part of the spectrum, by comparing it to insulin antibody stained tissue sections. Implementing our previously developed OPT post-processing routines (see Material and Methods) islet volumes could be segmented, and the islet numbers and their individual volumes calculated. Notwithstanding the distinct origin of OPT-analysed material (see Supplementary Dataset 1) and the relatively low AF signal intensity, the obtained number of islets and their distribution characteristics are well in line with comprehensive stereological assessments . Taken together, endogenous islet AF may be used to assess islet mass distribution in unprecedently large tissue biopsies of the human pancreas. www.nature.com/scientificreports/
# Discussion
Although mesoscopic imaging approaches has been demonstrated as useful modalities for pathological assessments of human tissues, these have largely relied on staining's of the investigated samples. For example, Glaser et al., demonstrated how an optimized light sheet microscope could be used for non-destructive pathological assessments of (non-pancreatic) clinical specimens stained by fluorescence emitting dye 29 , whereas Nojima et al., demonstrated the utility of LSFM in histopathological analyses of a wide range of antibody stained human tissues. In this short report, we demonstrate the utility of mesoscopic imaging of AF features in optically cleared pancreatic tissue specimens to assess pancreatic anatomy/pathology without the need for prior cell/ tissue labelling schemes. We propose that the method could become a useful complement to current routine histopathology in a wide range of pancreatic aberrations, since data can be obtained in a short time without adversely affecting tissue morphology for subsequent immunohistochemistry (see Figs. 2h-j and 3e,h,j). Notwithstanding the limited sample number, the demonstrated possibility to visualize the PDAC morphology in 3D solely based on AF properties of the tissue may have significant implications. Most importantly the developed imaging schemes may develop into a clinically useful tool for analyses of specific regions of interest to facilitate delineation of free resection margins after cancer surgery. In addition, an improved general understanding of 3D tumour morphology at the current resolution could be translated to better evaluation of non-invasive imaging approaches. Further, the possibility to study the individual vessel trajectories in relation to tumour volume may www.nature.com/scientificreports/ be highly useful to increase our understanding of PDAC vascularization and how to overcome problems related to drug delivery in these cancers. Although it has been described that islets grafted to the anterior chamber of the eye display AF properties up to 600 nm in vivo 31 , the high signal to noise ratio between islet AF and surrounding pancreatic tissue in the red to the NIR part of the spectrum has to the best of our knowledge not been described previously. In our study, we observed droplet like regions of islet AF over a broad range of the spectrum (excitation maxima tested between 480 and 710 nm). A plausible source candidate for this broad fluorescence profile are lipofuscin-like lipopigments, which are present in highly secretory endocrine cells. Notably, these photoinduced fluorescent granules provided sufficient signal to noise ratio for quantification of islet volumes with 3D imaging tools throughout the investigated tissue in the NIR spectrum, which was challenging at lower wavelengths. Large scale analyses of islet mass distributions in material from deceased diabetic donors (be it type 1 or type 2 diabetes) may contribute to a better understanding of the relationship between remaining islet mass and disease development. Despite the striking resemblance with stereological data from previous studies, the possibility to evaluate the accuracy of these analysis remains limited for large tissue volumes until reliable whole mount immunolabelling protocols for specimen on the current scale are developed.
Since the imaging technology required to perform the described analyses is either commercially available (LSFM) or built to a relatively low cost 32-34 (see also www.mesos pim.org), it should be possible to establish the www.nature.com/scientificreports/ proposed analysis schemes as a complement to routine procedures in most pathology laboratories. Given the dramatic development in tissue clearing procedures during the past decade (for review see Matryba et al., it is possible that these procedures can be further refined, facilitating studies of larger tissue samples with even shorter processing times and increased quality. Finally, the described protocols could be directly translated to study other organs and tissues, depending on their AF properties. Our preliminary data suggest that structures such as nerves, striated musculature and vessels may be studied also in other tissues without prior labelling schemes implementing the outlined procedures. In summary, the developed protocols enable a fast method to assess different anatomical structures such as; vessels, ducts and tumour delineation within mesoscopic-sized pancreatic samples without the need for prior labelling schemes and without negatively affecting histology or subsequent immunohistochemical assays.
## Data availability
Raw and processed imaging datasets acquired by NIR-OPT and LSFM on all samples displayed are available upon reasonable request. The software used for OPT image processing are available from the authors upon request subject to an MTA.
Received: 28 July 2020; Accepted: 5 October 2020 . OPT data of islet volume and number distributions correlate with stereological assessments. (a) Graphs displaying islet mass distribution from OPT measurements (displayed as islet diameters calculated from average length of the individual islets x, y and z axis, green) into size categories of a total of 7034 islets (n = 4 biopsies), compared to stereological assessments as described by Hellman et al., (red). (b) The same stereological data as in (a) but from individual pancreata. (c) The same OPT-data as in (a) but from individual biopsies. (d) The same data as in (a) but displayed as the number of islets falling within each size category. (e) The same stereological data as in (d) but from individual pancreata. (f) The same OPT-data as in (a) but from individual biopsies. Notwithstanding that the OPT material was collected from different regions of the pancreata (see methods), it is well in line with previous stereological assessments. The data in (a,d) are presented as mean ± SEM. www.nature.com/scientificreports/ |
Full-field optical coherence tomography for the diagnosis of giant cell arteritis
Histopathological examination of temporal artery biopsy (TAB) remains the gold standard for the diagnosis of giant cell arteritis (GCA) but is associated with essential limitations that emphasize the need for an upgraded pathological process. This study pioneered the use of full-field optical coherence tomography (FF-OCT) for rapid and automated on-site pathological diagnosis of GCA. Sixteen TABs (12 negative and 4 positive for GCA) were selected according to major histopathological criteria of GCA following hematoxylin-eosin-saffronstaining for subsequent acquisition with FF-OCT to compare structural modifications of the artery cell wall and thickness of each tunica. Gabor filtering of FF-OCT images was then used to compute TAB orientation maps and validate a potential automated analysis of TAB sections. FF-OCT allowed both qualitative and quantitative visualization of the main structures of the temporal artery wall, from the internal elastic lamina to the vasa vasorum and red blood cells, unveiling a significant correlation with conventional histology. FF-OCT imaging of GCA TABs revealed destruction of the media with distinct remodeling of the whole arterial wall into a denser reticular fibrous neo-intima, which is distinctive of GCA pathogenesis and accessible through automated Gabor filtering. Rapid on-site FF-OCT TAB acquisition makes it possible to identify some characteristic pathological lesions of GCA within a few minutes, paving the way for potential machine intelligence-based or even non-invasive diagnosis of GCA.
# Introduction
Giant cell arteritis (GCA) is a large vessel vasculitis that mainly affects the aorta and the branches of the external carotid, with a predilection for the temporal arteries. Even though we now have an accurate understanding of its complex pathogenesis, the causative agent of GCA is still unknown. Mostly occurring in northern European females between 50 and 80 years old, the predominant cranial phenotype is usually revealed by new-onset headache, temporal artery tenderness, jaw claudication, and partial or complete visual loss associated with possible systemic symptoms, notably fever, weight loss and weakness. The critical complications of GCA include anterior ischemic optic neuropathy, stroke, aortic aneurysm or dissection; these serious complications being responsible for the prognosis of the disease and the need for prolonged high-dose glucocorticoid treatment.
The diagnosis of GCA usually relies on the association of concurrent clinical, biological and pathological features of vasculitis that are revealed by temporal artery biopsy (TAB). Significant advances in the field of medical imaging have improved the assessment of the extent of vasculitis and refined non-invasive diagnosis and follow-up. For instance, the validity of hypoechoic thickening surrounding the temporal artery wall with color duplex sonography (CDS), also referred to as the halo sign, was confirmed at least three times in a meta-analysis for the diagnosis and follow-up of GCA. However, the combination of intense infiltration of mononuclear cells in the three layers of the artery, fragmentation of the internal elastic lamina (IEL), intimal hyperplasia and neoangiogenesis on TAB histological examination undoubtedly remains the gold standard for GCA diagnosis in all study group guidelines .
Apart from rare local complications, TAB is a safe procedure. Nevertheless, the segmental and focal nature of transmural inflammation in GCA generates skip lesionsand is responsible for a significant false-negative rate of up to 30%that makes it necessary to either increase biopsy lengthor to perform a contralateral TAB. These limitations emphasize the potential interest and need for an upgraded pathological procedure dedicated to the diagnosis of GCA.
Based upon an optimization of the technology described by Fujimoto and colleagues in the early 1990s, full-field optical coherence tomography (FF-OCT) exploits en face whitelight interference microscopy to provide not only ultra-high resolution images of biological structuresbut also subcellular metabolic contrast in the tissue depth. When compared to other modalities such as conventional OCT or even confocal microscopy, FF-OCT was demonstrated to significantly improve spatial resolution by a factor varying from five to ten depending on the acquisition axis. Until now, most groups have focused on the potential role of FF-OCT during oncologic interventions as new routine approach to surgical pathology, and, except for one preliminary study in which the superficial temporal arteries were imaged with dermal OCT, there has been no reported attempt to employ high definition interference microscopy for the pathological diagnosis of GCA. The present work pursues the hypothesis that FF-OCT could help both the clinician and pathologist to improve TAB performance, and compares, for the first time, FF-OCT and conventional histological examination for the pathological diagnosis of GCA.
# Materials and methods
# Ethics statement
Patients included in this study participated in two studies involving GCA patients (Clinicaltrials.gov NCT02158208 and NCT02857192) and gave both oral and written informed consent for the use of their temporal arteries for subsequent research in the field of GCA. This study was approved by the Institutional Review Board and the Ethics Committee of the Dijon University Hospital.
## Preparation of tab sections
All patients suspected of GCA and scheduled for TAB surgery at the Dijon University Hospital Ophthalmology department from January 2013 to December 2016 were included. TAB was performed according to standard procedure, and fresh biopsies were sent to the pathology department. A ten millimeter segment was used for conventional hematoxylin-eosin-saffron (HES) staining, and the other part of the artery segment was immediately frozen at -196˚C in fetal bovine serum and dimethyl sulfoxide (10%). The day of FF-OCT imaging, samples were slowly defrosted. The surrounding tissue was removed, and transversal 1 mm-thick sections were cut with a triangular-bladed scalped and placed in complete RPMI culture medium before placement on the sample holder for image acquisition.
## Histological tab selection
A total of sixteen TABs were selected for subsequent analysis with optical coherence microscopy. Twelve negative TABs were identified according to the absence of mononuclear cell infiltrate, IEL fragmentation or neoangiogenesis and defined as the control TABs. In these control samples, the pathologist studied the qualitative aspect of the temporal artery wall to distinguish between negative TAB with normal intima (thinner than media, referred to as niTAB.1 to 9, n = 9) and negative TAB with intimal hyperplasia (thicker than media, referred to as ihTAB.1 to 3, n = 3). These control TABs were compared to four specimens that met the major histopathological criteria for the diagnosis of GCA (referred to as gcaTAB.1 to 4, n = 4).
## Ff-oct imaging
FF-OCT images were acquired with a commercially available FF-OCT apparatus (Light-CTScanner, LLTech SAS, Paris, France). Briefly, illumination was provided by a LED source with short coherence length ensuring a sectioning ability or axial resolution of 1 μm. In the FF-OCT set-up, 10x microscope objectives are placed in the interferometer arms in the Linnik configuration, bringing a transverse resolution of 1.5 μm. Following full-field illumination of the axial TAB section, FF-OCT images were captured with a complementary metal oxide semiconductor camera. The theoretical penetration depth for the TAB specimen was approximately 200 μm. The TAB section was placed in the dedicated sample holder with its revolution axis perpendicular to the imaging plane so that one FF-OCT slice showed the architecture of the TAB section from the lumen to the outer wall. A series of FF-OCT slices with 1.5 μm spacing were recorded in depth, and ImageJ 1.52o software was used for axial reconstruction of TAB FF-OCT imaging following z-stacking of a minimum of 20 images.
## Image and statistical analysis
Quantitative FF-OCT image analysis and tunica thickness were accessible with a contrastbased ImageJ 1.52o protocol (Plot Profile Function) and calculated as the mean of three representative measurements throughout each TAB section. NDP.view software version 2.6.17, provided by Hamamatsu, allowed similar measurements from scanned glass slides following HES staining. Statistics were calculated using GraphPad Prism version 5. For intima-to-media ratios (I/M), values reported as medians and interquartile ranges were discriminated by Mann-Whitney tests. The Pearson r coefficient was calculated to evaluate the strength of the linear correlation between histology and FF-OCT measurements of media or intima thicknesses. Interval two-tailed P < 0.05 was considered statistically significant. Finally, orientation maps were calculated for a selection of both healthy and GCA-positive TABs following Gabor filtering of the axial reconstructed images with a custom-made software based on Matlab 2018b (Matworks, Natick, MA).
# Results
## Qualitative ff-oct imaging
Similar to histological preparation, TAB sections acquired with FF-OCT allow the identification of several important structures within the artery wall. Notably, the tripartite architecture is perceived with a clear separation between intima, media and adventitia. Interestingly, the physical junction between the intima and media appears as a thin hyporeflective serpentine band that most obviously corresponds to the IEL. Moreover, inobtained with FF-OCT, the vasa vasorum can be seen distinctly within the arterial wall and the red blood cells can be identified precisely, returning a spherical contrast highly similar to the one obtained with conventional histology. Indeed, the vasa vasorum display similar architecture with both techniques, revealing small (20 to 80 μm) blood vessels characterized by thin elastic walls and a round to oval shape directly inserted into the outlying thread of the temporal artery (i.e. mostly between the media and the adventitia layers), white arrow shows arterial thrombi). Magnification of the lumen of the vasa vasorum makes it possible to observe the red blood cells. These cells resemble partially transparent pink-colored ovoid structures following HES staining or a collection of iso-reflective dots with a surrounding hypo-reflective annulus on direct FF-OCT acquisition.
FF-OCT acquisition and histological images from negative TAB samples are compared indisplay a representative negative TAB specimen with a thin intimal layer (niTAB).show a negative TAB with intimal hyperplasia (hiTAB). No matter the group of negative TAB, the overall architecture of the vessels is preserved, and there is a clear distinction between intima, media and adventitia. Indeed, the tunica media displays a relative hyper-reflectivity on the image acquired with FF-OCT when compared with the tunica intima, whose thin muscle fibers mostly run parallel to the global circular orientation of the TAB section (see magnified region from. Similar conclusions regarding the differential contrast and circular symmetry within the two inner layers of the arterial wall separated by the IEL, which appears as a hypo-reflective strip in FF-OCT, can be drawn from the analysis of all negative TAB specimens (S1 and S2 Figs). Inwe can see that the tunica adventitia is constructed on a denser and more complex fibrous connective tissue that also seems to follow the overall circular symmetry of the system. When compared withthe FF-OCT-acquired TAB section from Fig 2C is characterized by an increased intima thickness (see magnified region). These observations can mostly be transposed for the comparison of all hiTAB specimens detailed in S1 and S2 Figs. The results obtained with FF-OCT analysis largely correlate with the data obtained after conventional histology. Indeed, the intima in Fig 2B appears much thinner than inwhich the intima is thicker than the media.
InTABs are positive for GCA. The conventional histopathological images show a relatively preserved media that is strongly infiltrated by T-cells, macrophages and multinucleated cells (see the magnified region from. By contrast, FF-OCT acquisition demonstrates a complete disruption of both regular reflectivity and circularity of the media and intima-associated connective tissue fibers due to the infiltration of inflammatory cells. This process remodels the structure of the artery into a denser, reticular, fibrous, collagen-rich structure responsible for both the progressive destruction of the media and the formation of a neo-intima (see the magnified region from.obtained with FF-OCT, confirms the fragmentation of the internal elastic lamina along with a rebalancing of the contrasts throughout the netlike fibrous structure connecting all three layers. Similar to the corresponding image obtained with conventional histology, there is no clear distinction between the intima and the media, which is consistent with the stage of the disease. The
## Quantitative ff-oct imaging
Given that FF-OCT images provide good quality spatial resolution, we hypothesized that proper image analysis could return quantitative information regarding both the thickness of the artery wall layers and the global architecture of the underlying connective tissue.show the main aspects of contrast-based ImageJ protocol along a linear profile drawn across the arterial wall of a negative TAB section. The protocol was designed to access the most precise measurements for each tunica of the vessel. The gray-scale plot profile fromconfirms a significant rupture in contrast between the intima and media, as well as between the media and adventitia, allowing concomitant measurements of the thickness of each artery wall layer. Software provided by Hamamatsu facilitated similar measurements from scanned glass slides following HES staining, as exploited elsewhere. In addition, Gabor filtering was applied to the same reconstructed negative TAB section in order to provide vector orientation maps and subsequent global analysis of the symmetry of the arterial section. As expected from the previous qualitative analysis, Gabor filtering of FF-OCTacquired negative TAB section returned a perfect orientation match from one point of the artery to its exact opposite following a 180-degree rotation, as demonstrated by the paired color system respecting an overall 180-degree rotational symmetry. A similar procedure was applied for the analysis of the gray-scale plot profile from positive TAB sections. Due to a high heterogeneous contrast within the whole TAB section of GCA patients,shows almost no possible distinction between the different layers composing the artery wall with a contrast oscillating between 500 and 2000 arbitrary units from the very inner to the outer layer. Subsequent Gabor filtering of the positive section proves the pathological loss of the 180 degree rotational symmetry-based vector orientation match, as illustrated by the relative chaotic color distribution within the layers of the artery resulting in a rainbow-like appearance.an example of a negative TAB for which FF-OCT images and conventional histology match perfectly from the inner to the outer layer of the biopsy. Regardless of intima thickness or GCA status (when quantifiable for GCA patients), quantitative analysis of both intima and media thickness confirms the absence of statistical difference between FF-OCTbased and histology-based measurements of I/M. Moreover, these results let to an accurate association of both quantitative classification and qualitative selection established by the pathologist for negative sections. Indeed, TABs with thin intima (S1 TABs with intimal hyperplasiaappear as two separate groups: one with normal I/M <1 and another that shows an intimal hyperplastic response with I/M between 1 and 2. When data were accessible for TABs with GCA lesions, quantification brought out a third GCA group defined by I/M largely > 2. Finally, data fromdemonstrate a significant correlation between the thickness of the intimaand mediameasured with FF-OCT and conventional histology.
# Discussion
The present work describes the first attempt to assess the potential of FF-OCT for the diagnosis of GCA in comparison with conventional histology. The first advantage of FF-OCT is that it provides rapid (within minutes) and on-site acquisition of TAB sections. We demonstrate, from the analysis of both healthy and GCA-positive TAB sections, that the high spatial resolution of FF-OCT technology makes it possible to visualize with precision several essential structures correlated with the diagnosis of GCA. Notably, we found that FF-OCT accurately returns both qualitative and quantitative information relative to the structure of the three arterial tissue layers and the IEL or vasa vasorum, with a significant correlation to histopathological imaging. When focusing on the FF-OCT analysis of healthy TAB sections, the inverted I/M can be interpreted as a reflection of the stage in human atherosclerosis. Moreover, we provide preliminary proof that automated Gabor filtering could deliver both immediate and essential structural information regarding the preservation of the regular circularity of the media and intima-associated connective tissues, paving the way for potential machine intelligence-based pathological diagnosis of GCA. FF-OCT acquisitions return additional and complementary information with focus on the appearance and structural orientation of the underlying fibrous supporting tissue within each layer of the temporal artery. When TABs from GCA patients were compared to the global circular symmetry of healthy TAB sections, FF-OCT imaging revealed the destruction of the media layer and the modification of the arterial wall structure, which was rearranged into a denser reticular fibrous neo-intima, distinctive of GCA pathogenesis. Despite the current success of non-invasive techniques like CDS, a precise FF-OCT-based analysis of the temporal artery wall on a meso-structural level remains of particular importance for the diagnosis of GCA. There is, however, a potential pitfall for GCA diagnosis with CDS since the atherosclerotic lesions responsible for significant increases in the thickness of the intima might mimic the halo sign, resulting in false positives.
We acknowledge several limitations that had an impact on the use of FF-OCT for rapid onsite pathological diagnosis of GCA in the current study. First, T-cells, macrophages and multinucleated cells, which are hallmarks of GCA, were not visible in the present set-up of FF-OCT, which used previously frozen TAB samples. Despite the high spatial resolution, the loss of information was due to the structural nature of contrast imaging, rendering direct black and white photographs of the specimen without any preparation or staining. However, this limitation is for the most part the result of using defrosted TAB samples with dead cellular material. This issue can be overcome by performing dynamic FF-OCT acquisition of fresh TAB sections, yielding complementary subcellular contrastand putative direct visualization of inflammatory infiltrates. Moreover, FF-OCT-based image analysis allows z-stacking of a varying number of slices up to a cumulative length approaching 200 μm, highly dependent on the overall quality and sharpness of the initial TAB axial section, that may potentially improve diagnosis accuracy by unveiling skip lesions. Such hypothesis would require additional experiments with a dedicated reproducible sampling technique allowing concomitant and iterative acquisition of the same slice with both FF-OCT and conventional histology throughout the whole length TAB section. Unfortunately, such experimental conditions remains to be found. Finally, the potential of en face white-light interference microscopy demonstrated in this work should encourage further investigations into the FF-OCT-based handheld acquisition probe, a promising technology dedicated to direct transcutaneous imaging and further noninvasive diagnosis of GCA.
# Conclusion
This preliminary study is the first to compare FF-OCT imaging to the gold standard histopathological procedure for the diagnosis of GCA. It brings conclusive proof regarding the potential of FF-OCT for both qualitative and quantitative structural visualization of underlying fibrous tissues and architectural changes in the arterial wall that occur throughout the inflammatory processes of GCA. After this first promising step, further investigations are warranted to confirm the potential of FF-OCT technology for rapid, on-site, non-invasive diagnosis of GCA.
## Supporting information
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Isokinetic and Isometric Assessment of the Knee Joint Extensors and Flexors of Professional Volleyball Players
Citation: Wilkosz, P.; Kabacinski, J.; Mackala, K.; Murawa, M.; Ostarello, J.; Rzepnicka, A.; Szczesny, L.; Fryzowicz, A.; Maczynski, J.; Dworak, L.B. Isokinetic and Isometric Assessment of the Knee Joint Extensors and Flexors of Professional Volleyball Players. Int. J. Environ. Res.
# Introduction
Volleyball is a non-contact team sport that requires players to alternately perform high-intensity actions involving passing a ball and scoring points against the opposing team on their court. The ball-passing actions also involve jumping activities that are performed near the net (e.g., sets, spikes, and blocks) and a jumping serve that is executed at one end of the court. These movement structures are critical for executing such actions and have a great impact on the final game performance. Additional factors for success in the game of volleyball are the quality of the jumps performed (vertical jump height), the technique of passing the ball, and the players' anthropometry (height of the body). Moreover, the repetitive loading of the joints from the jumping and landing actions-often executed more than 200 times in one match-exposes players to a high likelihood of injury. A characteristic of this jumping and landing is an extreme impact overload that exceeds a player's body mass by several times.
Knowing how many different jumps volleyball players perform during a game can provide new insight into the quality of their specific strength requirements. It can help design appropriate volleyball training and, most of all, monitor and control the improvement of training results. Current technological advances have made it possible to control the level of muscle strength, especially the force ratio between the knee flexors and extensors, which helps in the assessment of aspects of the quality of the jumps and thus in determining the load on the knee joint. Additionally, the second factor mentioned above can lead to different types of injuries. Statistics show that in volleyball, 60% of injuries occur during training sessions and 40% occur during competitions. Among the participants in this sport, injuries happen most frequently in the following joints: knees, ankles, shoulders, lumbar spine, and small joints of the hand. Therefore, the disturbing force ratio between the knee flexors and extensors could cause an accumulation of microtraumas and then the spread of chronic excessive trauma, such as patellar tendinopathy.
Maintaining the ability to perform jumps with a qualitatively explosive force-which is defined as the ability of the neuromuscular system of an individual to manifest the load in the shortest possible time-is considered one of the priorities in volleyball training. Therefore, according to McNitt-Gray, landing from an increased height increases the mechanical demand and eccentric force of the impact, which directly increases the peak extensor joint moments in the ankle, knee, and hip. In turn, these requirements are related to the assessment of the level of a player's muscle strength, which can be performed under isokinetic and isometric conditions. In isokinetic testing, the resistance is adjusted to the strength of the individual while the angular velocity is fixed. This allows the measurement of the net torque of the knee extensors and flexors.
Dauty et al.,conducted tests on volleyball players on the French national team. The Cybex 6000 system was used to analyze the peak torque (PT) during concentric and eccentric work. The values achieved by the participants were slightly higher than the population standard for the hamstring/quadriceps ratio (H/Q)-0.66 vs. 0.60. These researchers suggested that under functional conditions, a better indicator of the balance between the knee flexors and extensors would be the ratio measured during the eccentric work of the flexors and the concentric work of the extensors. Bittencourt et al.carried out tests on representatives of Brazil's junior teams-aged 19 to 21-using the Biodex System 3. The strength H/Q ratio for this group was low-at the level of 0.51. Continuing this analysis, an interesting comparison of the strength of the knee joint flexors and extensors in volleyball players and soccer players was presented by Magalhaes et al.. They found that the PT and H/Q ratios were only significantly lower in volleyball players at 90 - /s and that their lower limbs' values were more symmetric (dominant vs. non-dominant) than those of soccer players. Another research article by Markou et al.analyzed flexion/extension strength in the lower limbs, as well as internal/external rotation strength in the upper extremities. They used Cybex II+ and found a significant asymmetry between dominant and non-dominant upper limb strength (PT), but not for the lower extremities. Furthermore, they stated that this phenomenon occurred especially among volleyball players who occupied offensive positions.
Moreover, isokinetic testing is a great source of information on muscle strength deficiencies, which is vital in determining the quality of jumps and the risk of joint injury.
Therefore, this study evaluated the level of muscle strength by using isokinetic and isometric measurements-more specifically, the force ratio between the knee flexors and extensors (the values of the torques). Additionally, in order to evaluate the dynamic stabilization of the knee, the strength indexes of these muscles were assessed.
# Materials and methods
## Study design
This study was a laboratory experiment with a designated division into groups. The strength measurements were performed in two independent sessions during the mid-point of the volleyball season of the Polish League (February). The first session included the measurements of the concentric isokinetic torque of the hamstrings and quadriceps. In addition, on the day before the force measurements, height and body mass were measured. During the second session (2nd day), the isometric muscle strength of the knee extensors (KEs) that developed in a closed kinetic chain (CKC) was examined. The participants were instructed regarding the procedures of the isokinetic and isometric tests and several repetitions were performed for familiarization.
## Subjects
Fourteen elite male volleyball players in the division-one Polish Volleyball League (KS Poznan) and 14 male physical education students participated in the experiment. All of the participants were healthy. The volleyball team consisted of nine players who played in offensive positions (two middle blockers, three receivers, and four attackers) and five players who played in defensive positions (three setters and two liberos). The volleyball players were qualified for league matches by the appropriate league medical services. The main division criterion was participation in a high level of volleyball performance (at least five years of competitive experience: 7.5 ± 1.04 years) for the players and non-practice in any sport for the control group (the students). An additional criterion was that all participants were free from any injuries in the lower extremities that could affect their strength performance at the time of testing.
# Ethics statement
The review board approved this experiment. The Bioethical Commission of the Poznan University of Medical Sciences (No 203/08) approved the study design. The procedures were in accordance with the Code of Ethics of the World Medical Association (Helsinki Declaration of 1964). Before signing the informed consent forms, the participants were informed about the experiment's aim and the risk of injury.
# Strength measurement procedures
The isokinetic testing was performed at the mid-point of the volleyball season by utilizing the Biodex System 3. In order to obtain the maximum precision and the highest reliability, the tests were conducted in the laboratory of the Department of Biomechanics at the Poznan University School of Physical Education. Before each series of tests, both groups performed a warm-up by exercising for 10 minutes on a MONARK Ergomedic 874E bicycle ergometer. Moreover, during the testing procedure, to maintain optimum readiness between tests, the participants used a Pliant Centauri Omega electric treadmill as a warmup device. Each test was administered in groups of 3 or 4 persons in order to provide adequate rest between trials. Three angular velocities (ω) were used for the isokinetic tests: 60, 180, and 300 - /s. The range of knee joint motion throughout each measurement was fixed at 90 - for all participants. The initial position in which the leg was fully extended was set as 0 - . Before every test, participants performed three sub-maximal flexion/extension cycles. The participants then repeated the flexion/extension cycle five times with the goal of generating the maximum torque. The participants performed with their upper limbs crossed and resting on their chest. In order to isolate the work of the muscle groups acting on the knee joint, the torso, pelvis, thigh, and lower leg were stabilized each time. Additionally, a static test was performed (the knee-bend angle was 90 - /s) to determine the maximum values of the moments of the forces generated by the examined muscle groups. Measurements of the left and right lower limbs were performed during both the static and isokinetic studies.
# Statistical analysis
Descriptive statistics (the mean and SD) were calculated for all dependent variables. The Shapiro-Wilk test was used to assess the conformity of the statistical distributions of the analyzed variables with a normal distribution. The Mann-Whitney U test for two independent samples and the Wilcoxon signed-rank test for two related samples were used to examine the differences between the groups. Effect sizes were evaluated by calculating Cohen's d with 95% confidence intervals. Cohen suggested that d + 0.2 be considered a small effect size, 0.5 be considered a medium effect size, and 0.8 be considered a large effect size. The statistical power was determined to be 0.90 at the 0.05 alpha levels.
# Results
The results of the experiments with the twenty-eight participants who were divided into two groups-fourteen volleyball players and fourteen physical education studentswere analyzed. The results of the Shapiro-Wilk test showed that the variables were not normally distributed (p < 0.05).
The somatic parameters within the experimental group and the control group-given as arithmetic means and standard deviations-had the following values, respectively: height, 195.7 (±6.2) and 178.7 (± 6.7) cm; mass, 87.8 (±6.9) and 73.4 (±8.9) kg; BMI, 22.9 (±1.3) kg/m 2 , which was similar in both groups. The average ages within the experimental group and the control group amounted, respectively, to 23.9 (±3.8) and 23.6 (±1.9) years. A Mann-Whitney U test revealed a significant difference (p < 0.05) between the somatic characteristics of the two groups. The volleyball players were taller and heavier than the participants from the control group by 8.7% and 16.4%, respectively. This can be explained by the fact that the volleyball players were a carefully selected group of males with respect to height. Accordingly, their body mass was also greater. However, the age and BMI of both groups were similar.
The results of the PT tests presented in indicate that there were statistically significant differences between the two groups in terms of the PT of the knee joint extensors and flexors (p < 0.05). One exception was for the PT of the extensors of the right limb at ω = 60 - /s. The volleyball players achieved higher PT values than the control group for all conditions. For the extensors, the differences ranged from 17.1% (right limb at ω = 60 - /s) to 26.2% (right limb at ω = 300 - /s), whereas in the flexor group, the difference ranged from 27.7% (right limb at ω = 180 - /s) to 32.0% (left limb at ω = 60 - /s). . Peak torque of the knee joint extensors and flexors obtained during isokinetic tests with concentric movements (mean ± s) and joint angles at which the peak torque was obtained during these tests (mean ± s).
## Parameter
## Experimental group
Control Group In terms of the PT, the dominant limb was the right one. Except for the ω = 60 - /s test in the volleyball players, the knee extensors in the right limb were slightly stronger, reaching a maximum difference of about 3%. also provides information about the values of the joint angle at which the PT was generated. For the experimental group, there was a statistically significant difference between the extensor muscles of the left and right limbs at ω = 180 - /s (p < 0.05). There were statistically significant differences between the two tested groups in terms of the joint angle at which the knee joint flexor PT was generated (p < 0.05), except for the right limb at ω = 180 - /s and at ω = 300 - /s. The analysis of the isokinetic measurements of the knee joint extensors in both groups shows that the angle at which the PT value was obtained fluctuated between of 56.0 - and 62.8 - , and the differences between the two groups ranged between 1.9% and 5.1%. The range of motion for all measurements was 90 - with a full extension equal to zero. The PT for the extensors for both the players and the control group occurred at an average knee joint angle of 59 - , or approximately 34.4% of the range of motion. In the case of the isokinetic measurements of the knee joint flexors, in both groups, the angle at which the PT value was achieved fluctuated within the range of 40.0 - to 86.6 - of the bending angle, or approximately 4.6% and 31.4% of the range. However, the differences were within a wider range of 4.6% to 31.4%.
[formula] ω = 60 - /s ω = 180 - /s ω = 300 - /s ω = 60 - /s ω = 180 - /s ω = 300 - /s L R L R L R L R L R L R [/formula]
The static test results are shown in. The analysis revealed that the PT values of the extensors (L, R) and flexors (L) were significantly higher in the experimental group than in the control group (p < 0.05). For the extensor muscle group, the control group produced 249.1 Nm of torque, while the athletes produced 315.1 Nm-an increase of 26%. For the flexor muscle group, the control group produced 112 Nm and the athletes produced 143.3 Nm-an increase of 30%. When corrected for body weight, however, the difference in favor of the athletes was only 6%.presents the relationship of PT values generated by extensors and flexors with the angular velocity of the knee joint, which was determined during isokinetic concentric movement tests and the static tests on the players. The higher the contraction velocity was, the lower the tension in the muscle was. The flexor group followed the same pattern, but it was not as pronounced. Unexpectedly, the PT of the flexors when tested statically was lower than the PT in the concentric isokinetic measurements at ω = 60 - /s. The analysis of the results fromshows that at ω = 60 - /s, there was a statistically significant difference in the H/Q ratio between the left and right limbs of the athletes (p < 0.05). In addition, the H/Q ratio of the players was higher than that of the control group under both conditions. At ω = 60 - /s, the H/Q ratio of the athletes was 17.3% higher than that of the control group, and it was 5% higher under the static conditions. The analysis of the results fromshows that at ω = 60 °/s, there was a statistically significant difference in the H/Q ratio between the left and right limbs of the athletes (p < 0.05). In addition, the H/Q ratio of the players was higher than that of the control group under both conditions. At ω = 60 °/s, the H/Q ratio of the athletes was 17.3% higher than that of the control group, and it was 5% higher under the static conditions.
# Discussion
The main objective of this study was to evaluate the level of muscle strength through isokinetic and isometric measurements-in particular, the force ratio between the knee flexors and extensors (values of the torques). This evaluation will help in the assessment of aspects of the quality of jumps and, thus, in the determination of the load on the volleyball players' knee and their injury prevention status. Therefore, to confirm the validity of the aim of this research, it seems interesting to conduct a discussion in two interrelated areas. One area concerns the training load as an interpretation of the improvement of a volleyball player's motor skills and game effectiveness, and the other area is the protection of volleyball players against injuries resulting from the application of the above-mentioned training load.
The correct assessment of the application of the training load in specialized volleyball training, which consists of many components, is mainly based on the level of strength and. Flexor/extensor ratio obtained from an isokinetic test with concentric movements (ω = 60 - /s) and a static test (α = 90 - ) (mean ± s).
## Parameter
## Experimental group
Control Group
[formula] ω = 60 - /s isom α = 90 - ω = 60 - /s isom α = 90 - L R L R L R L R F/E (%) x [/formula]
# Discussion
The main objective of this study was to evaluate the level of muscle strength through isokinetic and isometric measurements-in particular, the force ratio between the knee flexors and extensors (values of the torques). This evaluation will help in the assessment of aspects of the quality of jumps and, thus, in the determination of the load on the volleyball players' knee and their injury prevention status. Therefore, to confirm the validity of the aim of this research, it seems interesting to conduct a discussion in two interrelated areas. One area concerns the training load as an interpretation of the improvement of a volleyball player's motor skills and game effectiveness, and the other area is the protection of volleyball players against injuries resulting from the application of the above-mentioned training load.
The correct assessment of the application of the training load in specialized volleyball training, which consists of many components, is mainly based on the level of strength and technical skills. A certain level of strength is necessary in order to carry out training to improve the power of the lower limbs-plyometrics. The jumping skills combined with the technical skills of volleyball players guarantee high performance. Therefore, the assessment of the strength level concerns the muscles of the flexors and extensors of the knee joint, is mainly isokinetic, and is based on torque-angle representations (Alexandre et al.. Polish volleyball players achieved significantly higher isokinetic PTs than those of Greek players. The H/Q ratio in the current study was about 83% of the standard value reported by Coombs et al.. This was higher than that of the Brazilian junior team by 7.1% and lower than that of Greek volleyball players by 16.9%.
With the knee extensor muscle group, we observed that as angular velocity increased, the PT decreased. This result was in accordance with the theory of A.V. Hill, who stated that the higher the load applied to the muscle is, the lower the contraction velocity will be. Based on this statement, we found some similarities and differences in the static PT values and H/Q ratios that we measured with respect to data found in the literature. With respect to the right knee extensors and flexors, the Polish players produced 31% and 3% higher torque than the group studied by Janiak et al., as well as 9.5% and 32% higher torque than other athlete males of a similar age. The H/Q ratio calculated for the PT of the right limb in the static tests. The H/Q ratio calculated for the PT values of the right limbs (in the static tests) was 0.47 for the players in the current study and 0.34 for the more prestigious Polish national team. Compared to the data provided by Freedson et al., the volleyball players in the current study generated higher PT values in the knee flexors and extensors. The one exception was the PT of the flexors. That value was only slightly higher. Additionally, in the isokinetic tests conducted on the Greek volleyball team, players from Poland achieved 18.2% higher PT values for the knee extensors, but 1.4% lower values for flexors. However, the PT of the flexors of the examined players at the speed of 180 - /s was slightly lower (5.5%) than that of the French national volleyball team. In turn, Pelegrinelli et al.claimed that the isokinetic parameters of the PT, TW, and MP were significantly higher for PRO group volleyball players than for U17 group players in terms of knee extension and flexion (for the knee flexors, only at 120 - /s). Kabacinski et al.compared female volleyball players with basketball players. They showed that the significantly greater H/Q ratio at 60 Ê/s in the volleyball athletes indicated better utilization of the hamstring muscle group in volleyball-specific movements or a weakening of the quadriceps relative to the hamstrings in comparison with the basketball players. Dauty et al., in their research on volleyball players, obtained slightly higher results for the hamstring-to-quadriceps ratio (H/Q)-0.66 vs. 0.60.14-than that indicated by the population norm. They believed that a better indicator of the balance between the knee flexor and extensor in functional conditions would be the ratio measured during the eccentric work of the flexors and the concentric work of the extensors. In turn, Bittencourt et al.conducted tests on Brazilian junior teams and found that the H/Q strength ratio for this group was low at 0.51. In recent years, the successes of male and female Polish volleyball players have inspired the emergence of research projects aimed at biomechanical aspects of dynamic loads in volleyball-namely, traumatogenicity, prevention, and strength training, including nonspecific methods and assessment. Within the group of the 14 players that were tested, three experienced negative consequences of knee joint injuries. These injuries happened in the past, before the research began. The most serious among them were damage to the lateral meniscus and anterior cruciate ligament or patellar tendinopathy. Increased training volume and exposure to matches can significantly affect modifiable risk factors that are associated with the development of patellar tendinopathy. The data presented by Visnes and Bahrconfirm this assumption, as they showed that for each additional hour of training (odds ratio = 1.72) and a set of matches played per week (odds ratio = 3.88), there is a significantly increased risk of injury. This is mainly related to the increased load on the lower limbs due to the increased number of take-offs and landings from different heights after the implementation of specialized volleyball movement structures. According to McNitt-Gray, landing from an increased height increases the mechanical demand and the eccentric impact force, which, in turn, has been shown to increase the ankle, knee, and hip extensor moments. Earlier findings by Charlton et al.regarding the increased volume of jumps and the greater vertical displacement of the volleyball player's body showed that these were also correlated with the presence of patellar tendinopathy. The research concerned both volleyball and basketball players. Therefore, the ability to accurately and more effectively monitor loads provides coaching and support staff with the opportunity to avoid training activities that have been shown to increase risk of injury.
From a practical point of view, the results obtained here can be valuable materials for analysis by volleyball coaches and trainers. This experiment showed that the correct isokinetic and isometric assessment of the strength of the knee joint extensors and flexors directly affects the selection of the type of strength training and the load when playing volleyball. Properly selected strength training can improve strength and power production (the application of plyometric training). In turn, from a clinical or physiotherapeutic standpoint, the strength training exercises that are selected should maintain a balance of muscular strength across the knee joints and between opposing muscle groups and, therefore, reduce the frequency of injuries and contribute to a faster recovery from them.
According to this statement, one of the limitations of this study was the fact that four to six weeks of strength training or power training were not included in the preparatory period. This would have allowed changes to be observed in the strength ratio, which would be expressed as the percentage growth of the muscle strength in relation to the measurement before the application of training. Additionally, this would help in the calculation of a strength index that would be defined as the ratio of the maximal torques of the flexors to the maximal torques of the extensors; this would be required for the assessment of the plyometric performance during training. Another limitation would be the small experimental group in this study. However, the athletes were professional volleyball players that represented the club in the highest volleyball league in Poland. It was not possible to recruit additional players from another club, and the main reason was that the club was implementing a different training program during this period.
# Conclusions
In all trials, volleyball players achieved significantly higher peak torque (PT) values for both the extensors and flexors (p < 0.05) than those of the control group. However, the strength ratio of the flexors to extensors (H/Q) in the experimental group was only 83% of the standard reported in the literature. There were larger differences in the values obtained in the static tests.
The most developed and dominating muscles in the knee joints of volleyball players are the extensors, accounting for the low strength ratio and dynamic instability of this joint.
Based on a proper assessment of the strength ratio of the knee flexors and extensors, properly selected and implemented resistance training can improve the maximum strength and power production and can reduce the incidence of injuries in volleyball. |
Influence of microbiome species in hard-to-heal wounds on disease severity and treatment duration
SequencingSuperficial wounds a b s t r a c t Background: Infections, mostly those associated with colonization of wound by different pathogenic microorganisms, are one of the most serious health complications during a medical treatment. Therefore, this study is focused on the isolation, characterization, and identification of microorganisms prevalent in superficial wounds of patients (n = 50) presenting with bacterial infection.Methods: After successful cultivation, bacteria were processed and analyzed. Initially the identification of the strains was performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on comparison of protein profiles (2-30 kDa) with database. Subsequently, bacterial strains from infected wounds were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequencing of 16S rRNA gene 108.Results: The most prevalent species was Staphylococcus aureus (70%), and out of those 11% turned out to be methicillin-resistant (mecA positive). Identified strains were compared with patients' diagnoses using the method of artificial neuronal network to assess the association between severity of infection and wound microbiome species composition. Artificial neuronal network was subsequently used to predict patients' prognosis (n = 9) with 85% success. b r a z j i n f e c t d i s . 2 0 1 5;1 9(6):604-613605Conclusions: In all of 50 patients tested bacterial infections were identified. Based on the proposed artificial neuronal network we were able to predict the severity of the infection and length of the treatment.
# Introduction
Skin has an important role in preventing the entry of undesirable substances, organisms, and bacteria into the bloodstream. [bib_ref] Antimicrobial susceptibility pattern of bacterial isolates from wound infection and their sensitivity..., Mama [/bib_ref] [bib_ref] In-vitro antimicrobial activity of selected honeys on clinical isolates of Helicobacter pylori, Ndip [/bib_ref] Loss of skin integrity leads to different types of wounds which have the humidity, warmth, and a nurturing environment ideal for colonization and proliferation of undesirable bacterial strains, changing the naturally occurring microbiome. Colonized sites are usually polymicrobial, i.e. contain more than one bacterium with pathogenic potential. [bib_ref] Topical antimicrobials for burn wound infections, Dai [/bib_ref] Wound infections are marked by disturbed host-bacteria equilibrium in a traumatized tissue environment favoring the pathogenic bacteria. A wound infection not only has the possibility to elicit a systemic response (sepsis), but is also able to inhibit the multiple processes involved in the orchestrated progression of normal wound healing. [bib_ref] Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects, Dai [/bib_ref] The concept of microbiome was first suggested in 2001 by McCray and was described as an ecological cohort of commensal, symbiotic, and pathogenic microorganisms sharing a body space. [bib_ref] Ome sweet' omics -a genealogical treasury of words, Lederberg [/bib_ref] Previously, it was estimated that as much as 20 to 60% of human-associated microbiome is hard-to-identify, which has likely resulted in an underestimation of microbiome diversity. [bib_ref] Molecular analysis of the bacterial microbiota in the human stomach, Bik [/bib_ref] One of the most frequent microorganisms in infected wound is Staphylococcus aureus. [bib_ref] Methicillin-oxacillin resistant strains of Staphylococcus aureus (MORSA) in infected burn wounds, Polaczek-Kornecka [/bib_ref] [bib_ref] Antibacterial activity of honey against strains of Staphylococcus aureus from infected wounds, Cooper [/bib_ref] [bib_ref] RNAIII-inhibiting peptide enhances healing of wounds infected with methicillin-resistant Staphylococcus aureus, Simonetti [/bib_ref] [bib_ref] Interventions for wound healing among diabetic patients infected with Staphylococcus aureus: a..., Lima [/bib_ref] According to numerous studies, [bib_ref] Epifluorescent microscopic visualization of an in-vitro biofilm formed by a Pseudomonas aeruginosa..., Ricotti [/bib_ref] [bib_ref] Pathotyping of Pseudomonas aeruginosa isolated from chronically infected wounds, Schmoldt [/bib_ref] [bib_ref] Specific protease activity indicates the degree of Pseudomonas aeruginosa infection in chronic..., Wildeboer [/bib_ref] another common organism in infected wounds is Pseudomonas aeruginosa with up to 10% occurrence, causing nosocomial infections together with S. aureus and other bacteria. In addition, the Enterobacteriaceae family is most often identified in connection with immunocompromised patients or those who have undergone abdominal surgery. [bib_ref] Antimicrobial susceptibility pattern of bacterial isolates from wound infection and their sensitivity..., Mama [/bib_ref] Bacterial infections, increasingly occurring in medical facilities, can seriously complicate the outcome of treated patients. [bib_ref] Comparison of the effects of silver phosphate and selenium nanoparticles on Staphylococcus..., Chudobova [/bib_ref] [bib_ref] Gram-negative and Gram-positive bacterial infections give rise to a different metabolic response..., Hoerr [/bib_ref] This is particularly connected with rising resistance of bacterial strains toward antibiotics or metals, [bib_ref] Effect of ampicillin, streptomycin, penicillin and tetracycline on metal resistant and non-resistant..., Chudobova [/bib_ref] ,17 thus significantly hindering treatment success. Although being highly debated the mechanism of resistance development has not been satisfactorily elucidated. [bib_ref] The impact of MRSA on vascular surgery, Nasim [/bib_ref] [bib_ref] Vascular graft infections, Young [/bib_ref] [bib_ref] Severe surgical site infection in community hospitals: epidemiology, key procedures, and the..., Anderson [/bib_ref] [bib_ref] Methicillin-resistant Staphylococcus aureus: vascular surgeons should fight back, Earnshaw [/bib_ref] The elevated occurrence of resistant bacterial strains is strictly linked with increased utilization of invasive surgical techniques, which are often performed in elderly, immunocompromised patients. Simultaneously, with the use of antibiotics, bacterial resistance can evolve in surgical sites, leading to bacteremia and sepsis, and thus significantly prolonging the healing phase of a patient. Although bacterial resistance presents a problem in healthcare facilities, there still exist few possibilities to eliminate the most frequent resistant strains that cause hospital-acquired infectionmethicillin-resistant S. aureus (MRSA), 22,23 e.g. highly potent glycopeptide vancomycin. [bib_ref] In vitro activity of telavancin compared with vancomycin and linezolid against Gram-positive..., Rolston [/bib_ref] However, for a correct choice of antibiotics one needs to accurately identify the microbiome composition of infected wounds. Knowledge of the bacterial ecology of wounds may thus lead to increase treatment success, coupled with curbing bacterial resistance as a result of inadequate utilization of antibiotics. [bib_ref] Identification of a novel antibiotic from myxobacterium stigmatella eracta WXNXJ-B and evaluation..., Wang [/bib_ref] [bib_ref] Chemical nose for the visual identification of emerging ocular pathogens using gold..., Verma [/bib_ref] [bib_ref] Isolation and characterization of faecal bifidobacteria and lactobacilli isolated from dogs and..., Strompfova [/bib_ref] [bib_ref] Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection..., Dulay [/bib_ref] Accordingly, this work is focused on identification of the microbiome associated with serious infections in hard-to-heal wounds with the aim to propose a prediction model, comprising both the microbiome composition and patientshealth conditions.
# Materials and methods
## Chemicals, preparation of deionized water and ph measurement
Chemicals used in this study were acquired from Sigma-Aldrich (St. Louis, MO, USA) in ACS purity unless noted otherwise. Deionized water was prepared using reverse osmosis equipment Aqual 25 (Aqual s.r.o., Brno, Czech Republic) and further purified using Milli-Q Direct QUV equipped with the UV lamp, with 18 M resistance. pH was measured using the pH meter WTW inoLab (Weilheim, Germany).
## Preparation of hospital samples and their cultivation
## Cohort of patients with bacterial infections
For evaluation, patients with superficial or deep wounds were selected according to infection severity. Detailed information concerning the patients is documented in S1. A total of 50 patients aged 19 through 93 years were enrolled into the clinical study, and 13 patients were 70-79 years old; 23 patients superficial wounds and 27 deep wounds. For all patients, the treatment duration was determined by the traumatologist based severity and extent of infection, associated diseases potentially interfering with treatment outcome and healing of wounds, and other factors such as patient age, concomitant medications, and previous medical history. For confirmation of the functionality of the neural network 9 blank samples from 9 patients identified by MALDI-TOF MS were used. Enrollment of patients into the clinical study was approved by the Ethics Committee of Trauma hospital in Brno.
## Collection of wound swabs from patients with bacterial infections
The smears, collected from infected wounds with the agreement of patients, were sampled by rolling motion at the wound using a sterile swab sampler. All patients were divided into two subgroups, on the grounds of infection severity: deep and superficial wound. A detailed description of comorbidities and duration of treatment was obtained. Patients were classified according to the Classification of surgical wounds -SSI (surgical site infections). [bib_ref] Surgical site infections: time to modify the wound classification system?, Eisenberg [/bib_ref] [bib_ref] An evaluation of surgical site infections by wound classification system using the..., Ortega [/bib_ref] [bib_ref] Capillary electrophoresis in the diagnosis of surgical site infections, Klodzinska [/bib_ref] Infected wounds were sampled by using disposable tampon swabs maximizing collection of representative microflora. Tampons were subsequently stored in transport medium (inorganic salts, sodium thioglycolate, 1% agar, activated charcoal). The important part of our workflow process comprised sampling in duplicates with further transport in both aerobic and anaerobic conditions to preserve bacterial viability.
## Cultivation of clinical specimens
Four types of selective nutrient media (blood agar enriched by 10% NaCl, Endo agar, blood agar without any other component, and blood agar with amikacin) we employed for further microbiological selection. Petri dishes, containing the above mentioned media were subsequently incubated according to conventional protocols, as described elsewhere, [bib_ref] Pilot-scale continuous recycling of growth medium for the mass culture of a..., Sing [/bib_ref] [bib_ref] Potential new method: a comparability study of M-coliblue24 and M-endo les agar..., Goetz [/bib_ref] [bib_ref] The influence of culture conditions on the identification of Mycobacterium species by..., Balazova [/bib_ref] [bib_ref] Investigation of toxin genes among methicillin-resistant Staphylococcus aureus strains isolated from a..., Lim [/bib_ref] to maintain suitable conditions for growth of all types of bacteria. These Petri dishes were incubated for 24-48 h at 37 - C supplemented by TGY medium (1 g L −1 glucose, 5 g L −1 tryptone, 2.5 g L −1 yeast extract). Subsequently, individual colonies were collected from each Petri dish and stored in 1 L of enriched media. These samples were processed as described in the DNA sequencing section and utilized for both -MALDI-TOF MS identification and PCR with subsequent sequencing.
## Maldi-tof ms identification of bacteria
The following extraction protocol was based on MALDI Biotyper TM 3.0 User Manual Revision 2, also used in a previous report. 36 500 L of bacterial culture, cultivated overnight, was centrifuged at 14,000 × g for 2 min. The supernatant was discarded and the pellet was re-suspended in 300 L of deionized water besides adding 900 L of ethanol. After centrifugation at 14,000 × g for 2 min, the supernatant was discarded and the obtained pellet was air-dried. The pellet was then dissolved in 25 L of 70% formic acid (v/v) and 25 L of acetonitrile and mixed. The samples were centrifuged at 14,000 × g for 2 min and 1 L of the clear supernatant was spotted in duplicate onto the MALDI target and air-dried at room temperature. Then, each spot was overlaid with 1 L of ␣-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (20 mg mL −1 ) in organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid, both v/v) and air-dried completely prior to MALDI-TOF MS measurement on UltrafleXtreme MS (Bruker Daltonik GmbH, Bremen, Germany). As matrix solution 2,5dihydroxybenzoic acid (DHB) was also used in the same concentration and solvent as HCCA. Spectral data were taken in the m/z range of 2000-30,000 Da, resulted from the accumulation of 240 laser shots targeted to different regions of the same sample spot. These data were analyzed with the Flex Analysis software (Version 3.4). Final preparation of dendrograms was carried out in the MALDI BioTyper TM 3.1 (Bruker Daltonik GmbH, Bremen, Germany).
## Dna sequencing
Bacterial cells were centrifuged at 4450 × g and 20 - C for 10 min. The pellet was resuspended in 400 L of lysis buffer (6 M guanidium hydrochloride, 0.1 M sodium acetate) and cells were lysed for 1 hour at 20 - C and 600 rpm on Multi RS-60 (Biosan, Riga, Latvia). Genomic DNA was isolated from lysed bacterial cultures via MagNA Pure Compact (Roche, Mannheim, Germany), using Nucleic Acid Isolation Kit I, protocol DNA Bacteria.
16S rRNA gene was amplified using Ta q PCR Mix (New England Biolabs, Ipswich, MA, USA) and MasterCycler realplex [bib_ref] Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects, Dai [/bib_ref]
## Amplification of s. aureus genes meca and fnba
Isolation of genomic DNA was performed using the same method as described in section DNA sequencing. The mecA and fnbA genes were amplified using polymerase chain reaction (PCR) as previously reported. [bib_ref] Hypolithic and soil microbial community assembly along an aridity gradient in the..., Stomeo [/bib_ref] The primers were synthesized by Sigma-Aldrich and the sequences of forward and reverse primers for mecA gene were 5 ′ -CCCAATTTGTCTGCCAGTTT-3 ′ , and 5 ′ -TGGCAATATTAACGCACCTC-3 ′ and for fnbA gene were 5 ′ -GATACAAACCCAGGTGGTGG-3 ′ , and 5 ′ -TGTGCTTGACCAT-GCTCTTC-3 ′ . The volume of PCR reaction mixture was 100 L containing 1× Ta q reaction buffer, 0.2 mM dNTP, 1.6 U of Ta q DNA polymerase (New England Biolabs) and 0.5 mM of each primer. The reaction profile was as follows: initial denaturation at 94 - C for 4 min, 30 cycles of denaturation at 94 - C for 30 s, annealing at 53 - C for 30 s and extension at 72 - C for 1 min with a final extension of 7 min. The amplification generated a 223 bp for mecA and 191 bp for fnbA.
Agarose gel (2% (v/v), high melt, Mercury, San Diego, CA, USA) was prepared with 1× TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM ethylenediaminetetraacetic acid) and ethidium bromide (5 L per 100 mL of the gel) as described elsewhere. [bib_ref] Isolation of Xis Gen fragment of lambda phage from agarose gel using..., Smerkova [/bib_ref] 100 bp DNA ladder (New England Biolabs) within the size range from 100 to 1517 bp was used to monitor the size of the analyzed fragment. The electrophoresis (Bio-Rad, Hercules, CA, USA) was run at 60 V and 6 - C for 160 min. The bands were visualized by UV transilluminator at 312 nm (Vilber-Lourmant, Marne-la-Vallée, France).
## Statistical processing of obtained results
Automated neuronal network was used as a predictive model. Classification analysis automated neuronal network was used for the estimation of categorical data. The dataset was randomly divided as follows: 80% for learning, 10% for testing, and 10% for validation. Following network types were tested using automated network search: multilayer perceptron network (MLP), and radial basis function (RBF). Number of hidden units to search was determined as follows: 8-24 and 8-11 for MLP and RBF, respectively. Total 1000 networks were trained, and activation functions were searched for identity, logistic, tanh, exponential. Weight decay of 0.0001-0.001 was used for hidden layer and output layer. Weight of input variables for learning was used based on MALDI-TOF classification score. Unless noted otherwise, p-value less than 0.05 was considered significant. Software Statistica 12 (StatSoft, CA, USA) was used for analysis.
# Results and discussion
We decided to employ a variety of cultivation approaches (in presence of O 2 , CO 2 or in microaerophilic conditions) to reveal the presence of real microbiota associated with superficial infections.
MALDI-TOF MS was explored as an accurate and rapid identification tool, using the protein mass patterns, which are compared with patterns from a commercial Bruker Daltonics database (BDAL) of MALDI Biotyper TM software. [bib_ref] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new possibility for the identification..., Nagy [/bib_ref] Due to a powerful software support, the method can be used for identification within few minutes, which is one of the advantages. [bib_ref] Classification and identification of bacteria by mass spectrometry and computational analysis, Sauer [/bib_ref] Moreover, sequencing of amplified 16S rRNA gene [bib_ref] Experimental and analytical tools for studying the human microbiome, Kuczynski [/bib_ref] was employed for identification independent of protein patterns. Finally, an artificial neural network (ANN) was developed as a predictive model for evaluation of infection severity and using developed ANN we attempted to find the relationship between disease severity and the microorganisms identified in clinical specimens.
## Identification of bacterial strains by maldi-tof ms and sanger sequencing
For the identification of bacterial entities we employed complementary methods for independent evaluation of different biomolecules -proteins and DNA. [bib_ref] Classification and identification of bacteria by mass spectrometry and computational analysis, Sauer [/bib_ref] [bib_ref] Metabolomic signatures in guinea pigs infected with epidemic-associated W-Beijing strains of Mycobacterium..., Somashekar [/bib_ref] [bib_ref] Proteomic analysis of clinical isolate of Stenotrophomonas maltophilia with bla(NDM-1), bla(L1) and..., Liu [/bib_ref] Sanger sequencing was utilized as a confirmation method, based on sequencing of 16S rRNA gene. This gene contains hypervariable regions, providing species-specific sequences, hence it can provide enough information for a confident discrimination, and thus became popular in medical microbiology to classify bacteria. [bib_ref] Identification of species by multiplex analysis of variable-length sequences, Pereira [/bib_ref] [bib_ref] Ribosomal DNA sequencing as a tool for identification of bacterial pathogens, Kolbert [/bib_ref] When compared to sequencing, MALDI-TOF MS offers much shorter analysis time. By using this technique, wound microbiome could be discriminated within one hour of incubation, and thus this will likely become the method of choice for future microbiome identification. Nevertheless, the classification is based on a still developing database [bib_ref] The influence of culture conditions on the identification of Mycobacterium species by..., Balazova [/bib_ref] ; hence MALDI-TOF MS identification of non-databased bacteria has still to be connected with other confirmation methods. From this reason we firstly employed MALDI-TOF MS with a condition: If score <2.00 = 16S rRNA sequencing.
As shown in S2, 108 bacterial strains were identified 37 of them had to be confirmed by sequencing and confirmed strains were immediately databased to increase future classification success. Strains of S. aureus were the most often identified (n = 35). Thus, methicillin-resistant S. aureus (MRSA) is highly associated with severe infections in post-surgical wounds [bib_ref] Meta-analysis of methicillin-resistant Staphylococcus aureus colonization and risk of infection in dialysis..., Zacharioudakis [/bib_ref] ; we further analyzed the mecA gene, encoding a modified penicillin binding protein (PBP) known as PBP2a, with decreased affinity toward -lactams. [bib_ref] Prevalence of methicillin resistant Staphylococcus aureus among Egyptian patients after surgical interventions, Ahmed [/bib_ref] The mecA positivity was determined in four isolates. Since 67% of patients had deep wound infections and were treated for more than 8 weeks after admission to infectious Department of Trauma Hospital of Brno, presence of mecA was shown to be a crucial microbiological factor, affecting patients prognosis. Further, we determined the presence of fnbA gene, responsible for adhesins production. Adhesion to human extracellular matrix components and serum proteins is an important facet in the interaction between bacteria and its host cells. [bib_ref] Surface adhesins of Staphylococcus aureus, Clarke [/bib_ref] Lim and coworkers identified the presence of fnbA in 96% of all isolated MRSA strains. [bib_ref] Investigation of toxin genes among methicillin-resistant Staphylococcus aureus strains isolated from a..., Lim [/bib_ref] In our case, fnbA presence was confirmed in all MRSA isolates and in 89% of methicillinsensitive S. aureus isolates. Similarly to mecA, fnbA was found to be associated with infection severity. In patients with negative fnbA and mecA the treatment duration was less than four weeks in 75% of cases, despite the fact that patients had deep wound infections. This finding suggests that the severity of staphylococcal infections does not depend solely on antimicrobial resistance, but also on adhesins expression, which enhance the interaction with the target host cells.
## Distribution of identified strains within various cohorts of patients
According to duration of treatment, the patients were divided into specific subgroups, where each sector represents one bacterial strain.
The subdivision of patients was based on surgical wounds classification SSI. As shown, patients were divided into two groups -deep and superficial wounds and the associated bacterial strains are depicted in and B.
As it is obvious from , in the more serious infections (deep) S. aureus was the main bacterium of microbiome composition (28% of identified strains), followed by Enterococcus faecalis (15%), and Escherichia coli (11%). On the other hand, E. coli was not so often identified in surficial wounds (5% -
## Phylogenetic analysis of protein alterations
As was shown by Rettinger and colleagues, [bib_ref] Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool..., Rettinger [/bib_ref] MALDI-TOF mass spectra-based phylogenetic analysis is considered equivalent to 16S rRNA gene sequencing. Therefore, we employed MALDI Biotyper TM for preparation of dendrograms for our groups, divided by treatment duration [fig_ref] Figure 2 -: Dendrograms from protein mass profiles of microorganisms in different groups based on... [/fig_ref]. Dendrograms showed similarity of same bacterial strains (low distance level), but in some cases larger differences were found -usually among bacterial strains from different patients. These differences were caused probably by modifications of bacterial proteins. Karger et al. found methylation as a cause of higher distance level in dendrogram between Burkholderia pseudomallei and other types of B. pseudomallei. [bib_ref] Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell matrix-assisted..., Karger [/bib_ref] Thus it can be concluded that not only changes in microbiome representatives affect treatment duration and success, but also small changes in protein posttranslation modifications can be highly important for patients' recovery. [fig_ref] Table 1 -: Characterization of neuronal network performance for the prediction of patient outcome [/fig_ref]
## Artificial neural network
Tw o neuronal networks were created: (1) for the prediction of time-to-heal, and (2) for the prediction of infection severity. The following input parameters were used for the construction of networks: from 2000 networks five were retained and one was used for further final custom neuronal network. The settings of the network created using automated algorithm and used for the custom final learning were Multilayer perceptron 89-13-3 (input-hidden-output neurons), Broyden-Fletcher-Goldfarb-Shanno (BFGS) training algorithm, sum of squares error function, identity function for hidden layer, and then for output layer. The design of the network is displayed in [fig_ref] Figure 3 -: Design and performance of the neuronal networks [/fig_ref]. With stopping conditions enabled [fig_ref] Figure 3 -: Design and performance of the neuronal networks [/fig_ref] , a final network was created in the 17th training cycle with performances of 91.4%, 85.7%, and 71.4% for training, testing, and validation (accuracy in prediction up to 85% - [fig_ref] Figure 3 -: Design and performance of the neuronal networks [/fig_ref] , respectively.
Consequently, a second neuronal network for the prediction of infection severity was created using an automated algorithm. The best-performing network was trained under following settings: multilayer perceptron 89-19-2 (inputhidden-output neurons) [fig_ref] Figure 3 -: Design and performance of the neuronal networks [/fig_ref] BFGS training algorithm, cross entropy error function, and exponential and softmax activation function for hidden and output layer. The training process is depicted in [fig_ref] Figure 3 -: Design and performance of the neuronal networks [/fig_ref] (accuracy in prediction up to 85% - [fig_ref] Figure 3 -: Design and performance of the neuronal networks [/fig_ref].
The performances of the network were 100.0%, 85.7%, and 85.7% for training, testing, and validation [fig_ref] Table 1 -: Characterization of neuronal network performance for the prediction of patient outcome [/fig_ref] , respectively. The accuracy for individual cases is displayed in [fig_ref] Table 2 -: Performance of the network [/fig_ref].
Sensitivity analysis of input variables for both networks was carried out. For the prediction of infection severity, the mean sensitivity level was 4.66, ranging from 0.63 to 20.1, and a total sensitivity = 179.9 [fig_ref] Figure 4 -: Sensitivity analysis of all factors for prediction of time-to-heal and infection severity [/fig_ref] The highest level of sensitivity (thus highest impact on prediction of a network) was observed for hypertension
## Conflicts of interest
The authors declare no conflicts of interest.
[fig] Figure 2 -: Dendrograms from protein mass profiles of microorganisms in different groups based on treatment duration. Created in MALDI Biotyper TM . (A) Treatment duration less than four weeks. (B) Treatment duration 4-7 weeks. (C) Treatment duration eight and more weeks. (D) Exitus. [/fig]
[fig] Figure 3 -: Design and performance of the neuronal networks. (A) Design of classification network for the prediction of time-to-heal. The number of neurons/inputs is indicated by n. *Note the number of input and hidden neurons is not displayed exactly. (B) Training process of the classification network with stopping conditions activated. (C) Accuracy of the final network for classification of time-to-heal. (D) Design of classification network for the prediction of infection severity. (E) Training process for creation of this network with stopping criteria activated. (F) Accuracy of the network for the prediction of infection severity. [/fig]
[fig] Figure 4 -: Sensitivity analysis of all factors for prediction of time-to-heal and infection severity. Sensitivity of individual factors depicted as a percentage of total sensitivity. (A) Sensitivity of individual factors for the prediction of time-to-heal. (B) Sensitivity of individual factors for the prediction of infection severity. [/fig]
[table] Table 1 -: Characterization of neuronal network performance for the prediction of patient outcome. Performance displayed in % for training, testing, and validation samples. The number of training cycle for custom network training is displayed in training algorithm column. BFGS, Broyden-Fletcher-Goldfarb-Shanno training algorithm; SOS, sum of squares. [/table]
[table] Table 2 -: Performance of the network: verification of the test and validation cohort. Analysis for both networks for prediction of infection severity and time-to-heal. Test cohort was employed for stopping conditions. Validation sample was used to test final network. "target" indicates input data, network output reflects calculated result from the neuronal network. id, identification of patient; w, week.Targ et Network output Accuracy Conf. level Targ et Network output Accuracy Conf. level [/table]
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# Introduction
Trypanosoma cruzi, the etiological agent of Chagas' disease, has a complex life cycle, alternating between an intermediate invertebrate host and a definitive mammalian host [bib_ref] Cell Biology of Trypanosoma cruzi, De Souza [/bib_ref]. Trypanosoma cruzi has a high requirement for iron for proliferation, in vitro and in vivo, mobilizing heme and nonheme iron [bib_ref] Role of Iron in Trypanosoma Cruzi Infection of Mice, Lalonde [/bib_ref]. Trypanosoma cruzi can hijack Fe-proteins from a mammalian host. The addition of deferoxamine, an Fe chelator, or transferrin-free serum can inhibit the proliferation of amastigote cells in culture, an indication that Fe is an obligatory nutrient [bib_ref] Trypanosoma cruzi Receptors for Human Transferrin and Their Role, Lima [/bib_ref]. This form of the parasite presents receptors for human transferrin, which bind exogenous transferrin. Transferrin bound to amastigote cells is not removed by acid treatment, indicating a possible internalization and utilization of this transferrin [bib_ref] Trypanosoma cruzi Receptors for Human Transferrin and Their Role, Lima [/bib_ref]. In epimastigotes, transferrin uptake occurs through the cytostome, a specialized structure composed of a membrane invagination in the anterior region, close to the flagellar pocket [bib_ref] Trypanosoma Cruzi Epimastigote Endocytic Pathway: Cargo Enters the Cytostome and Passes Through..., Porto-Carreiro [/bib_ref] , an early observation showing that Fe-carrying molecules are important for the parasite in its different morphological stages and, therefore, for the establishment, maintenance, and evolution of the infection in the vertebrate host. Heme is also utilized as an Fe source for T. cruzi; it can stimulate T. cruzi proliferation in culture in a dose-dependent manner [bib_ref] Heme Requirement and Intracellular Trafficking in Trypanosoma cruzi Epimastigotes, Lara [/bib_ref]. Moreover, heme/porphyrin translocates in epimastigote forms of T. cruzi, possibly mediated by an ABC transporter protein [bib_ref] The Heme Uptake Process in Trypanosoma cruzi Epimastigotes Is Inhibited by Heme..., Cupello [/bib_ref]. However, the limiting step for the utilization of heme by pathogenic trypanosomatids is the initial heminic ring hydrolysis for Fe liberation [bib_ref] Iron Metabolism in Trypanosomatids, and Its Crucial Role in Infection, Taylor [/bib_ref] , since there is no heme oxidase gene in the T. cruzi genome [bib_ref] The Genome Sequence of Trypanosoma Cruzi, Etiologic Agent of Chagas Disease, El-Sayed [/bib_ref].
Before direct Fe incorporation by cells, Fe 3+ (the predominant redox form in nature) has to be reduced to Fe 2+ , catalyzed by an Fe-reductase [bib_ref] Characterization of the Quinone Reductase Activity of the Ferric Reductase B Protein..., Sedlaćek [/bib_ref]. In this way, the identification of an Fe-reductase activity in Leishmania chagasi [bib_ref] Leishmania Chagasi: Uptake of Iron Bound to Lactoferrin or Transferrin Requires an..., Wilson [/bib_ref] , Leishmania amazonensis, and more recently, T. cruzi [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref] is strongly indicative of a Fe 2+ transport mechanism in trypanosomatids. Due to the Fe low redox potential (E°= −0.04 V for the reaction Fe 3+ + 3e − ! Fe), it becomes a suitable element for redox catalysis reactions [bib_ref] Termodynamics of Ion and Electron Transport, Atkins [/bib_ref] , where it can act as an electron donor and receptor. The ability to pass readily through oxidation/reduction cycles leads to the inherent toxicity of Fe, since it catalyzes the formation of reactive O 2 species (ROS) as hydroxyl radical (OH - ), which provides a high redox potential (E°= +2.33 V for the reaction OH - + e − + H + ! H 2 O) [bib_ref] Overview of Reactive Oxygen Species, Krumova [/bib_ref] , via the Fenton reaction.
Regarding Fe metabolism in T. cruzi, there is strong evidence that it is important in the catalysis by antioxidant defenses [bib_ref] Oxidative Stress Fuels Trypanosoma cruzi Infection in Mice, Paiva [/bib_ref]. Superoxide dismutase isoforms (SODs) are metalloproteins that can dismutate O ·− 2 in H 2 O 2 and O 2 . Metallic ions are present in the catalytic center from SODs, e.g., Cu-Zn-SOD in eukaryotes [bib_ref] Structure and Mechanism of Copper, Zinc Superoxide Dismutase, Tainer [/bib_ref] and Mn-SOD in bacteria, such as Escherichia coli [bib_ref] The Manganese and Iron Superoxide Dismutases Protect Escherichia Coli From Heavy Metal..., Geslin [/bib_ref] , whereas trypanosomatids exclusively have Fe-SOD, with Fe as the cofactor [bib_ref] Functional Characterisation of the Iron Superoxide Dismutase Gene Repertoire in Trypanosoma brucei...., Wilkinson [/bib_ref]. However, there is little information regarding Fe uptake in T. cruzi [bib_ref] Crusade for Iron: Iron Uptake in Unicellular Eukaryotes and Its Significance for..., Sutak [/bib_ref] and its role in i) the differentiation process that is required for the infection of the vertebrate host and ii) the stimulus of oxidative stress within the parasite, which favors this differentiation process.
We have identified a sequence putatively annotated as the Zn/ Fe transporter. In-silico analysis demonstrated its homology to the Fe transporters described in L. amazonensis [bib_ref] A Leishmania amazonensis ZIP Family Iron Transporter Is Essential for Parasite Replication..., Huynh [/bib_ref] and Arabidopsis thaliana [bib_ref] IRT1, an Arabidopsis Transporter Essential for Iron Uptake From the Soil and..., Vert [/bib_ref]. It is the first identification of an Fe transporter (TcIT) homologous to LIT1 possibly responsible for Fe uptake in T. cruzi, working coupled to the Fe-reductase TcFR recently described in our laboratory [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref]. We hypothesize that this transporter may be important in the maintenance and progression of T. cruzi infection.
# Materials and methods
## Epimastigote growth and metacyclogenesis
Epimastigotes of T. cruzi (Dm28c strain) were maintained at 28°C in stationary phase by using brain heart infusion (BHI) medium supplemented with 10% FBS, 30 mM hemin, and 1% penicillin-streptomycin cocktail (regular medium, RM). Irondepleted medium (IDM) was prepared using BHI medium without hemin and supplemented with 10% iron-free FBS, following the protocol described in [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref]. The viability of the parasites was assayed by evaluating the mitochondrial transmembrane potential. When this potential is generated and maintained, the 3-(4,5-dimethylthiazol)-2,5diphenyltetrazolium (MTT) bromide (Sigma-Aldrich, Saint Louis, MO, USA) is converted to insoluble formazan. The concentration of formazan was spectrophotometrically determined at 570 nm after its solubilization with Triton X-100.
For the assay of epimastigote proliferation, the parasites were inoculated (10 6 cells/ml) on the 6th day of culture into the BHI medium (RM or IDM, with or without G418). Cell proliferation was assessed every day by counting the number of cells in a hemocytometer. Metacyclogenesis was induced as described in [bib_ref] In Vitro Differentiation of Trypanosoma cruzi Under Chemically Defined Conditions, Contreras [/bib_ref] and [bib_ref] Golgi UDP-GlcNAc:polypeptide O-a-N-Acetyl-D-Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma..., Koeller [/bib_ref]. Briefly, epimastigotes in transition from logarithmic to stationary phase were adjusted to 5 × 10 8 parasites/ml in triatomine artificial urine (TAU) medium [190 mM NaCl, 17 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 0.035% (w/v) NaHCO 3 , and 8 mM phosphate buffer at pH 6.0]. After 2 h at 28°C, the cultures were diluted 100-fold in 10 ml TAU medium supplemented with 10 mM L-proline, 50 mM glutamic acid, 2 mM aspartic acid and 10 mM glucose (TAU3AAG), and 500 µg/ml G418 (Sigma-Aldrich) and transferred to T25 flasks-lying at an angle of 45°t o increase the area in contact with O 2 -and maintained at 28°C to promote metacyclogenesis. After 3-5 days, the parasites were quantified by hemocytometry, and the percentage of metacyclic trypomastigotes was estimated by their morphology after Giemsa staining.
The percentage of trypomastigotes was also quantified by cytometry as previously described [bib_ref] Expression and Cellular Trafficking of GP82 and GP90 Glycoproteins During Trypanosoma Cruzi..., Bayer-Santos [/bib_ref]. Briefly, live parasites (4 × 10 7 ) were incubated for 30 min on ice with the monoclonal antibody 1G7 against GP90, diluted in 1% bovine serum albumin in phosphate-buffered saline (BSA/PBS). Later, the cells were washed in PBS and were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min. Then, after washings in PBS, the parasites were incubated with Alexa Fluor 488-conjugated anti-mouse IgG diluted in 1% BSA/PBS for 1 h at room temperature. Subsequently, after two more washes, fluorescence was determined on a FACSCalibur II cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and data analysis was performed using the CellQuest software (Becton Dickinson).
# In-silico analysis
From the in-silico analysis of the genome of the Dm28c strain of T. cruzi (available through the TriTryp database under the accession no. TCDM_06386) [bib_ref] Trypanosoma cruzi Clone Dm28c Draft Genome Sequence, Grisard [/bib_ref] , we found a homolog (TcIT) to the Fe transporter of L. major (available from the TriTryp database under the accession no. LmjF.31.3070-LIT). The model of TcIT was constructed using the protein structure prediction PHYRE (www.sbg.bio.ic.ac.uk/phyre/) [bib_ref] Protein Structure Prediction on the Web: A Case Study Using the Phyre..., Kelley [/bib_ref] , which is based on the model of the SERCA ATPase and visualized with the standard molecular viewer PyMOL 2002 (PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA; http://pymol. sourceforge.net/). Phylogenetic analysis used the MEGA 7 software. The evolutionary history was inferred using the neighbor-joining method [bib_ref] The Neighbor-Joining Method: A New Method for Reconstructing Phylogenetic Trees, Saitou [/bib_ref]. Amino-acid multiple sequence alignments were obtained by using Clustal W and Clustal X software version 2.0 (http://www.ebi.ac.uk/Tools/ msa/clustalw2).
## Cloning and overexpression of tcit in trypanosoma cruzi
The full-length TcIT coding region was amplified from Dm28c gDNA, by using designated primers FTcIT (5′-GGATCCAT GAACAACGTTGAGTCAAGTGACGCG-CACCT) introducing the BamHI restriction site at the 5′-end and RTcITHA (5′-AAGCTTTTAAGCGTAATCTGGAACATCGTATGGG TACGCCCACTTCCCAAGGAGCGTCATAA), with the addition of HindIII restriction site with the hemagglutinin (HA) epitope tag at the 3′-end, thus generating the TcIT-HA insert. The amplicon was subcloned into pCR2.1-TOPO vector (Thermo Fisher Scientific, Waltham, MA, USA), released by digestion with BamHI and HindIII (sites underlined above), and ligated into similarly digested expression vector pTEX [bib_ref] A Shuttle Vector Which Facilitates the Expression of Transfected Genes in Trypanosoma..., Kelly [/bib_ref]. The shuttle vector pTEX-TcIT-HA, which replicates in E. coli and T. cruzi, was used as the vehicle for the expression of TcIT-HA in T. cruzi. Cell electroporation was performed with an Amaxa Nucleofector II device with human T-cell buffer (Lonza, Basel, Switzerland). A total of 5 × 10 7 epimastigotes were transfected with pTEX-TcIT-HA or empty pTEX (pTEX-Ø) vectors (10 mg DNA). After electroporation, the cells were cultured for 48 h in standard medium and 500 µg/ml G418 (Sigma-Aldrich) was added. Non-DNA control cells died after 3 to 4 weeks. Cultures were 5-fold diluted with fresh G418-containing medium after 5-10 days. Stable resistant cells were obtained~30 days after transfection, indicating resistance to G418.
## Intracellular iron concentration determination
The concentration of intracellular Fe accumulated under different conditions and by different strains (wild type and mutants obtained as described above) was determined by a colorimetric assay based on the use of ferrozine. Suspensions containing 10 8 parasites were collected from different cultures and washed three times with PBS pretreated with 5 g/100 ml Chelex resin (Sigma-Aldrich). The cells were lysed with 100 ml 50 mM NaOH, followed by the addition of 100 ml 10 mM HCl; the release of ionic Fe bound to intracellular structures was achieved by adding 100 ml of a mixture of 1.4 M HCl and 4.5% (w/v) KMnO 4 (1:1) to the cell lysate, followed by incubation at 60°C for 2 h. Then, 30 ml Fe detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid) was added. After 30 min of incubation at room temperature, the absorbance of the sample was recorded at 550 nm. The concentration of Fe was determined using a standard curve with known FeCl 3 concentrations (0-75 mM) [bib_ref] Iron Uptake Controls the Generation of Leishmania Infective Forms Through Regulation of..., Mittra [/bib_ref].
## Membrane fraction preparation
Mutant epimastigotes (5 × 10 9 cells) in late log phase of growth were harvested by centrifugation and washed three times in cold PBS. The plasma membrane fraction/PM was obtained as previously reported [bib_ref] Evidence for Distinct 5'-and 3'-Nucleotidase Activities in the Surface Membrane Fraction of..., Gottlieb [/bib_ref] [bib_ref] Leishmania amazonensis: Biological and Biochemical Characterization of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activities, Pinheiro [/bib_ref] [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref] , with slight modifications. The washed organisms, overexpressing pTEX-TcIT or pTEX-Ø, were resuspended in Tris-EDTA buffer (10 mM Tris-HCl at pH 8.0, 125 mM sucrose, 3 mM MgCl 2 , 2 mM EDTA, and 1 mM phenylmethanesulfonyl fluoride) and maintained on ice for 30 min. The parasites were mixed with glass beads (1:4) and disrupted by abrasion for 10 min on an ice bath. After grinding, glass beads, unbroken cells, and large cell debris were removed by centrifugation at 1,000×g for 15 min at 4°C. The supernatant (total homogenate/HG) was centrifuged at 200,000×g for 1 h. The resulting pellet (total membranes/TM) was resuspended in 50 mM Tris-HCl (pH 8.0) and subsequently applied to a continuous density gradient of 18% Percoll in 0.25 M sucrose and 12 mM Tris-HCl (pH 7.4), to obtain the PM-enriched fraction. After centrifugation at 40,000×g for 1 h, the bands were removed by aspiration, analyzed for 5′-nucleotidase activity [the marker of PM in epimastigote forms of T. cruzi [bib_ref] Purification of an Adenylyl Cyclase-Containing Plasma Membrane Fraction from Trypanosoma Cruzi, Zingales [/bib_ref] and Fe-reductase activity, and the two fractions (TM and PM) were kept at −80°C, along with aliquots of HG, for further assays. Protein concentration was determined by the Lowry method [bib_ref] Protein Measurement With the Folin Phenol Reagent, Lowry [/bib_ref] , using BSA as standard. Supplementary [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref] depicts the assays of 5′-nucleotidase and Fe-reductase activities, demonstrating i) the enrichment of the PM fraction with both enzymes and ii) that these activities were barely detectable in the supernatant recovered after centrifugation of HG at 200,000×g for 1 h (the cytosolic fraction/C).
## Western blotting and immunolocalization
For Western blotting detection, the proteins of the fractions obtained as described above (30 µg/lane) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA), which were blocked with 5% milk in PBS plus 0.1% (w/v) Tween 20, probed overnight at 4°C with the primary rabbit anti-HA antibody (1:1,000, Sigma-Aldrich), and detected using an HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA). Ponceau red was used as loading control.
Immunofluorescence was assayed as previously described, with minor modifications [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref]. The mutant T. cruzi epimastigotes (10 7 cells, pTEX-TcIT or pTEX-Ø) were washed three times in 0.5 ml PBS and the suspension was fixed with 500 ml formaldehyde 4% (w/v) in PBS for 1 h at room temperature. The samples were washed twice with 0.5 ml PBS, suspended in 40 ml PBS, and settled on poly-L-lysine-coated coverslips for 15 min. The coverslips were incubated in 0.4% (w/v) saponin in PGN [PBS at pH 7.2, supplemented with 0.2% (w/v) gelatin and 0.1% (w/v) NaN 3 ] for 15 min to allow parasite permeabilization. The samples were incubated with anti-HA antibody raised in rabbit (1:1,000, Sigma-Aldrich) and mouse anti-TcSMP (surface membrane proteins) antibody (1:100), prepared and used as described by [bib_ref] Molecular Characterization of a Novel Family of Trypanosoma cruzi Surface Membrane Proteins..., Martins [/bib_ref] in PGN with 0.1% saponin, and incubated overnight at 4°C. After washing with PGN, the samples were incubated with anti-rabbit FITC conjugated (1:100, Sigma-Aldrich) and anti-mouse Alexa 594 (1:1,000, Sigma-Aldrich) in PGN for 1 h at room temperature. Coverslips were washed in PBS and incubated with 0.1 µg/ml 4′,6diamino-2-phenylindole (DAPI, Sigma-Aldrich) for 30 min. After washing, coverslips were mounted on slides in Miowol (Antifade) reagent. Images were taken with a Leica TCS-SPE confocal microscope and processed with Leica confocal software.
## Transmission electron microscopy
Cells were fixed by using 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature and postfixed using an osmium-thiocarbohydrazide-osmium (OTO) protocol [bib_ref] The Use of Osmium-Thiocarbohydrazide-Osmium (OTO) and Ferrocyanide-Reduced Osmium Methods to Enhance Membrane..., Willingham [/bib_ref]. Briefly, the cells were incubated in a post-fixative solution [1% (v/v) OsO 4 , 0.8% (v/v) potassium ferrocyanide, and 5 mM CaCl 2 in 0.1 M cacodylate buffer (pH 7.2)] for 40 min, washed twice in water, and then incubated in a solution of 1% (w/v) thiocarbohydrazide (TCH, Sigma-Aldrich) in water for 5 min. After three washes in water, the cells were incubated again in the post-fixative solution for 3 min. Samples were dehydrated in an acetone series and embedded in epoxy resin. Ultrathin sections (70 nm) were stained post-embedding with 5% (w/v) uranyl acetate and lead citrate and observed by using a Tecnai Spirit electron microscope (FEI Co., Hillsboro, OR, USA) operating at 120 kV.
## Real-time pcr
Total T. cruzi RNA was extracted using a Direct-zol RNA MiniPrep Kit (Zymo Research, Orange, CA, USA) from epimastigotes maintained at RM or IDM for 6 days, or mutants pTEX-TcIT or pTEX-Ø (as indicated in the figure legends). Total RNA was subjected to reverse transcription using the High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). For RT-PCR, 100 ng/ml cDNA per well was used (15 ml total volume), along with 5 mM primer mix and 7 ml PowerUp SYBR green master mix (Thermo Fisher Scientific). The primers 5′-TCTGGTCGC-TTCTCTTCTCG and 5′-TAAAGA-CTC CGGCACACAGT were used to amplify a 152-bp fragment of the TcIT gene. The primers 5′-AGCGCGCGTCTAAGACTTACA and 5′-TG-GAGCTGCGGTTGTCATT that amplify the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) constitutive gene were used to provide an endogenous control.
## High-resolution respirometry in different respiratory states
Oxygen consumption was measured using intact epimastigotes (5 × 10 7 parasites/chamber; pTEX-TcIT or pTEX-Ø). Analyses used an O2k-system high-resolution oxygraph (Oroboros Instruments, Innsbruck, Austria). The cells were suspended in 2 ml respiration solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris-HCl (pH 7.2) at 28°C with continuous stirring, and 50 mM digitonin was added to permeabilize the parasites. Oxygen concentrations and O 2 consumption were recorded using DatLab software coupled to Oxygraph-2K (basal O 2 consumption). Subsequently, 10 mM succinate and 200 mM ADP were added. Uncoupled respiration was stimulated after adding 3 mM FCCP and respiration was inhibited by adding 2.5 mg/ml antimycin A to measure residual O 2 consumption [bib_ref] Heme Modulates Trypanosoma cruzi Bioenergetics Inducing Mitochondrial ROS Production. Free Radic, Nogueira [/bib_ref]. In a series of experiments, titration with substrates and inhibitors was not carried out, and O 2 consumption was measured only in the presence of endogenous substrates (basal consumption) over all recordings.
## Superoxide dismutase activity
Superoxide dismutase (SOD) activity was measured as described in [bib_ref] The Estimation of Red Cell Superoxide Dismutase Activity, Winterbourn [/bib_ref] , with modifications, based on SOD inhibiting the reduction of nitro blue tetrazolium (NBT) by O ·− 2 . Epimastigote cells were harvested by centrifugation, washed three times in cold PBS, and disrupted by freeze-thaw. The protein concentration of the total homogenate was quantified by the Lowry method [bib_ref] Protein Measurement With the Folin Phenol Reagent, Lowry [/bib_ref]. The homogenates (using known quantities of protein in the range of 10-50 µg) were incubated in reaction medium (200 µl, final volume) containing 45 mM potassium phosphate buffer (pH 7.8), 6.5 mM EDTA, and 50 mM NBT. The reaction was initiated by adding 2 mM riboflavin. After 12 min in a light box, the absorbance of the sample was recorded at 560 nm. The percent inhibition was measured for each amount of protein, and SOD activity was expressed as the amount of enzyme inhibiting NBT reduction by 50%.
## Intracellular atp quantification
The intracellular ATP was quantified by using an ATP bioluminescent somatic cell assay kit (Sigma-Aldrich). Briefly, mutant epimastigotes (10 7 parasites per tube, 0.1 ml) were incubated in a solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris-HCl (pH 7.2 adjusted with HCl). Cellular extracts were prepared by mixing 0.1 ml epimastigotes with 0.1 ml somatic cell ATP releasing reagent and the mixture was left on ice for 1 min. Half of the cellular extract (0.1 ml) was transferred to MTS-11C minitubes (Axygen, Union City, CA, USA) containing 0.1 ml ATP assay mix and stirred for 10 s at room temperature. The total amount of light emitted was measured with a GloMax Multi JR detection system (Promega, Madison, WI, USA). Total intracellular ATP concentration per cell number was calculated using a standard ATP curve, prepared, and analyzed in each experiment .
## Amplex red peroxidase assay
The production of H 2 O 2 in mutants overexpressing TcIT was assayed by the rate of H 2 O 2 reduction to H 2 O, which is stoichiometrically coupled (1:1) to the simultaneous oxidation of the non-fluorescent Amplex ® Red probe to the fluorescent resorufin (Dos-Santos et al., 2019). Briefly, assays containing 0.1 mM H 2 O 2 were incubated with 10 7 parasites/ml for 30 min at room temperature in 5 mM Tris-HCl (pH 7.4), 1.7 mM Amplex ® Red (Invitrogen, Carlsbad, CA, USA), and 6.7 U/ml horseradish peroxidase (Sigma-Aldrich) in a final volume of 100 ml. The evolution of fluorescence was followed at excitation/emission wavelengths of 563/587 nm (slit 5 nm).
## Interaction of trypomastigotes with llc-mk2 cells
Metacyclogenesis was induced as described above in section 2.1. After 3 days, the parasites were collected and purified using the ion-exchange chromatography technique Sepharose membrane-DEAE [bib_ref] Purification of Trypanosoma cruzi Metacyclic Trypomastigotes by Ion Exchange Chromatography in Sepharose-DEAE,..., Cruz-Saavedra [/bib_ref] to obtain a preparation enriched with metacyclic trypomastigotes. Then, the parasites were incubated with LLC-MK2 cells (CCL-7; ATCC, Rockville, MD, USA) (50 parasites:1 cell) in RPMI 1640 medium supplemented with 10% FBS in a 96-well PS F-bottom microplate (Greiner Bio-One Brazil Ltd., Americana, Brazil) for 24 h at 37°C under 5% CO 2 in air. After removing the medium containing parasites not adhered to the cells, adding the fresh medium, and incubating further (48 h), the cells were fixed with 4% paraformaldehyde in PBS for 10 min. After incubation with Hoechst 33342 (Invitrogen) (1:5,000 dilution) for 60 min at room temperature in the dark, the wells were washed with Milli-Q deionized water and analyzed in a high-content screening system ImageXpress Micro XL (Molecular Devices, San Jose, CA, USA) using the MetaXpress 6.0 software. The interaction parasites:cells was quantified by counting the respective nuclei.
# Statistical analysis
Data are presented as mean ± SE. Means were compared by using unpaired Student's t-test and GraphPad Prism 7.0 (San Diego, CA, USA). When more than two means were compared, one-way ANOVA followed by Tukey's test or two-way ANOVA with multiple comparisons was used, as indicated in the text or in the figure legends.
# Results
## Identification and analysis of iron transporter sequence in trypanosoma cruzi
A putative zinc-iron (Zn-Fe) transporter sequence was found in the genome database of T. cruzi (TriTrypDB: TCDM_06386) following a BLAST search, using the Fe transporter LIT1 from L. amazonensis (TritrypDB: LmjF.31.3060) [bib_ref] A Leishmania amazonensis ZIP Family Iron Transporter Is Essential for Parasite Replication..., Huynh [/bib_ref] as target. The TCDM_06386 was named TcIT and has 1,119 bp, and the deduced amino-acid sequence of the peptide comprises 372 residues, thus resulting in a predicted molecular mass of 39.8 kDa. The deduced protein TcIT has a structural model [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref] in which its eight possible transmembrane domains can be seen (TMpred Server) [bib_ref] TMbase-A Database of Membrane Spanning Proteins Segments, Hofmann [/bib_ref]. TcIT has a highly conserved domain corresponding to the zinc iron permeases (ZIP) superfamily domain (permeases classified as 2.A.5 in the Transporter Classification Database/TCDB, Saier Lab. Bioinformatics Group). We encountered the domain in Zn and Fe transporter proteins by using the Conserved Domain Database (CDD, National Center for Biotechnology Information/NCBI). This domain has conserved amino acids corresponding to Zn/Fe binding, such as those labeled H105, H223, S224, H249, and E253 in [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref] , which are located along the inner surface of the channel formed by the transmembrane domains and bind the metal during the transport process [bib_ref] Functional Characterization of LIT1, the Leishmania amazonensis Ferrous Iron Transport, Jacques [/bib_ref].
TcIT alignment had 34% identity and 53% similarity to the LIT1 from L. amazonensis and 28% identity and 46% similarity to the IRT1 from A. thaliana [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref] , with 30% identity between the sequences of L. amazonensis and A. thaliana [bib_ref] Functional Characterization of LIT1, the Leishmania amazonensis Ferrous Iron Transport, Jacques [/bib_ref]. Analysis showed that all three sequences have eight transmembrane domains (underlined in [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref] and the same residues for Zn/Fe binding (residues H105, H223, S224, H249, and E253, red boxes in [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref]. To analyze the phylogenetic relationship with the ZIP superfamily, we compared the amino-acid sequence of TcIT with sequences of putative and functionally characterized Fe transporters from several organisms, using MEGA 7 software [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref].
## Low iron availability induces the expression of tcit
Impaired growth was seen in epimastigotes maintained in IDM compared with those grown in RM . The decrease in cell number was not due to cell death: MTT assays showed that viability of the cells was similar . Although cells in IDM possess TcIT transcripts that increased with Fe removal from the culture medium , this was insufficient for maintaining Fe intracellular content: epimastigotes from IDM had 50% lower intracellular Fe content compared with epimastigotes from RM . In addition, Fe depletion lowered basal O 2 consumption (representative recordings in , comparatively quantified in .
## Plasma membrane localization of tcit
By using immunofluorescence assays, we determined that the labeling of the tagged protein TcIT-HA colocalizes with the labeling of the plasma membrane protein TcSMP, indicating that the Fe transporter is localized in the plasma membrane of epimastigotes.
As negative control, parasites transfected with pTEX-Ø did not stain with HA. Mutants overexpressing TcIT had a higher Fe intracellular content compared with the basal content of the mutant transfected with pTEX-Ø, together with very high transcription levels of TcIT. In agreement with the immunofluorescence results, the plasma membrane-enriched fraction (PM) from pTEX-TcIT had a 39-kDa band corresponding to TcIT.shows a representative Western blot image, where the arrow points to the 39-kDa band; no bands were observed in pTEX-Ø epimastigotes. The densitometric analysis of pTEX-TcIT Western blots (n = 3) of the HG, total membranes, and PM gave, respectively, the following values in arbitrary units: 52.9 ± 8.6, 59.6 ± 2.4, and 133.0 ± 2.9. One-way ANOVA followed by Tukey's test demonstrated that the enrichment of TcIT in PM with respect to HG (160%) and TM (130%) was highly significant (C) Evolutionary relationships of ZIP family members. The evolutionary history was inferred using the neighbor-joining method [bib_ref] The Neighbor-Joining Method: A New Method for Reconstructing Phylogenetic Trees, Saitou [/bib_ref]. The optimal tree with the sum of branch length = 6.09506879 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown at the beginning of each branch [bib_ref] Confidence Limits on Phylogenies: An Approach Using the Bootstrap, Felsenstein [/bib_ref]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and given
## Tcit transporter increases mitochondrial respiratory rates in trypanosoma cruzi
In , we showed that Fe depletion in culture decreases O 2 consumption by T. cruzi. [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref] and [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref] (quantification of O 2 consumption in different respiratory states) show that higher respiration rates are encountered-in phosphorylating and uncoupled conditionswhen TcIT is overexpressed. Permeabilization with digitonin did not alter the profile of basal respiration (data not shown). After adding succinate (the LEAK O 2 consumption), there was no difference in O 2 consumption between pTEX-Ø and pTEX-TcIT epimastigotes, but the subsequent addition of ADP (corresponding to the oxidative phosphorylation-OXPHOS-state) that caused a significant increase of respiration by the two classes of mutants, O 2
## A b
C D E F FIGURE 2 | Growth and respiration of wild-type epimastigotes are dependent on Fe content in culture medium and Fe depletion increases the expression of TcIT.
(A) Influence of culture medium Fe content on T. cruzi proliferation. Wild-type epimastigotes of T. cruzi at mid-log phase were harvested, washed twice, seeded in fresh medium, and grown for the indicated times in regular medium [RM: brain heart infusion medium (BHI) supplemented with 30 µM hemin and 10% fetal bovine serum (FBS)] (filled circles) or in iron-depleted medium (IDM: BHI without hemin supplementation and treated with Chelex for ionic Fe depletion plus 10% irondepleted FBS) (empty circles); ***P < 0.001 (n = 12). (B) Viability of epimastigotes incubated in RM (filled circles) or IDM (empty circles) (5 × 10 7 epimastigotes/ml) was assayed using the MTT assay. (C) Quantification of the TcIT transcript in wild-type T. cruzi epimastigotes. Quantitative PCR was done using 100 ng cDNA from epimastigotes in mid-log phase when maintained in RM (filled circles) or IDM (empty circles); *P < 0.05 (n = 5). The housekeeping GADPH gene was used to normalize qPCR. consumption of TcIT mutants was higher compared with pTEX-Ø mutants. There were a further increase and uncoupled respiration rates (EST, electron transfer system uncoupled from phosphorylation) after the addition of FCCP (a H + ionophore), again higher in overexpressing TcIT. The addition of antimycin A completely abolished mitochondrial respiration in both mutants. Higher mitochondrial O 2 consumption can be coupled to both increased oxidative phosphorylation and higher ROS production. Therefore, we measured total intracellular ATP concentration [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref] and H2O2 formation [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref] in both mutants. Parasites overexpressing TcIT had higher intracellular ATP (ATPi) content, as was expected, and a 2-fold higher content of H 2 O 2 . Nevertheless, there was no difference between the mutants in their activity of the antioxidant enzyme SOD [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref].
Possible ultrastructural changes in epimastigotes that overexpress TcIT were analyzed by transmission electron microscopy [fig_ref] FIGURE 5 |: Ultrastructural changes in TcIT-superexpressing parasites [/fig_ref]. As control, we used parasites transfected with pTEX-Ø. Control epimastigotes presented normal cell and organelle morphology and positioning [fig_ref] FIGURE 5 |: Ultrastructural changes in TcIT-superexpressing parasites [/fig_ref]. We could not find any alteration in the major cellular organelles, including kinetoplast, nucleus, and organelles of the endocytic pathway such as cytostome-cytopharynx complex and reservosomes. Most organelles had normal morphology and positioning in epimastigotes that overexpress TcIT [fig_ref] FIGURE 5 |: Ultrastructural changes in TcIT-superexpressing parasites [/fig_ref] , except for the mitochondria. In mutant parasites, we frequently observed mitochondrial swelling, increase in cristae density, inner membrane vesiculation [fig_ref] FIGURE 5 |: Ultrastructural changes in TcIT-superexpressing parasites [/fig_ref] , and mitochondrial disorganization [fig_ref] FIGURE 5 |: Ultrastructural changes in TcIT-superexpressing parasites [/fig_ref]. These findings may reflect the increase in mitochondrial activity found in the case of parasites overexpressing TcIT.
## Identification of the trypanosoma cruzi epimastigote phenotype overexpressing the tcit transporter: stimulus of differentiation to trypomastigote and replication amastigote rate
Overexpression of TcIT in T. cruzi epimastigotes did not interfere with their in-vitro proliferation [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref] , since these mutants proliferate at the same rate as the epimastigotes transfected with the empty vector pTEX-Ø. However, when epimastigotes transfected with pTEX-TcIT were subjected to in-vitro differentiation in TAU medium, almost 40% of the forms present after 96 h were trypomastigotes, whereas transfection with pTEX-Ø for the same time led to only 20% of the organisms as trypomastigotes [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref]. This indicates that overexpression of the TcIT . The values represent the mean ± SEM. In (C-E), using Student's t-test assessed differences between mean values. In (B), differences were assessed by two-way ANOVA followed by Tukey's test. In all cases, the uncoupled respiration (with FCCP) was significantly higher than the respiration in phosphorylating conditions (with ADP). Both stimulated respiratory states were also significantly higher than those obtained only after addition of succinate (at least P < 0.05). No differences were found between the respiration of pTEX-Ø pTEX-TcIT epimastigotes in the presence of endogenous substrates or after the addition of succinate.
transporter favors in-vitro differentiation. The cytometric analysis [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref] using the 1G7 antibody against the marker of metacyclogenesis GP90 demonstrated, after the 4th day in TAU, a higher number of GP90(+) cells in parasites transfected with TcIT than in those transfected with pTEX-Ø (54.5% vs. 46%). In contrast, the number of GP90(−) cells was higher in pTEX-Ø parasites (54% vs. 46.4%). The hypothesis that TcIT overexpression stimulates metacyclogenesis is also supported by the fact that trypomastigotes have higher levels of TcIT mRNA than epimastigote forms [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref]. In fact, LLC-MK2 cells infected with metacyclic trypomastigotes derived from epimastigotes pTEX-TcIT present a higher replication rate of amastigotes (parasites/LLC-MK2 cells) when compared with those LLC-MK2 cells infected with pTEX-Ø metacyclics [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref]. Representative images of the amastigote forms inside LLC-MK2 cells are shown in [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref].
# Discussion
Trypanosoma cruzi has a high requirement for Fe, mobilizing heme or non-heme Fe, for both in-vitro proliferation of epimastigotes and in-vivo proliferation in mice [bib_ref] Role of Iron in Trypanosoma Cruzi Infection of Mice, Lalonde [/bib_ref]. In mammalian cells, Fe is complexed with proteins, e.g., ferritin, which has the highest number of bound Fe per molecule of protein; hemoglobin, which carries the largest amount of Fe in the body; lactoferrin, mainly found in mucosae; and finally, transferrin (Tf), a seric protein responsible for transporting Fe to all cells [bib_ref] Iron Availability and Infection, Weinberg [/bib_ref]. The identification of a Tf receptor in T. cruzi amastigote forms [bib_ref] Trypanosoma cruzi Receptors for Human Transferrin and Their Role, Lima [/bib_ref] and the fact that these forms can take up human transferrin directly by the endocytosis pathway via the cytostome [bib_ref] Trypanosoma Cruzi Epimastigote Endocytic Pathway: Cargo Enters the Cytostome and Passes Through..., Porto-Carreiro [/bib_ref] [bib_ref] Attachment of Flagellum to the Cell Body Is Important to the Kinetics..., Rocha [/bib_ref] indicate that T. cruzi amastigotes can use Tf as the major Fe source. Trypanosoma cruzi may take up hemin through an ABC transporter [bib_ref] Heme Requirement and Intracellular Trafficking in Trypanosoma cruzi Epimastigotes, Lara [/bib_ref] [bib_ref] The Heme Uptake Process in Trypanosoma cruzi Epimastigotes Is Inhibited by Heme..., Cupello [/bib_ref]. Before heme can be utilized, its degradation by heme oxidase (HO) is necessary; however, the HO gene is absent in the T. cruzi genome [bib_ref] The Genome Sequence of Trypanosoma Cruzi, Etiologic Agent of Chagas Disease, El-Sayed [/bib_ref] , limiting Fe removal from the heme ring [bib_ref] Iron Metabolism in Trypanosomatids, and Its Crucial Role in Infection, Taylor [/bib_ref]. In this way, reducing Fe 3+ to Fe 2+ releases the ion from the extracellular hemin, thus facilitating Fe uptake by heavy-metal transporters, as proposed for Leishmania parasites [bib_ref] Iron-Saturated Lactoferrin and Pathogenic Protozoa: Could This Protein be an Iron Source..., Ortıź-Estrada [/bib_ref]. Identifying Fe-reductase TcFR in T. cruzi plasma membranes [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref] opened up the possibility of the same mechanism being present in the parasite. Ferric ion reduction to Fe 2+ by Fe-reductases is usually coupled to Fe 2+ transport in bacteria, yeasts, plants, and animal cells (Huynh cruzi at mid-log phase were harvested, washed twice, seeded into fresh medium, and grown for the indicated times in regular medium under the antibiotic pressure of G418 (50 mg/ml) (n = 6). (B) Transfected epimastigotes with empty vector pTEX-Ø (white circles) or pTEX-TcIT (gray circles) were exposed to in-vitro differentiation medium TAU. Each day, epimastigotes and trypomastigotes were differentially counted to determine the percentage of trypomastigotes in culture; *P < 0.05 (n = 4). (C) Cytometric analysis of parasites after the 4th day in TAU (B) using the 1G7 antibody against the marker of metacyclogenesis GP90. (D) Quantification of the TcIT transcript in T. cruzi epimastigotes (EPI) or trypomastigotes (TRYP). Quantitative PCR used 100 ng cDNA from mutants, as indicated on the abscissa; ***P < 0.001 (n = 3). (E) Index of association estimated by total number of intracellular parasites per infected LLC-MK2 cells. ***P < 0.001 (n = 6). (F) Representative images of pTEX-Ø or pTEX-TcIT parasites infection in LLC-MK2 cells, quantitatively compared in (E). Except for (C), using Student's t-test assessed the differences between mean values. In , the differences were assessed by comparing time-matched determinations. In (C), the comparisons were performed by using two-way ANOVA. **P < 0.01 (n = 3).
and Andrews, 2008). The identification of an Fe-reductase in L. chagasi [bib_ref] Leishmania Chagasi: Uptake of Iron Bound to Lactoferrin or Transferrin Requires an..., Wilson [/bib_ref] , L. amazonensis, and recently, in T. cruzi [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref] strongly suggests that trypanosomatids have Fe 2+ transport systems. In this way, the identification of a putative Fe transporter in the T. cruzi genome, TcIT (TriTryDB: TCDM_06386) modeled in [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref] , which is homologous to the newly described Fe transporter in L. amazonensis, LIT (TritrypDB: LmjF.31.3060), and the Arabidopsis thaliana Fe transporter IRT1 (GenBank: AAB01678.1), supports this idea. Indeed, analysis of the predicted amino-acid sequence shows eight transmembrane domains, as shown for TcIT, LIT, and IRT1 sequences [fig_ref] FIGURE 1 |: Model for the iron [/fig_ref]. Both Leishmania LIT1 and Arabidopsis IRT1 are Fe 2+ transporters from the ZIP family, and the amino-and carboxyl-terminal ends are located on the extracellular side of the plasma membrane. By alignment analysis, we found that, as in LIT1 and IRT1, T. cruzi TcIT has the same features. The most conserved portion of ZIP family proteins is also present; the amphipathic helix of the putative transmembrane domain IV contains a His residue and an adjacent semipolar Ser residue, essential components for heavy-metal binding sites [bib_ref] The ZIP Family of Metal Transporters, Guerinot [/bib_ref] [bib_ref] A Leishmania amazonensis ZIP Family Iron Transporter Is Essential for Parasite Replication..., Huynh [/bib_ref]. Thus, TcIT is the first member of the ZIP family to be identified in T. cruzi. A wide range of biological processes, such as electron and O 2 transport and DNA synthesis, depend on Fe content, making Fe homeostasis an ensemble of processes that are essential for all cells, including pathogenic protozoa [bib_ref] Iron Transport & Homeostasis Mechanisms: Their Role in Health & Disease, Nadadur [/bib_ref] [bib_ref] Iron-Saturated Lactoferrin and Pathogenic Protozoa: Could This Protein be an Iron Source..., Ortıź-Estrada [/bib_ref]. Upregulation of TcIT mRNA levels in response to Fe depletion indicated that the parasite has a compensatory mechanism, although insufficient for sustaining the Fe requirement , possibly because the Fe content is very low in IDM. Low O 2 consumption in parasites maintained in Fe-depleted medium suggests that FIGURE 7 | Proposed model of functional coupling between TcFR and TcIT in the plasma membrane of T. cruzi, which allows Fe 3+ reduction, Fe 2+ uptake, its delivery to the cytosol, and through reaching the mitochondria either via an unknown iron transporter present in the inner mitochondrial membrane or bound to a carrier protein or metalloprotein. Once in the mitochondria, Fe 2+ stimulates H 2 O 2 formation from an excess of the substrate O ·− 2 , and differentiation to trypomastigote forms, as depicted in the amplified graphical insert of a mitochondrial structure. Green filled circles represent Fe 2+ ions entering the mitochondrial matrix via internalized TcIT molecules that could insert in the mitochondrial internal membrane after their cycling into the cytosol. H 2 O 2 -induced mitochondrial remodeling could be responsible for the activation of differentiation, as recently proposed for other scenarios where epimastigotes differentiate into trypomastigotes [bib_ref] Starvation and pH Stress Conditions Induced Mitochondrial Dysfunction, ROS Production and Autophagy..., Pedra-Rezende [/bib_ref]. Created with BioRender.com.
Fe depletion arrests mitochondrial function. Plasma membrane immunolocalizationand enrichment in the plasmamembrane fractionalso strongly suggest that TcIT is responsible for Fe uptake in T. cruzi epimastigotes, ensuring differentiation to infective trypomastigotes. TcIT could be translocated to the inner mitochondrial membrane via an unknown mechanism involving membrane fusion despite the evident immunolocalization in the parasite plasma membrane. In addition, Fe 2+ could reach the mitochondria through an unknown iron transporter present in the inner mitochondrial membrane or bound to a carrier protein or metalloprotein. This could be one of the ways to allow Fe to interact with the mitochondrial complexes, thus stimulating respiration and H 2 O 2 formation.
Parasites overexpressing TcIT [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref] have a higher capacity for differentiating into trypomastigotes in vitro [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref]. These forms express proteins (such as gp83 glycoprotein, cruzipain, oligopeptidase B, and P21) that are involved with host-cell invasion [bib_ref] Signaling and Host Cell Invasion by Trypanosoma Cruzi, Burleigh [/bib_ref] [bib_ref] Trypanosoma cruzi: Parasite and Host Cell Signaling During the Invasion Process, Yoshida [/bib_ref] [bib_ref] The Recombinant Form of Trypanosoma cruzi P21 Controls Infection by Modulating Host..., Martins [/bib_ref]. In this regard, and as an example of maintenance of chronic infection, it is of interest that P21 was recently demonstrated to be important for intracellular confinement of T. cruzi amastigotes [bib_ref] The Recombinant Form of Trypanosoma cruzi P21 Controls Infection by Modulating Host..., Martins [/bib_ref]. In addition, parasites that overexpress TcIT display an increase in the average number of intracellular parasites per LLC-MK2-infected cells after 48 h post-infection [fig_ref] FIGURE 6 |: TcIT overexpression influences T [/fig_ref] , demonstrating the importance of Fe transporter in increasing the rate of amastigote replication.
In Leishmania, upregulation of LIT1 expression activates promastigote to amastigote differentiation, mainly via Fe-SOD activity. Fe-SOD activity converts O ·− 2 to H 2 O 2 , a molecule that triggers the generation of infective amastigotes [bib_ref] Iron Uptake Controls the Generation of Leishmania Infective Forms Through Regulation of..., Mittra [/bib_ref]. Although H 2 O 2 production is very high in parasites that overexpress TcIT [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref] , Fe-SOD activity is not modulated by the presence of the gene [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref] , indicating that elevated levels of H 2 O 2 are the result of accelerated dismutation of an elevated O ·− 2 formation. In T cruzi, the significant increase in H 2 O 2 production seems to be due to mitochondrial activity [fig_ref] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity [/fig_ref]. Indeed, the mitochondria were the only organelles where significant changes were observed [fig_ref] FIGURE 5 |: Ultrastructural changes in TcIT-superexpressing parasites [/fig_ref]. We propose that the 200% increase in H 2 O 2 could be key for T. cruzi infectivity and virulence, as demonstrated by the persistence of amastigotes in macrophages of infected mice [bib_ref] Oxidative Stress Fuels Trypanosoma cruzi Infection in Mice, Paiva [/bib_ref]. Since heme induces epimastigote proliferation via mitochondrial ROS production without altering ATP production [bib_ref] Heme Modulates Trypanosoma cruzi Bioenergetics Inducing Mitochondrial ROS Production. Free Radic, Nogueira [/bib_ref] , it may be that TcIT overexpression induces epimastigote to trypomastigote differentiation through a different signaling pathway. Therefore, we conclude that this work presents evidence regarding the possible participation of TcIT in Fe metabolism, proliferation/differentiation processes, infectivity virulence, and maintenance of T. cruzi infection. Since the lack of correlation between the increase of TcIT expression in IDM and the Fe content in the parasite raises the question of whether the protein is the only mechanism responsible for Fe uptake in T. cruzi, further transport or generation and analysis of mutant cell lines need to be performed in the future.
Finally, presents a hypothetical mechanistic model for the functional coupling between TcFR [bib_ref] A Ferric Reductase of Trypanosoma cruzi (TcFR) Is Involved in Iron Metabolism..., Dick [/bib_ref] and
TcIT for Fe 2+ uptake across the plasma membrane of T. cruzi, which increases mitochondrial H 2 O 2 formation and stimulates parasite differentiation, thus allowing invasion of the host cells.
# Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
# Author contributions
CD conceived, designed, and performed the experiments; analyzed the data; and wrote the manuscript. NR-M, AD-S, LC-K, and CA contributed to the experimental process. CA and NC-S analyzed the data and revised the manuscript. JM-F and AV conceived the experiments, analyzed the data, and revised the manuscript. All authors contributed to the article and approved the submitted version.
[fig] FIGURE 1 |: Model for the iron (Fe) transporter TcIT: alignment and phylogenetic analysis for TcIT and ZIP family members from different species. (A) Structural model of Fe transporter from T. cruzi (TcIT). The model was constructed using the protein structure prediction PHYRE (www.sbg.bio.ic.ac.uk/phyre/)(Kelley and Sternberg, 2009) and visualized with the standard molecular viewer PyMOL 2002 (PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA; http://pymol.sourceforge.net/). The eight transmembrane helices are shown in pink, and labels highlight the five key residues mentioned in the text. (B) Alignment of amino-acid sequences from T. cruzi TcIT, DM28c strain (TriTryDB: TCDM_06386), L. major LIT1 (TritrypDB: LmjF.31.3060), and Arabidopsis thaliana IRT1 (GenBank: AAB01678.1). Conserved residues responsible for Fe transport in IRT1 and LIT1(Jacques et al., 2010) are indicated in red boxes. Predicted transmembrane domains are underlined in black. Alignment used the ClustalW algorithm. Identical amino acids are indicated with an asterisk (*). Conservation between amino acid groups of strongly similar properties is indicated by a colon (:). Conservation between amino acid groups of weakly similar properties is indicated by a period (.). [/fig]
[fig] FIGURE 3 |: (D) Intracellular Fe content in epimastigotes maintained in RM (black bars) or IDM (empty bars); **P < 0.01 (n = 6). (E) Representative recordings of O 2 consumption rates vs. time (pmol/s per cell; lower traces in both panels) and decrease of O 2 concentration (nmol/ml; upper traces in both panels): 5 × 10 7 epimastigotes/chamber from RM (upper panel) or IDM (lower panel) were harvested, washed twice, and used to measure O 2 consumption in reaction medium (2 ml) containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 50 mM HEPES-Tris (pH 7.2). (F) Quantification of O 2 consumption by epimastigotes incubated in RM (black bars) or IDM (empty bars) (***P < 0.001; n = 6). In all cases, using unpaired Student's t-test assessed differences between mean values. In (A), the differences were assessed by comparing time-matched determinations. (A) Immunofluorescence of epimastigotes shows TcIT expression in plasma membranes. Trypanosoma cruzi epimastigotes were transfected with empty vector (pTEX-Ø) or TcIT tagged with hemagglutinin (pTEX-TcIT). DIC: differential interference contrast microscopy. TcSMP: images using antisera for this surface membrane protein and the secondary anti-mouse-Alexa 546 (red). HA: TcIT expression using anti-HA and secondary anti-rabbit FITC (green). DAPI: nuclei stained with DAPI (blue). In merge, the green fluorescent staining for TcIT overlaps with the red fluorescent signal for TcSMP, suggesting the proteins are in the same region of the plasma membrane; ×60 magnification, scale bar 4 mm. (B) Intracellular Fe content is higher in epimastigotes transfected with pTEX-Ø (empty bar) or pTEX-TcIT (gray bar); **P < 0.01 (n = 6). (C) Quantification of the TcIT transcript in T. cruzi epimastigotes. Quantitative PCR was carried out using 100 ng cDNA from mutants pTEX-Ø (empty bar) or pTEX-TcIT (gray bar); ***P < 0.001 (n = 4). In both cases, using Student's t-test assessed differences between mean values.(D) Representative Western blot using homogenate (HG), total membrane (TM), plasma membrane (PM), and cytosol (C) fractions from pTEX-TcIT epimastigotes, as indicated in the upper part of the image, and the antibody anti-HA. (E) Representative Western blot using HG, TM, PM, and C fractions from pTEX-Ø epimastigotes, as indicated in the upper part of the image, and the antibody anti-HA. The predicted size of TcIT is 39.8 kDa. (F, G) Ponceau red loading controls for representative Western blots of membranes from pTEX-TcIT and pTEX-Ø epimastigotes, as indicated in the upper left corners. [/fig]
[fig] FIGURE 4 |: Effect of TcIT overexpression on epimastigote mitochondrial physiology and Fe-SOD activity. (A) Representative recordings of O 2 consumption and decrease of O 2 concentration. Trypanosoma cruzi epimastigotes transfected with pTEX-Ø empty vector (upper panel) or pTEX-TcIT plasmid (lower panel) were initially tested for respiration of intact epimastigotes. Epimastigotes were then digitonin-permeabilized, as described inNogueira et al. (2017), to measure mitochondrial activity after the successive additions indicated by arrows: Rot (rotenone), Succ (succinate), ADP, FCCP, and AA (antimycin A). (B) Graphic representation of O 2 consumption after the additions shown on the abscissa: SUCC, ADP, FCCP, and AA; **P < 0.01, *P < 0.05 (n = 4). (C) Intracellular ATP in T. cruzi epimastigotes transfected with pTEX-Ø empty vector (empty bar) or pTEX-TcIT plasmid (gray bar) was measured as described in M&M section. *P < 0.05 (n = 3). (D) Production of H 2 O 2 by living cells of T. cruzi epimastigotes transfected with pTEX-Ø empty vector (empty bar) or pTEX-TcIT plasmid (gray bar); **P < 0.001 (n = 3). (E) SOD activity was assessed from epimastigotes transfected with pTEX-Ø empty vector (empty bars) or pTEX-TcIT plasmid (gray bars) [/fig]
[fig] FIGURE 5 |: Ultrastructural changes in TcIT-superexpressing parasites. pTEX-Ø and epimastigotes overexpressing TcIT were processed and observed by transmission electron microscopy. (A-C) Control pTEX-Ø epimastigotes show normal morphology of major cellular organelles: kinetoplast (K), nucleus (N), Golgi complex (GC), cytostome-cytopharynx complex (Cyt), and reservosomes (R). Mitochondrial branches are seen all along the cell body, juxtaposing the cell membrane (arrowheads). F (flagellum). (D-H) Epimastigotes overexpressing TcIT. (D) Mutants also present normal organelle morphology and positioning when compared with controls, except for the mitochondria. (E) Swollen mitochondrial branches (arrowheads) are observed in overexpressing parasites. (F) Increased magnification of the area delimited by the rectangle in (E) shows an increase in mitochondrial cristae and inner membrane vesiculation (arrowheads). (G) Mitochondrial inner vesiculation was observed at the kinetoplast (K) region (arrowheads). (H) Areas of mitochondrial inner membrane disorganization were also observed (arrows). Bars: (A-C, E, F, H) 500 nm; (D) 1 µm; (G) 200 nm. [/fig]
[fig] FIGURE 6 |: TcIT overexpression influences T. cruzi differentiation to trypomastigotes, but not proliferation. (A) T. cruzi epimastigotes transfected with pTEX-Ø empty vector (white circles) or pTEX-TcIT plasmid (gray circles) proliferation. Epimastigotes of T. [/fig]
|
Entorhinal cortex and parahippocampus volume reductions impact olfactory decline in aged subjects
Introduction: Pathological abnormalities first appear in the medial temporal regionsincluding entorhinal cortex and parahippocampus in patients with Alzheimer's disease. Previous studies showed that olfactory decline in elderly subjects was associated with volume reductions in the left hippocampus and left parahippocampus without cognitive impairment. The aim of this study is to investigate the link between olfaction and volume reductions in the medial temporal regions including the parahippocampus, entorhinal cortex, and hippocampal subfields.Method: 27 elderly subjects and 27 young controls were measured olfaction acuity, cognitive function, and structural magnetic resonance imaging. Image processing and gray matter volumetric segmentation were performed with FreeSurfer. Volume data were analyzed with SPSS Statistics software.Results:Interesting results of this study were that volume reduction in the entorhinal cortex was not directly linked with declining olfactory ability. Volume reduction in the left entorhinal cortex was correlated with volume reduction in the left parahippocampus and dentate gyrus. However, left parahippocampus volume reduction had the greatest impact on olfactory decline, and the entorhinal cortex and dentate gyrus might additionally contribute to olfactory decline.Conclusion:Our results indicate that olfactory decline may be directly reflected in the medial temporal regions as reduced parahippocampus volumes, rather than as morphological changes in the entorhinal cortex and hippocampus. The parahippocampus may play an important role in the association between memory retrieval and olfactory identification.K E Y W O R D Sbrain volume, entorhinal cortex, memory, olfactory decline, parahippocampus
## | introduc ti on
Olfactory impairment is the first sign of Alzheimer's disease (AD) and Parkinson's disease [bib_ref] Olfactory dysfunction in parkinsonism: General deficit unrelated to neurologic signs, disease stage,..., Doty [/bib_ref].
Neuroimaging studies have revealed that pathological changes in these patients begin in the amygdala (AMG) and hippocampus (HI) and especially the parahippocampus (para-HI) and entorhinal cortex (ENT), which having an important role in olfactory recognition and identification [bib_ref] Olfactory dysfunction in parkinsonism: General deficit unrelated to neurologic signs, disease stage,..., Doty [/bib_ref] [bib_ref] Olfaction in neurodegenerative disease: A meta-analysis of olfactory functioning in Alzheimer's and..., Mesholam [/bib_ref]. In addition to neurodegenerative symptoms, patients with mild cognitive impairment (MCI) have also olfactory [bib_ref] Association between olfactory dysfunction and amnestic mild cognitive impairment and Alzheimer disease..., Roberts [/bib_ref] as well as healthy elderly subjects [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref].
The ability to identify a specific odor involves two capacities.
First, the chemical signature must be transmitted from the olfactory bulb to the ENT, which has direct projection to the HI [bib_ref] Hippocampal-neocortical interaction: A hierarchy of associativity, Lavenex [/bib_ref]. The second is olfactory identification ability, that is, the ability to categorize the odor by a single name and recall the memory of the odor through engaging the neural network connecting the HI and para-HI to the orbitofrontal cortex (OFC), which is a center for odor recognition [bib_ref] The rules of formation of the olfactory representations found in the orbitofrontal..., Rolls [/bib_ref]. Olfactory impairment could be caused by abnormalities in the "input" or "output" of this system or both factors, depending on the functional state and connectivity in the brain.
A previous study reported that the detection threshold for odor was intact in patients with AD, but recognition of the odor was specifically impaired [bib_ref] Markers of brain illness may be hidden in your olfactory ability: A..., Masaoka [/bib_ref]. Accordingly, we hypothesized that the "output' portion of olfactory ability might be impaired and the "input' ability remained intact. Based on this hypothesis, we used structural neuroimaging data to investigate whether volume changes in olfactory areas, AMG, HI, OFC, para-HI, rectus, and middle frontal cortex (MFC) were associated with olfactory decline in aged subjects with normal cognitive function [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref].
Although normal elderly subjects had reduced bilateral AMG, HI, OFC, and MFC and right rectus volumes compared with younger subjects, these reductions were not correlated with olfactory ability.
In contrast, no difference was found in the para-HI volume between the two age groups. However, a significant correlation was observed between para-HI volume and olfactory ability in aged subjects, indicating that para-HI volume changes exhibit individual differences in elderly subjects. Therefore, we suggest that declining olfactory ability begins with para-HI volume reduction [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref].
The para-HI may be the key area for olfactory identification; however, the ENT is anatomically near the para-HI and has been reported to exhibit initial pathological changes in patients with dementia [bib_ref] Molecular drivers and cortical spread of lateral entorhinal cortex dysfuntion in preclinical..., Khan [/bib_ref] [bib_ref] PET Tau and Amyloid-β Burden in Mild Alzheimer's Disease: Divergent relationship with..., Koychev [/bib_ref]. Indeed, the ENT has a direct projection to the HI including the dentate gyrus (DG), CA3 and CA1 regions and subiculum [bib_ref] The three -dimensional organization of the hippocampal formation: A review of anatomical..., Amaral [/bib_ref].
In turn, the CA1 region and subiculum project back to the ENT [bib_ref] Macaque monkey retrosplenial cortex: II. Cortical afferents, Kobayashi [/bib_ref] [bib_ref] Hippocampal efferents reach widespread areas of cerebral cortex and amygdala in the..., Rosene [/bib_ref] , playing the role of the hub of the hippocampal formation. Regarding the olfaction site, the ENT is one of the primary olfactory areas that directly projected from the olfactory bulb [bib_ref] Time course of odorant-induced activation in the human primary olfactory cortex, Sobel [/bib_ref]. Accordingly, pathological abnormalities in the ENT will impair transmission of information from the olfactory bulb (olfactory input) and the relay of signals to the HI for memory formation as well as projection back to the neocortex (output abilities). However, it remains unclear whether olfactory impairment is caused by disruption of the relationship between the ENT and hippocampal subfields or association between the ENT and para-HI in the beginning of olfactory decline before the onset of dementia.
A previous study [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref] reported that the para-HI volume, which included the ENT volume, predicted olfactory recognition ability. In this study, we separately measured the para-HI and ENT volumes, as well as that of the hippocampal subfields, and investigated a relationship between volume changes in these structures and olfactory ability in elderly subjects. To explore this idea, we hypothesized a link between olfactory identification and the olfactory circuit based on our path analysis results.
## | me thods
## | participants
The subjects included in this study comprised a subset of subjects from a previously published study [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref] wma.net/polic ies-post/wma-decla ratio n-of-helsi nki-ethic al-princ iples -for-medic al-resea rch-invol ving-human -subje cts/).
## | olfactory and cognitive assessments
T&T olfaction test was used to test all subjects' odor detection acuity and odor recognition acuity (Takasuna Co., Ltd.). The T&T test is a Japanese standardized olfactory ability test and often used in otorhinolaryngology for diagnosing patients with anosmia and hyposmia in Japan. The T&T test has been used for patients with neurodegenerative disorder including Parkinson's and Alzheimers' disease [bib_ref] Markers of brain illness may be hidden in your olfactory ability: A..., Masaoka [/bib_ref]. The test had correlation with the University of Pennsylvania Smell Identification Test [bib_ref] A study of the relationship between the T&T olfactometer and the University..., Kondo [/bib_ref]. The test has five odors. Odor A is the odor of rose (β-phenyl ethyl alcohol); odor B is the odor of caramel (methyl cyclopentenolone); odor C is the odor of rotten food (iso-valeric acid); odor D is the odor of peach (γ-undecalactone); odor E is the odor of lichen refuse (skatole). Each odor was dissolved in propylene glycol with eight different concentrations. Subjects were tested beginning with the lowest concentration and were repeated with higher concentrations. The odors were presented randomly but at the same concentration in each trial. The odor was perceived but not identified was considered the "odor detection." As increasing odor concentration, the subject could identify the odor. The subjects were required to naming each odor and describe the type of odor. Odor was first identified con-
## | mri data acquisition
All MRI data were measured at Ebara Hospital using a Siemens 3
Tesla MAGNETOM Prisma fit (Siemens) with a 64-channel phasedarray coil. The anatomical scan was measured following parameters: T1-weighted 3D MPRAGE sequence (9 degree flip angle; TR 2300 ms; TE 2.98 ms; matrix size 256 × 256; field of view 256 mm;
176 slices with a voxel size of 1 mm 3 .)
## | mri data analysis
Images were processed using FreeSurfer (Version 6.0) automated neuroanatomical segmentation software (http://surfer.nmr.mgh.
harva rd.edu). Gray matter volumes were determined on each T1weighted scan with the recon-all script on FreeSurfer. FreeSurfer automatically performs volumetric segmentation [bib_ref] Whole brain segmentation: Automated labeling of neuroanatomical structures in the human brain, Fischl [/bib_ref] , cortical surface reconstruction [bib_ref] Cortical surface-based analysis. II: Inflation, flattening, and a surface-based coordinate system, Fischl [/bib_ref] , and parcellation [bib_ref] An automated labeling system for subdividing the human cerebral cortex on MRI..., Desikan [/bib_ref] In addition to volume measurements of the medial temporal regions, olfactory sulcus volume was also measured because it has been reported to reflect olfactory bulb volume.
For analyses of the brain volume, intracranial volume (ICV) was used as a covariate. In the previous report [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref] , we compared the results of volumetric group comparison between analysis using ICV measured with FreeSurfer and ICV measured with the Statistical Parametric Mapping 12 (SPM12) program [bib_ref] Unified segmentation, Ashburner [/bib_ref]. The results indicated no significant difference in the ICVs calculated by the two methods. In this study, we used the ICV calculated by FreeSurfer as a covariate for the statistical analysis.
# | data analysis
Group differences in left and right hippocampal subfields were assessed using analysis of covariance (ANCOVA) with sex, years of education, and ICV included as covariates. Group comparisons of olfactory sulcus volumes were also performed using ANCOVA, with sex, years of education, and ICV as covariates. Volume data analyzed with FreeSurfer were entered into SPSS Statistics software (IBM SPSS Statistics, version 23.0, IBM Corp).
Before performing path analysis, partial correlation and multiple regression analyses were performed to refine our hypothesis.
Partial correlation tests within-group were conducted between olfactory recognition scores and the para-HI, ENT, and hippocampal subfields, covarying with differences in age, years of education, and sex. Then, multiple regression analysis was performed using the para-HI as an independent variable and the ENT and hippocampal subfields as dependent variables. Next, path analysis was A GFI close to 1 and a Bollen-Stine bootstrap p value > .05 were adopted for the model.
## For exploratory analyses, partial correlations between olfactory
sulcus volumes and olfactory detection or recognition scores were analyzed within groups, with age, years of education, and sex as covariates. shows the demographic data. There were significant differences in age, years of education, and MoCA-J between HCs and elderly subjects (the detailed statistical results were reported in [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref]. Lower olfactory detection ability was observed in elderly subjects than young HCs. Impaired olfactory recognition was observed in five elderly subjects, and other 22
## | re sults
subjects showed lower olfactory recognition abilities than young
HCs [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref]. All elderly subjects had hyposmia, which is a decreased sense of smell. In the current study, two types of hyposmia were observed. Five elderly subjects who impaired olfactory recognition were able to detect odors, but had difficulty identifying each odor, even when they were tested with higher odor concentrations. The other 22 subjects were able to both detect and identify odors, but their detection and recognition abilities were significantly worse than those of young HCs. That is, these 22 elderly subjects were only able to detect and recognize odors at higher odor concentrations compared with the young HCs. But all subjects had no complain about declining or loss of olfactory ability in daily life.
Comparisons of volumes of olfactory brain regions AMG, HI, OFC, rectus, and MFC volumes were reported in the previous study [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref]. Elderly subjects had smaller intracranial, whole brain, right rectus, bilateral AMG, HI, OFC, and MFC volumes than younger subjects (the detailed statistical results were reported in [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref]. There were also no correlations between right olfactory sulcus volumes and olfactory detection scores (young HCs, r = −.13, p = .5, elderly subjects, r = −.05, p = .83), or between right olfactory sulcus volumes and olfactory recognition scores (HCs, r = .21, p = .32, elderly subjects, r = .25, p = .28).
## Brain regions
## Ta b l e 2
Comparison of brain regions between healthy controls and elderly subjects 3.1 | Partial correlation and multiple regression analyses were performed to refine our hypothesis Partial correlations between olfactory recognition scores and para-HI, ENT, and hippocampal subfield volumes covaried with differences in age, sex, and years of education in elderly subjects (N = 22, impaired olfaction subjects were not included). All statistical results are included in . In brief, the left para-HI was negatively corre-
# | path analysis
Finally, path analyses were performed to test the predictive ability of interactions of each brain region' volume for olfactory impairment in elderly subjects . The model fitness was confirmed by the following indices: Bollen-Stine bootstrap, p = .07, GFI, 0.9.
Decreased olfactory recognition ability was predicted by a small left para-HI volume . The ENT volume indirectly impacted olfactory recognition via the para-HI. This indirect path had a greater effect on olfactory recognition than the direct path from the para-HI.
The volume change of the DG predicted ENT volume, and olfactory recognition was affected via ENT and para-HI volume changes. All statistical details were shown in . Before finalizing the path model, nonsignificant paths were eliminated. A model that was created before the final path included the important nodes (CA1, CA3, GC-DC, and subiculum) where a direct link to the DC and ENT was indicated (see and [fig_ref] Table 2: shows statistical differences for each brain region between elderly and young HCs [/fig_ref] , which includes the statistical results). Young HCs were tested with the same steps and showed no significant paths (all path, p > .05).
## | d iscuss i on
A previous study showed that olfactory decline without cognitive impairment was predicted by left HI and left para-HI volume reductions [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref]. We investigated in this study the link between olfaction and volume reduction in the medial temporal regions including the para-HI, ENT, and hippocampal subfields.
The most interesting finding in this study was that volume reduction in the ENT was not directly associated with a decline in olfactory ability. ENT volume reduction was associated with para-HI volume reduction, and the ENT indirectly affected the olfactory ability via the para-HI .
## | medial temporal regions as a center of olfactory processing
The role of the medial temporal regions in memory function has been established [bib_ref] The entorhinal cortex of the monkey. I. Cytoarchitectonic organization, Amaral [/bib_ref] [bib_ref] The three -dimensional organization of the hippocampal formation: A review of anatomical..., Amaral [/bib_ref] [bib_ref] Perirhinal and parahippocampal cortices of the macaque monkey: Cortical afferents, Suzuki [/bib_ref] , and these regions act as a center of olfactory processing. Olfactory input is transmitted directly to the medial temporal regions, especially the ENT, which receives direct olfactory input [bib_ref] Time course of odorant-induced activation in the human primary olfactory cortex, Sobel [/bib_ref] and
acts as a gateway to the HI for memory processing [bib_ref] The entorhinal cortex of the monkey. I. Cytoarchitectonic organization, Amaral [/bib_ref].
Indeed, two abilities are required for human olfaction, including detection of olfactory molecules and identification and naming of the target through memory retrieval. Damage to the ENT in AD has been reported to cause inability to detect odor and impairment of memory retrieval processing. We conjectured that volume reduction of the ENT might be related to olfactory decline, even in healthy elderly individuals. However, consistent with our previous study [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref] , that the left para-HI volume change impacted crucial on olfactory recognition rather than volume change of the ENT.
The para-HI is connected to the hippocampal formation, particularly via the ENT. Neocortical inputs from the OFC, medial prefrontal cortex, and insular cortex are sent to the ENT, which predominately provides cortical input to the DG [bib_ref] The entorhinal cortex of the monkey. I. Cytoarchitectonic organization, Amaral [/bib_ref]. However, the F I G U R E 2 Path diagram and standardized path coefficients of elderly subjects. The solid lines indicate significant standardized direct effects. The dotted lines indicate significant standardized indirect effects. *p < .05, **p < .01, ***p < .0001 Statistical details were indicated in . DG, dentate gyrus, ENT, entorhinal cortex, para-HI, parahippocampus para-HI converges inputs from multiple modalities and projects to the ENT [bib_ref] Perirhinal and parahippocampal cortices of the macaque monkey: Cortical afferents, Suzuki [/bib_ref]. The para-HI also receives efferent projections from the ENT and plays a role as a hub for input and output pathways [bib_ref] Perirhinal and parahippocampal cortices of the macaque monkey: Cortical afferents, Suzuki [/bib_ref]. According to these anatomical and functional relationships, we suggest that impairment of olfactory recognition might result from disruption of the association between olfactory information and the context of memory retrieval, in which the para-HI participates in convergence of information from multiple sources.
## | relationship between the ent and dg in olfaction
Additionally, volume reduction of the left DG was directly linked to volume reduction of the left ENT. Overall, volume reduction of the hippocampal subfields was observed in aged subjects, but the ENT volume was relatively resistant to aging. Therefore, volume reduction in the left ENT and left para-HI was individualized and separated from hippocampal subfield volume reduction. This finding is consistent with that of [bib_ref] Hippocampal subfield volumes: Age, vascular risk, and correlation with associative memory, Shing [/bib_ref] , who showed volume reduction associated with age in the CA1 region, DG, and CA3 region with relative preservation of the ENT volume.
However, among the hippocampal subfields, the left DG volume had positive correlation with the left ENT volume and indirectly affected olfactory recognition. Therefore, the degree of volume reduction of the left DG may exhibit individual differences. The ENT is directly connected to the DG, CA3 region, CA1 region, and subiculum [bib_ref] The three -dimensional organization of the hippocampal formation: A review of anatomical..., Amaral [/bib_ref]. Atrophy of the DG and CA3 region is correlated with age-related memory loss [bib_ref] Hippocampal subfield volumes: Age, vascular risk, and correlation with associative memory, Shing [/bib_ref]. The pathway between the DG and ENT has important role for associative memory information.
Our study only investigated the structural changes related to olfaction ability, and determination of a bi-directional cause and effect relationship between the DG and ENT was difficult. However, reduced input from the ENT to the DG might cause decreased neurogenesis.
Newborn neurons are generated within DG and the olfactory bulb in the adult mammalian brain . In the DG, granule cells are generated throughout the life span [bib_ref] Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats, Altman [/bib_ref] [bib_ref] Postnatal neurogenesis in the guinea-pig, Altman [/bib_ref] [bib_ref] Time of neuron origin in the hippocampus and dentate gyrus of normal..., Caviness [/bib_ref] [bib_ref] Subgranular zone of the dentate gyrus of young rabbits as a secondary..., Gueneau [/bib_ref] [bib_ref] Neurogenesis in the dentate gyrus of the adult rat: Age-related decrease of..., Kuhn [/bib_ref]. In addition, neurogenesis of new cells was observed in the adult human brain [bib_ref] Neurogenesis in the adult human hippocampus, Eriksson [/bib_ref]. Because the ENT has a direct link to the DG, the production of neurons in the DG is transiently increased by stimulation of the ENT [bib_ref] Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory, Stone [/bib_ref]. If the production of neurons is reflected as the DG volume, this volume might be affected by the functional decline of the ENT.
In addition to the left para-HI volume reduction, a weak DG-ENT association may lead to decreased memory processing, which might indirectly contribute to the decline in olfactory recognition in older adults.
One question may arise that olfactory decline could be caused by abnormality of the olfactory bulb. The lateral ENT may receive direct projections from the olfactory bulb [bib_ref] Hippocampal-neocortical interaction: A hierarchy of associativity, Lavenex [/bib_ref].
In Olfactory bulb, new neurons area also generated during adulthood [bib_ref] Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats, Altman [/bib_ref]. Possibly, less newborn neurons in the olfactory bulb may affect on the level of olfaction. However, all elderly subjects could detect odors in this study; therefore, olfactory decline may be caused by a central factor that is associated with prior volume changes of the ENT and para-HI.
## | laterality
The HI volume reduction associated with olfaction decline was always observed in the left hemisphere in elderly subjects [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref] , and in patients with first episode of schizophrenia [bib_ref] Hippocampus and parahippocampus volume reduction associated with Impaired olfactory abilities in subjects..., Kubota [/bib_ref]. Another study reported that decrease of left HI volume showed an impaired visuospatial memory in patients with schizophrenia spectrum disorders [bib_ref] Hippocampal subfields and visuospatial associative memory across stages of schizophrenia-spectrum disorder, Wannan [/bib_ref]. A meta-analysis of structural MRI studies on the HI reported that HI asymmetry was observed in healthy control subjects, and the left hemisphere volume was lower than that of the right hemisphere [bib_ref] Bilateral hippocampal volume reduction in adults with post-traumatic stress disorder: A meta-analysis..., Smith [/bib_ref].
Our results were consistent with prior findings that the left HI volume was smaller than the right HI volume in elderly and young
HCs (elderly, z = −3.2, p = .001, young HCs, z = −4.3, p < .0001).
This trend was not observed in the AMG in both groups; only elderly subjects had a smaller left AMG than right AMG (elderly, z = −3.26, p = .001, young HCs, z = −1.77, p = .75). It is unknown why the left HI and AMG are smaller than those of the right side, and variation in volume reduction was shown on the left side in the elderly subjects.
Further study is required to investigate the functional meaning of laterality and to clarify the differences in sides.
# | limitations
Our results were obtained from a small number of HC subjects, and additional data including patients with MCI and AD are needed to confirm our findings. In the present study, we were unable to investigate the volume of the olfactory bulb itself because the images did not follow the protocol for olfactory bulb analysis. However, previous reports have suggested that the size of the olfactory sulcus indirectly reflects the size of the olfactory bulb. We therefore compared olfactory sulcus volumes between the two groups to investigate any association between olfactory sulcus volume and olfactory ability.
There were no differences in olfactory sulcus volumes between the two groups, and olfactory sulcus volume was not correlated with olfactory ability. These results indicate that the olfactory bulb may not be associated with the olfactory decline that was observed in elderly subjects in this study. It would be of interest to explore how olfactory bulb volume might relate to the central olfactory system in individuals with impaired olfaction; this concept requires further research.
In addition, the ENT was not divided into subregions in this study.
The ENT has been divided into two subregions, the medial ENT and lateral ENT [bib_ref] Hippocampal-neocortical interaction: A hierarchy of associativity, Lavenex [/bib_ref]. [bib_ref] Molecular drivers and cortical spread of lateral entorhinal cortex dysfuntion in preclinical..., Khan [/bib_ref] reported that the lateral ENT was affected by preclinical disease, and the dysfunction can then spread cortically. The differences in damage to the lateral ENT between AD and the preclinical condition and how this damage relates to the para-HI and olfactory impairment will be investigated in further studies.
## Ack n owled g em ents
This study was supported by a JSPS KAKENHI Grant (Number 20K03482) and the Kao Corporation. We thank Katsutoshi Murata (Siemens Healthcare, Japan) for assisting the MRI sequence prototype used in this study. We thank Lisa Kreiner, PhD, from Edanz Group (https://en-autho r-servi ces.edanz group.com/ac) for editing a draft of this manuscript.
## Co n fli c t o f i nte r e s t
The authors declare that this study was funded by Kao Corporation.
HS was employed by Kao Corporation and was involved for MRI measurements and analysis of olfaction and MoCA data.
## Auth o r co ntr i b uti
## Pe e r rev iew
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.2115.
## Data ava i l a b i l i t y s tat e m e n t
Datasets generated for this study are available on request from the corresponding author.
## O rci d
Yuri Masaoka https://orcid.org/0000-0002-0498-1825
[table] Table 2: shows statistical differences for each brain region between elderly and young HCs. The left para-HI, left ENT, and right ENT had no differences between the elderly subjects and young HCs. There were statistically lower volumes in most hip- [/table]
|
Novel gene signatures for stage classification of the squamous cell carcinoma of the lung
The squamous cell carcinoma of the lung (SCLC) is one of the most common types of lung cancer. As GLOBOCAN reported in 2018, lung cancer was the first cause of death and new cases by cancer worldwide. Typically, diagnosis is made in the later stages of the disease with few treatment options available. The goal of this work was to find some key components underlying each stage of the disease, to help in the classification of tumor samples, and to increase the available options for experimental assays and molecular targets that could be used in treatment development. We employed two approaches. The first was based in the classic method of differential gene expression analysis, network analysis, and a novel concept known as network gatekeepers. The second approach was using machine learning algorithms. From our combined approach, we identified two sets of genes that could function as a signature to identify each stage of the cancer pathology. We also arrived at a network of 55 nodes, which according to their biological functions, they can be regarded as drivers in this cancer. Although biological experiments are necessary for their validation, we proposed that all these genes could be used for cancer development treatments.OPEN
As GLOBOCAN reported in 2018, lung cancer was the first cause of deaths and new cases by cancer worldwide [bib_ref] Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for..., Bray [/bib_ref]. Squamous cell carcinoma of the lung (SCC) is one type of lung cancer which comprises approximately 30% of all lung cancer cases. The available molecular targets for use in the treatment of SCC of the lung are behind of other types of cancer [bib_ref] Squamous cell lung cancer: from tumor genomics to cancer therapeutics, Gandara [/bib_ref] [bib_ref] Targeting FGFR in squamous cell carcinoma of the lung, Hashemi-Sadraei [/bib_ref] [bib_ref] Mouse models in squamous cell lung cancer: impact for drug discovery, Singh [/bib_ref]. Recent advances in the treatment have been achieved using immunotherapy as nivolumab and pembrolizumab and some clinical trials are being conducted to test molecular targets [bib_ref] Targeting FGFR in squamous cell carcinoma of the lung, Hashemi-Sadraei [/bib_ref] [bib_ref] Mouse models in squamous cell lung cancer: impact for drug discovery, Singh [/bib_ref]. Some efforts to understand the basis of the disease have been made using gene expression profiles, DNA sequencing and SNP arrays 2 . However, there are few preclinical murine models, some SCC of the lung cell lines have errors in their classification and molecular targets usually found in other types of lung cancer as lung adenocarcinoma are rarely present in SCC of the lung [bib_ref] Mouse models in squamous cell lung cancer: impact for drug discovery, Singh [/bib_ref]. Lung cancer is classified into two wide groups as follows: Small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC represents 85% of all lung cancer cases. From this group the most prevalent are the adenocarcinoma and the squamous cell carcinoma of the lung [bib_ref] Squamous cell lung cancer: from tumor genomics to cancer therapeutics, Gandara [/bib_ref] [bib_ref] Squamous-cell carcinomas of the lung: emerging biology, controversies, and the promise of..., Drilon [/bib_ref]. Lung cancer survival is less than 5% after 5 years and most of them metastasize. Most of the time lung cancer is detected in advanced stages in which treatment is less effective. The best treatment is surgery although the effectivity of the treatment is linked to early stages of the disease [bib_ref] Genetic changes in squamous cell lung cancer: a review, Heist [/bib_ref] [bib_ref] Treatment of advanced squamous cell carcinoma of the lung: a review, Derman [/bib_ref] [bib_ref] The biology and management of non-small cell lung cancer, Herbst [/bib_ref] [bib_ref] Lung cancer: current therapies and new targeted treatments, Hirsch [/bib_ref]. Smoking is considered as a risk factor associated to lung cancer development 2 . Network analysis is widely used in different areas including biological sciences with a wide variety of results. There are different metrics that can be obtained from networks as the hubs which are commonly referred as the most connected nodes which lead to network instability if they are perturbed [bib_ref] Statistical mechanics of complex networks, Albert [/bib_ref] [bib_ref] Error and attack tolerance of complex networks, Albert [/bib_ref] [bib_ref] The large-scale organization of metabolic networks, Jeong [/bib_ref] [bib_ref] Drug-target network, Yıldırım [/bib_ref]. Besides, other network measures as betweenness and multivariate entropy have been used to analyze cancer networks to find putative potential targets for cancer disease [bib_ref] Molecular signaling network complexity is correlated with cancer patient survivability, Breitkreutz [/bib_ref] [bib_ref] Multivariate entropy characterizes the gene expression and protein-protein networks in four types..., Juarez-Flores [/bib_ref]. We previously identified a set of nodes which due to its biological and network properties we called them network gatekeepers [bib_ref] Original Article Squamous cell carcinoma of the lung: gene expression and network..., Juarez-Flores [/bib_ref]. The latter was done by visual inspection. Gatekeepers have few nearest-neighbor interactions with other proteins, but these proteins have plenty of interactions. Gatekeepers might not be detected by standard differential gene expression analyses.
In this work, we use clustering centrality as a metric for a better and quicker identification of gatekeepers [bib_ref] Original Article Squamous cell carcinoma of the lung: gene expression and network..., Juarez-Flores [/bib_ref]. Machine learning algorithms have been applied to a wide variety of phenomena. Health sciences have a special interest in the applications of these techniques due to the vast data publicly available with the objective to achieve better diagnosis and treatments of diseases. Some of the analyzed data with these approaches include analysis of histopathological images [bib_ref] Machine learning in medicine, Deo [/bib_ref] [bib_ref] Machine learning approaches for pathologic diagnosis, Komura [/bib_ref] [bib_ref] eDoctor: machine learning and the future of medicine, Handelman [/bib_ref]. In this article, we make an analysis of the carcinogenic process of the squamous cell carcinoma of the lung using cutting-edge techniques as network and machine learning analyses to obtain sets of genes which could function as a signature to aid in the classification of patient tumor samples into one of the
# Results
Carcinoma-stage classification model derived from machine learning. Data collected in GSE33479 was used to train a supervised machine learning algorithm. A logistic regression model was trained to classify the eight stages of the small cell lung carcinoma. Logistic regression models have been shown to provide accurate non-linear classification models of complex data [bib_ref] Logistic regression and artificial neural network classification models: a methodology review, Dreiseitl [/bib_ref]. A parameter reduction procedure was applied to the trained model. For this, the parameters of the model were standardized so that the parameters follow a standard normal distribution. The parameters whose value was beyond 0.78 from the mean were selected as relevant parameters. The procedure of model training and parameter selection was applied two times. On first parameter reduction, a total of 800 genes out of 41,067 were selected; on the second round a total of 15 relevant genes were selected. When using the subset of 800 genes a logistic regression model was trained and tested with records of all 122 patients records with all correctly classified, when considering the set of 15 genes the trained model was able to correctly classify the healthy stage and the first stages of small cell carcinoma and presented 7 cases of misclassification on later stages. A neutral control was designed by considering a total of 500 random sets of 15 genes, on each random set a logistic regression model was trained, and its accuracy was measured using the Jaccard index and compared with the Jaccard index of a model trained with the selected set of 15 genes. The Jaccard index measures the proportion of correctly categorized cases by the model. The Jaccard index from the set of genes derived from the parameter reduction method was of 0.92 whereas for the random sets the maximum Jaccard index was 0.29. When considering the set of 15 selected genes coupled with the 26 genes identified from previous analysis on PPI networks resulted in a trained model with a Jaccard index of 1. APID PPI data was used for network analysis of the results from the implemented machine learning technique for the first glance results of approximately 800 genes. APID was used due to better coverage of most part of the 800 genes.
Differential gene expression analysis. The first step was to carry out an exploratory network analysis which is shown in 2a,b. These networks are obtained from joining the results from the Differential Gene Expression (DGE) to Mentha network database and then the application of Eq. 1 to highlight the network gatekeepers. [fig_ref] Figure 2: DGE-PPI network [/fig_ref] shows in the inset the color scale, which was applied in [fig_ref] Figure 2: DGE-PPI network [/fig_ref] ,b. The minimum degree (number of connections of a node) value is 1 which is yellow in color, the most connected nodes are in navy blue purple whose value is 75. [fig_ref] Figure 2: DGE-PPI network [/fig_ref] shows in red the connections that every gatekeeper has, and they are marked by bigger yellow circles. It can be observed that all of them are connected to other nodes, but they, at first glance, do not appear to be of importance because of the few connection they have. In [fig_ref] Figure 2: DGE-PPI network [/fig_ref] can be observed in red, not only the gatekeeper's connections but also the connections of the first connected nodes and how they have much more connections which comprises most of the network. The nodes at which gatekeepers are connected are hubs due to the highly connections they have. www.nature.com/scientificreports/ In [fig_ref] Figure 3: DGE-PPI network zoom [/fig_ref] , a zoom of the graph of [fig_ref] Figure 2: DGE-PPI network [/fig_ref] is presented and each node is tagged with its HGNC name tag. It can be observed to which nodes some of the gatekeepers are connected. For example, a connection (red) to MYC protein (purple) can be observed. This protein is considered as an oncogene frequently associated with poor outcomes; a gatekeeper is linked to other proteins as MEOX2 whose possible function in some cancers is to be a suppressor gene [bib_ref] Targeting oncogenic Myc as a strategy for cancer treatment, Chen [/bib_ref] [bib_ref] Over-expression of MEOX2 promotes apoptosis through inhibiting the PI3K/Akt pathway in laryngeal..., Tian [/bib_ref]. Every one of the gatekeepers are linked to highly connected nodes which have relevant biological functions. summarizes in a list the results of the network analysis and the application of the machine learning algorithm in the GEO data set. A list of 26 gatekeepers' proteins is displayed in the first column which were the proteins with a clustering centrality of 1 obtained by the network analysis. This set of genes were used as an input list for the machine learning algorithm in which the results showed that they can be used to identify each carcinogenic stage with great accuracy. Second and third columns are two lists that contain a reduced set obtained only from the machine learning algorithm to classify each sample into its corresponding stages. Second column contains the probe tag used by the chip. Using two different methods we obtained two list of potentially gene sets that could be used as an aid to help classify samples and whose biological functions denote their potential use as targets for therapy. Further experiments are needed to probe its potential use.
An enrichment test was performed using the gatekeepers list to discover the main pathways associated with them as shown [fig_ref] Table 2: Gatekeepers [/fig_ref]. The first characteristic is that every category is overrepresented, which means that in each presented category there are more genes from the input list than it can be expected (using as reference the Homo sapiens REFLIST) and most of the processes are involved in cell cycle-related specially in mitosis.
The next step was to search in distinct databases the list of genes obtained by the machine learning algorithm. We selected two pathway databases: the reactome pathways and the KEGG pathways. In [fig_ref] Table 3: Machine learning selected genes [/fig_ref] , it can be observed a list of 8 genes for which information was available. The first column corresponds to its name, the second column to the related pathways in Reactome and the third to KEGG pathways. Some of the related pathways are usually altered in some types of cancer as Beta-catenin independent WNT-signaling, SMAD2/ SMAD3, tight junction, ABC transporters, etc. [bib_ref] Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer..., Tian [/bib_ref] [bib_ref] β-catenin-independent regulation of Wnt target genes by RoR2 and ATF2/ATF4 in colon..., Voloshanenko [/bib_ref] [bib_ref] Tight junctions and the tumor microenvironment, Salvador [/bib_ref] [bib_ref] Role of ABC transporters in cancer chemotherapy, Sun [/bib_ref].
A network was made based in the results obtained from the machine learning algorithm first glance which comprised approximately 800 genes. It was observed a big component (when a significant proportion of the nodes in a graph are connected) created by some nodes as seen in [fig_ref] Figure 4: Big component network [/fig_ref].
An interesting characteristic of the identified network by the machine learning algorithm was that some of the network gatekeepers identified by the DGE analysis were connected to this big component as seen in [fig_ref] Figure 4: Big component network [/fig_ref]. Some biological functions of some nodes in this network are well known to be relevant for cancer progression as PTEN which is a tumor suppressor altered in some types of cancer, as well as others like MCL1 which is an anti-apoptotic protein altered in some types of cancers. Also, MCL1 is being studied as a target for cancer patient treatment in small cell lung cancer [bib_ref] PTEN: tumor suppressor and metabolic regulator, Chen [/bib_ref] [bib_ref] MCL1 inhibition is effective against a subset of small-cell lung cancer with..., Yasuda [/bib_ref]. FAR1 is observed to play an essential role in the production of ether lipids/ plasmalogens whose synthesis requires fatty alcohol. ABCA1 catalyzes the translocation of specific phospholipids from the cytoplasmic to the extracellular/luminal leaflet of membrane coupled to the hydrolysis of ATP. In cancer it was observed that its inhibition plays an important role for cancer survival due to an increase of mitochondrial www.nature.com/scientificreports/ cholesterol. The function of DMRT3 is not clear. It is thought to function as a transcription factor. In a study of lung cancer, the dysregulations of DMRT3 along with other two proteins were considered specific for lung squamous cell carcinoma [bib_ref] Landscape of transcriptional deregulation in lung cancer, Zhang [/bib_ref]. AAK1 is a kinase that participates in the regulation of clathrin-mediated endocytosis. It was discovered that in β-Catenin-dependent WNT signal a negative feedback loop is created by its expression. ASF1B Is a histone chaperone which facilitates histone deposition, exchange, and removal during nucleosome assembly/disassembly; in cervical cancer it was observed that it functions as an oncogene accelerating cancer cells proliferation. APOC1 functions as an inhibitor of lipoprotein binding to the low-density lipoprotein (LDL) receptor. In gastric cancer it was proposed as a potential diagnostic and prognostic biomarker; in colorectal cancer evidence points out to have a promoting role in carcinogenesis. ADRA1B is an alpha-adrenergic receptor whose action is mediated by association with G proteins; in gastric cancer it was found a methylation promoter and it could be frequently involved in development and gastric cancer progression. These mentioned proteins are other examples of gene protein products whose functions are or could be involved with cancer disease [bib_ref] Landscape of transcriptional deregulation in lung cancer, Zhang [/bib_ref] [bib_ref] Anticancer activity of the cholesterol exporter ABCA1 gene, Smith [/bib_ref] [bib_ref] WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of LRP6 and..., Agajanian [/bib_ref] [bib_ref] ASF1B promotes cervical cancer progression through stabilization of CDK9, Liu [/bib_ref] [bib_ref] Apolipoprotein C1 (APOC1) as a novel diagnostic and prognostic biomarker for gastric..., Yi [/bib_ref] [bib_ref] Apolipoprotein C1 (APOC1) promotes tumor progression via MAPK signaling pathways in colorectal..., Ren [/bib_ref] [bib_ref] Frequent reduced expression of alpha-1B-adrenergic receptor caused by aberrant promoter methylation in..., Noda [/bib_ref].
# Discussion
Lung cancer is the deadliest type of cancer, most of the diagnosed cases are made in the last stages of the disease and there are little available treatment options which could have an important effect. Small cell carcinoma of the lung comprehends a great part of all lung cancers. Our present results provide a better comprehension of the underlying components of the disease. The detection of genes and proteins that could be implicated in the carcinogenic process is urgently needed to provide better options for treatments and diagnosis. Herein, we performed a thorough search of genes and proteins that could be used to offer better treatments and diagnosis options. We made a comprehensive analysis of all the carcinogenic process and observed that some set of genes could be used as an aid for small cell carcinoma of the lung stage classification. We employed two pathways to identify relevant genes for diagnosis. The first was based in a classic method as DGE analysis with the aid of more www.nature.com/scientificreports/ recent techniques as network analysis with a novel concept as the network gatekeepers which are encountered by using clustering centrality. DGE analysis was used as an exploratory analysis to look for possibly patterns in the gene expression for each stage. Although a general panorama of the carcinogenic process was obtained, we wanted to summarize it into a small meaningful set of genes with high involvement in cancer development. To make this possible we used the output data of the DGE as the input for the network analysis and then search for the network gatekeepers. The other pathway was based in another cutting-edge technique, machine learning algorithms. Hitherto, machine learning applications on cancer have been for assessing cancer prediction and prognosis [bib_ref] Applications of machine learning in cancer prediction and prognosis, Cruz [/bib_ref]. These results are based on the analysis of a wide set of variables including biomarkers and clinical factors such as age, location and type of cancer, and size of tumor [bib_ref] The future of prognostic factors in outcome prediction for patients with cancer, Fielding [/bib_ref] [bib_ref] Prediction of outcome for patients with cutaneous melanoma, Cochran [/bib_ref] [bib_ref] Prostate cancer outcome: epidemiology and biostatistics, Burke [/bib_ref]. The results presented in here are not intended for cancer prevention or survivability directly, rather they provide a set of specific genetic biomarkers whose analysis can lead to an immediate diagnosis about the stage of development of small cell carcinoma in a patient. The analysis of the proposed genetic biomarkers can differentiate even the earliest stages of cancer development and lead a physician to administer the required treatment when the probabilities of survivability of the patient are higher. For each method we found a set of genes which we proposed to be useful as an aid for stage classification and due to the important biological roles in which they are involved they could also be useful for further validation as possible targets for treatment. The biological roles of the gatekeepers proposed set are marked as cell cycle regulation, DNA-repair breaks, nucleosome assembly and processes that occur in the mitotic phase. It was first observed in the gatekeeper network that most of them exhibit scarce connections but their first neighbor nodes which are directly connected are hubs (highly connected nodes). This along with the processes they are involved may permit an access for prior processes regulated by the hubs. It is known that network hubs are of high importance for network stability, but in this work, we are observing that it can be of great importance to use the network gatekeepers as a measure to find key components in a biological context. The machine learning algorithms are usually used in other fields to improve the understanding of a wide variety of processes. In the case of cancer its aim is to find new targets and possible key proteins that regulate cancer. We found a reduced set of genes that can be used for stage classification in a set of microarray data and this also can be done with the set of gatekeepers. The biological functions of each of the identified genes are relevant for normal stages and as previously observed for cancer development. For example, in the case of the reduced set . Gene list from network gatekeepers and machine learning algorithm. Some Id are labeled as ** which means is a Missing Id. The first column corresponds to Gatekeepers list with 26 genes and the second column to the probe tag id in the microarray chip for 15 genes found with the machine learning method. The third column are the HGCN tags for each probe id of the second column. Second and third columns list finished when blank fields were present. www.nature.com/scientificreports/ obtained with machine learning, ARPC5 is a protein whose normal function is involved in EPH-Ephrin signaling and tight junction regulation and they are involved in cancer processes as adhesion, migrations, invasion or growth. This protein was recently proposed to be a prognostic biomarker for patients with multiple myeloma [bib_ref] The expression of actin-related protein 2/3 complex subunit 5 (ARPC5) expression in..., Xiong [/bib_ref].
In the case of ABCA1 is a protein whose inhibition promotes cancer progression [bib_ref] Anticancer activity of the cholesterol exporter ABCA1 gene, Smith [/bib_ref]. Genes identified in the big component obtained from the machine learning first set was also analyzed and observed that some of them were previously studied in cancer and that their functions are involved with them. It is necessary to study these proteins in the context of squamous cell carcinoma of the lung, as it is known that the function is dependent of the type of tissue, microenvironment, and type of cancer. Our combined approach of DGE analysis plus the use of the metric of clustering centrality together with the application of machine learning algorithms, will facilitate the identification of relevant components in biological networks as the ones derived from cancer data.
# Conclusions
We found a small set of genes possibly involved in the development of the disease. We propose two sets of genes which could help in the classification of tumor samples. These findings can increase the available options for experimental assays and molecular targets that could be used in novel treatment development. Although further experimental research is needed to validate their utility in the clinical setting.
# Methods
A graphical flowchart that summarizes the methods and the data bases is shown in [fig_ref] Figure 5: Workflow [/fig_ref]. Data collection was made by using various databases: Gene expression Omnibus for gene expression patients set, GEO accession: GSE33479, which comprises 122 patient samples representing the carcinogenic stages. Samples were divided as: 13 normal histology and normo-fluorescent, 14 with normal histology and hypo-fluorescent, those were grouped as the control group, 15 metaplasia samples, 13 mild dysplasia, 13 moderate dysplasia, 12 severe dysplasia, 13 carcinoma in situ, and 14 for squamous cell carcinoma of the lung. The gene expression platform was Agilent-014850 Whole Human Genome, Microarray 4 × 44 K G4112F. Processing and differential gene expression analysis were performed using R v3.5.1 software (http://www.R-proje ct.org). Processed data retrieval was performed by GEOquery R package. Hgug4112a.db R package was used to annotate each gene ID to the data [bib_ref] GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor, Davis [/bib_ref]. Using limma package differential gene expression (DGE) analysis was used to compare each stage vs the normal. Limma package fits a generalized linear model before comparisons and then calculate a moderate t-statistic for each contrast [bib_ref] limma powers differential expression analyses for RNA-sequencing and microarray studies, Ritchie [/bib_ref] [bib_ref] How many biological replicates are needed in an RNA-seq experiment and which..., Schurch [/bib_ref]. A p-value is obtained which is adjusted based in Benjamini and Hochberg False Discovery Rate correction [bib_ref] limma powers differential expression analyses for RNA-sequencing and microarray studies, Ritchie [/bib_ref] [bib_ref] Controlling the false discovery rate: a practical and powerful approach to multiple..., Benjamini [/bib_ref]. A list from the DGE was obtained for each comparison, results were merged to obtain a new list with all differentially genes. Full Human Interactome was downloaded from Mentha and APID database [bib_ref] mentha: a resource for browsing integrated protein-interaction networks, Calderone [/bib_ref] [bib_ref] APID interactomes: providing proteome-based interactomes with controlled quality for multiple species and..., Alonso-López [/bib_ref]. Protein-protein interactions (PPI) level 0 data (all reported proteins pairs) was obtained from APID. Cleaning process for both networks was made using Cytoscape software (Networks for Figs. 2 and 3 were created using this software) which comprised: deletion of repeated interactions, deletion of protein interactions detected in other organism, deletion of self-loop interactions in proteins [bib_ref] Cytoscape: a software environment for integrated models of biomolecular interaction networks, Shannon [/bib_ref]. Both databases are public and free to use. The merged list resulted from DGE (using a filter of p < 0.05 and Fold change < − 1.5 & > 1.5) from the microarray data were coupled with Mentha PPI dataset which allowed to create a new network of PPIs which were used as a template to identify the network gatekeeper's proteins using clustering centrality measure Eq. (1). Mentha PPI data was used due to the better coverage of the genes that appeared in list of DGE analysis. To calculate clustering centrality measure we used the following Eq. (1):
where C i is the clustering coefficient of a node i and is defined as the fraction E i of existing connections among its k i nearest neighbors divided by the total number of possible connections. www.nature.com/scientificreports/
[formula] C i = 2E i k i (k i − 1) [/formula]
[fig] Figure 1: (a) Confussion matrix of the model trained with the 15 genes selected using the parameter reduction method. On the x-axis is the true classification and on the y-axis is the predicted classification for each of the 122 patient records. (b) Histogram of the Jaccard indexes from 500 trained models with random sets of 15 genes; In red the Jaccard index of the trained model with the 15 genes selected using the parameter reduction method. Figures were made using the library matplotlib of Python. Scientific Reports | (2021) 11:4835 | https://doi.org/10.1038/s41598-021-83668-1 [/fig]
[fig] Figure 2: DGE-PPI network. (a) It represents a gathering and merging of the DGE analysis results and the Human Protein-Protein Interaction network from the Mentha Database. Red lines represent the connections of the gatekeepers. Color scale is presented in the left top corner. It represents the color scale applied to show graphically the values in the centrality measure for each node in the networks. (b) The red lines represent the connections of the gatekeepers plus the connections of its first neighbors (direct connected nodes to gatekeepers). The red lines comprise most of the network connections. (a,b) The proteins with less connections are marked in yellow and the most connected proteins are marked in navy blue purple. The size of each node represents the value of the clustering centrality measure; The bigger, the more value it has. Scientific Reports | (2021) 11:4835 | https://doi.org/10.1038/s41598-021-83668-1 [/fig]
[fig] Figure 3: DGE-PPI network zoom. It can be observed with more detail some nodes with their respective connections. Red lines denote the connections of the network gatekeeper (nodes with a clustering centrality of 1) and some of the pointed nodes, in darker color, are associated to highly connected nodes.Scientific Reports | (2021) 11:4835 | https://doi.org/10.1038/s41598-021-83668-1 [/fig]
[fig] Figure 4: Big component network. (a) A network with 52 nodes is displayed. The network is a big component observed in the exploratory network analysis of a network created by the Machine learning algorithm. Nodes are displayed in red color; connections are in blue. (b) A network of 55 nodes of which 3 nodes are from the identified gatekeepers. Figures were made with the library NetWorkX of Python. [/fig]
[fig] Figure 5: Workflow. A general panorama of the methodology and the databases. Figure made with Microsoft Visio. Scientific Reports | (2021) 11:4835 | https://doi.org/10.1038/s41598-021-83668-1 [/fig]
[table] Table 2: Gatekeepers: Enrichment test-Reactome pathways. Main reactome pathways are shown if False discovery rate value was less than 0.05. [/table]
[table] Table 3: Machine learning selected genes: Reactome and KEGG pathways involved. The pathways that could be related to squamous cell carcinoma of the lung are shown in Reactome column. Most of the genes do not have a pathway related to KEGG database, they are labeled as null. If there were more than three pathways available in either database just three pathways or less were selected when its biological function could be useful in cancer progression, growth, or maintenance. If just one pathway was available, it was written in the corresponding field. Null is used when no hits were found in the database. Only. genes that do not appear in either database were not presented. [/table]
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Immunotherapy Combined With Radiation Therapy for Genitourinary Malignancies
Immunotherapy drugs have recently been approved by the Food and Drug Administration for the treatment of several genitourinary malignancies, including bladder cancer, renal cancer, and prostate cancer. Preclinical data and early clinical trial results suggest that immune checkpoint inhibitors can act synergistically with radiation therapy to enhance tumor cell killing at local irradiated sites and in some cases at distant sites through an abscopal effect. Because radiation therapy is commonly used in the treatment of genitourinary malignancies, there is great interest in testing the combination of immunotherapy with radiation therapy in these cancers to further improve treatment efficacy. In this review, we discuss the current evidence and biological rationale for combining immunotherapy with radiation therapy, as well as emerging data from ongoing and planned clinical trials testing the efficacy and tolerability of this combination in the treatment of genitourinary malignancies. We also outline outstanding questions regarding sequencing, dose fractionation, and biomarkers that remain to be addressed for the optimal delivery of this promising treatment approach.FIGURE 1 | Mechanisms underlying synergy of radiotherapy and immunotherapy. Radiation promotes the ability of antigen-presenting cells to present tumor antigens to naive T cells through antigen release, stimulation of calreticulin, and downregulation of CD47. MHC-1 expression and the subsequent antigen presentation leads to interaction with T-Cell Receptors (TCR). Moderate doses of radiation also activate a type I interferon response through the sensing of cytoplasmic DNA via cGAS-STING. Radiation can upregulate PD-L1 and CTLA-4, and therefore immunotherapy can augment radiation efficacy by targeting these pathways. (Created with BioRender.com).Ukleja et al.
# Introduction
Prostate, bladder, and kidney/renal pelvis cancers rank fourth, seventh, and eighth, respectively, in estimated cancer-related deaths in the United States in 2020. Radiation therapy is a wellestablished treatment modality for genitourinary malignancies, with clinical utility in the definitive, adjuvant, and palliative settings. In localized prostate cancer, for example, radiation therapy is a curative treatment option with survival outcomes that have been shown to be equivalent to those of radical prostatectomy. In bladder cancer, radiation is a critical part of bladder-preserving trimodality therapy, which has comparable outcomes to radical cystectomy in well-selected patients. Renal cell carcinoma has traditionally been considered relatively radioresistant, but recent advances in radiation delivery and image guidance technologies have led to the development of stereotactic body radiotherapy (SBRT), which enables the focal and conformal delivery of ablative radiation doses sufficient for the definitive treatment of primary renal cancer. In patients with metastatic cancer, palliative radiotherapy is frequently used to alleviate pain from bone metastases. In addition, emerging data suggests that in oligometastatic cancers with five or fewer metastatic lesions, the aggressive use of SBRT to ablate all sites of metastatic disease can lead to improved clinical outcomes.
The last several years have also seen the rapid availability of immunotherapy drugs that increase overall survival in patients with a variety of cancers, including genitourinary malignancies. Immunotherapy utilizes the patient's immune system to induce tumor cell killing and can be either active or passive in nature. Active immunotherapy directly targets tumor cells and includes antibody therapy and chimeric antigen receptor T-cell therapy. In contrast, passive immunotherapy enhances the ability of the immune system to eradicate tumor cells and includes checkpoint inhibitors and cytokines. Among these approaches, immune checkpoint inhibitors have shown some of the most promising clinical activity to date. Currently available checkpoint inhibitors target two immune checkpoints: PD-1/PD-L1, which modulates Tcell activity resulting in immune response inhibition, and CTLA-4, an immunoglobulin expressed by activated T cells that downregulates immune response. FDA-approved therapies that target these immune checkpoints include atezolizumab, durvalumab, pembrolizumab, nivolumab (PD-1/PD-L1), and ipilimumab (CTLA-4), among others.
Recent data suggest that radiotherapy and immunotherapy may act synergistically, and there has been mounting excitement about the possibility of combining these modalities to further improve outcomes in patients with genitourinary cancers. In this review, we discuss the pre-clinical mechanistic rationale for combining radiotherapy with immunotherapy, as well as emerging data from ongoing and planned clinical trials testing the efficacy and tolerability of this combination in genitourinary malignancies.
## Biological rationale for combining radiotherapy and immunotherapy
## Radiotherapy can augment immunotherapy
Several lines of evidence suggest that radiation can stimulate the tumor immune microenvironment, a concept that underlies a key rationale for combining radiotherapy with immunotherapy. In many cancers, the immune microenvironment becomes altered from a state of immune recognition/antagonism towards a state of immune escape, where the immune system becomes incapable of combatting the tumor. Biological changes commonly associated with immune escape include reduced MHC-class 1 expression, upregulated inhibitory ligands and cytokines, and increased numbers of myeloid-derived suppressor cells. Although the primary mechanism by which radiation causes local cell death is through the induction of DNA double-strand breaks, radiation has been shown to be immunogenic through the direct and indirect activation of innate and adaptive immune response . Local cell death caused by radiation instigates the direct release of tumor antigens and promotes the priming and activation of cytotoxic T cells. In addition, radiation can promote the ability of antigenpresenting cells to present tumor antigens to naive T cells through the stimulation of calreticulin, a calcium-binding protein that promotes phagocytosis. Conversely, radiation has also been found to downregulate the presence of CD47, a protein that signals down-regulation of phagocytosis. High radiation doses have been shown to increase MHC-1 expression, increasing the likelihood of tumor-specific peptide presentation by antigen-presenting cells to naïve T cells. This phenotype, in conjunction with increased expression of death receptors such as Fas, facilitates the immune system's ability to kill tumor cells by enhancing the visibility of the tumor to cytotoxic T cells. Moderate doses of radiation have also been shown to activate a type I interferon response in tumor cells through the sensing of cytoplasmic DNA derived from tumor micronuclei via the cGAS-STING pathway. Through these different processes, radiation therapy ultimately creates a proinflammatory microenvironment that instigates immune activation in a manner that may be synergistic with immunotherapy.
## Immunotherapy may augment radiotherapy
Not all tumors will respond to radiation, despite administration of definitive doses. Although the reason for radioresistance remains unclear, one hypothesis is that immune-mediated mechanisms may be involved. It is important to note that although radiation can be immunogenic, it can also be immunesuppressive. Radiation can directly kill immune cells in or near the tumor through DNA double strand breaks and apoptotic cell death, which in turn may negatively impact T cells in peripheral circulation. For example, a retrospective study of prostate cancer patients treated with (N=36) or without (N=95) pelvic nodal irradiation demonstrated a higher risk of radiation-related lymphopenia with pelvic nodal irradiation. Indirectly, while activation of type 1 interferon through cGAS-STING induces recruitment of effector T cells and antigen presenting cells, it can also upregulate transforming growth factor b (TGF-b), which triggers an immune-suppressive environment. Radiation can also drive the recruitment of myeloid-derived suppressor cells (MDSCs), which serve as critical mediators of immunosuppression and inhibit effector T cells as well as induce Tregs. Increased infiltration of Tregs into the tumor microenvironment through radiation can downregulate the immune response. As a result, radiation's impact on MDSCs and T cells may promote tumor growth, local invasion, and subsequent metastases. Thus, therapies that counteract this effect by augmenting T-cell function may lead to improved control of the tumor. Radiation can also alter the balance of key immune checkpoint pathways including PD-L1 and CTLA-4. Radiation temporarily upregulates PD-L1 in mice with bladder cancer. The binding of the PD-L1 protein to the inhibitory checkpoint molecule PD-1 reduces the proliferation of antigenspecific T cells in lymph nodes. Similarly, radiation can upregulate the CTLA-4 receptor in T cells, leading to a downregulated immune response. Thus, an important rationale for incorporating immunotherapy into radiotherapy regimens is to augment the efficacy of radiation by selectively targeting these immune suppressive effects.
## Radiotherapy and immunotherapy are synergistic
Compared to other cancer treatments, tumor response to immunotherapy is often slower and may result in transient increases in tumor burden, even in patients who have an effective immune response. Radiotherapy could potentially greatly reduce the growth of such tumors, thus enabling patients to respond to the immunotherapy for longer periods of time.
In a similar vein, radiation can be used to prime the tumor for immunotherapy by increasing the susceptibility of tumor cells to immune-mediated treatment. Moreover, combining immune modulating agents and radiation may induce protective immunologic memory, which could prevent disease recurrence. Finally, reports in the literature suggest that combining immune checkpoint inhibitors and radiotherapy may result in increased frequency of the "abscopal effect," the immunogenic cell killing of untreated distant tumors. Although the potential mechanism for the abscopal effect may include radiation-induced stimulation of systemic recognition of tumor-related antigens, the overall rarity of clinical cases necessitates further investigation.
## Clinical evidence for combining radiotherapy and immunotherapy
## Non-genitourinary cancers
Several clinical studies have demonstrated a benefit for the combination of radiotherapy and immunotherapy in nongenitourinary cancers, as reviewed comprehensively elsewhere. For example, in lung cancer, the PACIFIC trial enrolled 709 non-small cell lung cancer (NSCLC) patients previously treated with platinum-based chemoradiation and randomized them in a 2:1 ratio to receive either adjuvant durvalumab or placebo. Treatment with durvalumab resulted in an increase in median progression-free survival and 2-year overall survival (66.3% vs 55.6%, P=0.005). In a secondary analysis of the KEYNOTE-001 phase 1 trial of pembrolizumab in NSCLC, patients who had previously received radiation therapy prior to receiving pembrolizumab experienced an increased median progression-free survival and overall survival compared to patients without previous radiotherapy. In the PEMBRO-RT Phase 2 randomized trial, 76 NSCLC patients received either pembrolizumab and SRBT (3 x 8 Gy within 7 days prior to the first cycle) or pembrolizumab alone. The study found that pembrolizumab preceded by SBRT resulted in a doubling of the overall response rate at 12 weeks (36% vs 18%, P=0.07) without any significant increase in toxicity, although this did not meet the prespecified endpoint for meaningful clinical benefit. Interestingly, subgroup analyses showed the largest benefit from the addition of radiation in patients with PD-L1 negative tumors.
## Prostate cancer
Although numerous clinical trials are investigating the combination of radiotherapy and immunotherapy in genitourinary cancers, only a few randomized trials have been published to date with mature results. In prostate cancer, a multicenter phase 3 trial investigated the use of ipilimumab vs. placebo after bone-directed radiotherapy (8 Gy x 1 fraction) in 799 men with metastatic castration-resistant prostate cancer that progressed after docetaxel. Ipilimumab therapy was associated with a trend towards increased overall survival that was not statistically significant (P=0.053). However, subgroup analyses suggested that patients with favorable prognostic features such as the absence of visceral metastasis or anemia and normal alkaline phosphatase did have a significant improvement in survival with the addition of ipilimumab. In a phase 2 trial, 49 patients with oligometastatic hormone-sensitive prostate cancer were randomized to receive either the autologous cellular immunotherapy sipuleucel-T preceded by radiotherapy (30 Gy to a single metastatic site) or sipuleucel-T alone. Median progression-free survival was higher with the addition of radiotherapy (3.65 vs. 2.46 months, P=0.06), but this was not statistically significant. Overall, radiotherapy did not significantly enhance the humoral and cellular responses associated with sipuleucel-T.
Although these clinical trials have not demonstrated a definite benefit for the addition of radiotherapy to immunotherapy, results from additional ongoing clinical trials in prostate cancer are pending, including those testing PD-1/PD-L1 checkpoint inhibitors in combination with radiotherapy. For example, in an ongoing phase 2 study (NCT03795207), 96 oligometastatic prostate cancer patients are randomized to either SBRT with durvalumab or SBRT alone in a 2:1 manner. Durvalumab (1500 mg/cycle) is administered one month prior to SBRT (3 x 9 Gy or 3 x 11 Gy) and continued until progression with a maximum of 12 months. The primary endpoint of the trial is 2-year progression-free survival.
## Kidney cancer
Immune checkpoint inhibition has become a standard of care treatment for patients with metastatic renal cell carcinoma (RCC). Multiple clinical trials are currently evaluating whether the addition of radiotherapy to immunotherapy will further improve outcomes in this disease. Early results have been presented for the RADVAX RCC single arm phase 2 trial (NCT03065179), in which 25 metastatic RCC patients received nivolumab and ipilimumab (N/I) with SBRT (50 Gy in 5 fractions) between the first and second doses of N/I. Partial responses were observed in 14/25 patients, for an objective response rate of 56%, which is higher than the expected response rate of 40%. The regimen was noted to have acceptable safety, although 10 (40%) patients required prednisone for immune-related adverse events. These results are encouraging for further investigation, although the study is limited by its small sample size and single-site design. Preliminary results of the NIVES single arm phase 2 multicenter study (NCT03469713) have been presented recently, in which patients with metastatic RCC that progressed on up to two prior systemic therapies were treated with nivolumab for 6 months, in combination with SBRT (10 Gy x 3 fractions) to one metastatic lesion given 7 days after initiation of nivolumab. At a median follow-up of 15 months, the objective response rate was 17.4% (12/68 patients). Although tolerability was acceptable [most frequent grade 3/4 toxicities were diarrhea (5.8%), elevated amylase/lipase (4.3%), and fatigue (4.3%)], the study did not meet its primary endpoint of improving response rate to 40%.
Overall, the available results for combining immunotherapy with radiotherapy are mixed in RCC. Additional data from ongoing clinical trials are anticipated to clarify whether changing the timing or target site of SBRT will further improve outcomes. For example, to test the strategy of targeting the primary kidney lesion with SBRT rather than targeting metastases in this context, the CYTOSHRINK phase 2 trial (NCT04090710) will randomize up to 78 untreated advanced RCC patients to receive ipilimumab/nivolumab plus SBRT to the primary lesion (30-40 Gy in 5 fractions) between cycles 1 and 2, or ipilimumab/nivolumab alone.
## Bladder cancer
Although muscle-invasive bladder cancer has historically been treated with radical cystectomy, bladder-preserving trimodality therapy consisting of transurethral tumor resection, radiotherapy, and chemotherapy is now considered a standard treatment option according to consensus clinical guidelines. Several clinical trials are examining the potential role of adding immunotherapy to further improve outcomes of these patients. A phase Ib study (NCT02891161) demonstrated the safety of combining the anti-PD-L1 checkpoint inhibitor durvalumab with radiation therapy to the bladder (64.8 Gy in 36 fractions) in 6 patients with locally advanced bladder cancer, with no patients experienced dose limiting toxicity. A follow-up randomized phase 2 study (ECOG-ACRIN/NRG 8185; NCT04216290) is examining the addition of durvalumab to chemoradiation therapy in patients with clinically node-positive (N1-2) muscle-invasive bladder cancer. A large cooperative group randomized phase 3 study (SWOG/NRG 1806; NCT03775265) with a planned accrual of 475 patients is investigating the addition of the anti-PD-L1 inhibitor atezolizumab to chemoradiation in patients with localized muscle-invasive bladder cancer. Safety data from the first 73 patients of this study were recently presented, showing no grade 3 or higher immune-related adverse events to date. Another study is exploring the potential of this strategy for the management of non-muscle invasive bladder cancer, using the combination of radiotherapy with Bacillus Calmette-Guerin (BCG) and durvalumab (ADAPT-bladder; NCT03317158).
## Considerations surrounding combining radiotherapy and immunotherapy sequencing
The optimal timing and sequencing of radiotherapy and immunotherapy for maximum efficacy of combination therapy remain unknown, although these may vary depending on tumor histology and type of immunotherapy. Interestingly, in a post-hoc analysis of the PACIFIC trial, patients who received durvalumab within 14 days after completing chemoradiation had better progression free survival than those who received durvalumab after 14 days, suggesting that immunotherapy should be started soon after radiation. Similarly, in a retrospective review of 758 patients with a range of cancer diagnoses who received radiotherapy and immunotherapy (either anti-CTLA-4 or anti-PD-1/PD-L1), patients who received concurrent therapy had better overall survival. Moreover, of those who received concurrent therapy, patients who received induction immunotherapy starting more than 30 days before radiation had improved overall survival compared to those who started less than 30 days before radiation. These studies suggest that careful consideration needs to be given to timing and sequencing of radiotherapy and immunotherapy in the design of clinical trials.
## Dose and fractionation
The optimal radiation dosing and fractionation strategy to maximize immunogenicity remains controversial. Most lines of evidence suggest that higher doses of radiation (>6-8 Gy per fraction) are more immunogenic than typical doses used in conventional fractionation (1.8-2 Gy per day). Moderately high doses of 8-12 Gy seem to optimally activate the type I interferon response via cGAS/STING, while very high doses (20-30 Gy in 1 fraction) result in a decline in radiationinduced STING activation, in part due to negative feedback inhibition by Trex1 exonuclease which reduces accumulation of cytoplasmic DNA. Ultimately, the various fractionation schemes incorporated into ongoing clinical trials will yield insights into the optimal radiation dosing and fractionation needed for the effective combination with immunotherapy.
## Biomarkers of efficacy and toxicity
The efficacy of immunotherapy varies greatly across patients and cancer types, and biomarkers that can identify the tumors that would be most responsive to specific immunotherapies are an area of active investigation. Candidate biomarkers of efficacy including PD-L1 expression, mutational burden, neoantigens, tumor infiltrating lymphocytes, and radiographic characteristics are under active study. Whether these same biomarkers will also predict responses to the combination of radiotherapy and immunotherapy remains an open question that should be actively addressed in ongoing and planned clinical trials. There is also a need for biomarkers that can predict the occurrence of severe toxicity after the combination of immunotherapy and radiotherapy. Immune stimulatory drugs can cause immune-related adverse events (IrAEs) including fatigue, rash, skin disorders, and GI issues. Several large cohort studies (e.g. NCT03984318) are seeking to discover the underlying mechanisms responsible for severe IrAEs and identify predictive biomarkers. Biomarker candidate for IrAE prediction currently under investigation include cytokines, immune-cell subsets, autoantibodies, human leukocyte antigen haplotype, and radiomic characterization. Other studies are investigating the reduction of immunotherapy-related side effects through the use immunosuppressive drugs such as rituximab (anti-CD20) and tocilizumab (anti-IL-6) (NCT04375228). Radiotherapy is associated with its own set of toxicities, but can also cause adverse events similar to IrAEs through non-tumor specific antigens released into the tissue microenvironment by irradiation, potentially priming auto-reactive T cells to attack normal tissue. Predictors of these and other adverse events related to the combination of immunotherapy and radiotherapy need further study.
# Conclusion
A growing body of preclinical and clinical evidence indicates a potential synergy between radiotherapy and immunotherapy, lending support for the combination of these two treatment approaches. Unanswered questions remain regarding the optimal sequencing of treatment, dose fractionation, and biomarkers of response and toxicity. Within genitourinary cancers, multiple clinical studies are ongoing with early indications of both promising as well as negative results, suggesting that specific details regarding the protocol by which treatment is delivered may impact the overall success of the approach. These efforts are exemplified by the SWOG/NRG 1806 phase 3 study testing the addition of atezolizumab to chemoradiation in muscle-invasive bladder cancer. Should these initial trials show promise, confirmatory trials may be necessary given increased FDA scrutiny of immunotherapy in light of recent voluntary withdrawal of drugs that received accelerated approval in bladder cancer. Continued research efforts are needed to fully evaluate and optimize this promising combination of radiotherapy and immunotherapy.
# Author contributions
JU, EK, and DM acquired, analyzed, and interpreted the data. JU, EK, and DM drafted and revised the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.
# Funding
This work was funded in part by the Cygnus Montanus Foundation founded by the Svanberg family and the Massachusetts General Hospital. |
An Accidental Intestinal Myiasis Caused by Cochliomyia macellaria
Intestinal myiasis is recognized as pseudomyiasis or accidental myiasis caused by dipteran fly larvae transmitted to humans via contaminated food or water. A case of intestinal myiasis acquired via contaminated food is reported in this case study. e patient is a 4-year-old boy who had frequent episodes of crampy abdominal pain and diarrhoea and the passage of many live worms at each time. As the child had the habit of eating ripe guava from his garden, the infection source was suggested as ripe guava, and the possibility was explored. All larvae collected from faeces and fruit were morphologically similar, and it has been identified as Cochliomyia macellaria. e treatment with several antihelmintics failed, and the recovery was achieved with a simple measure of abstinence from eating guava that came from his garden.
# Introduction
Infestation of live human or any vertebrate host with dipteran fly larvae is called myiasis. Human myiasis can manifest as cutaneous myiasis, wound myiasis, oracular myiasis, and nasopharyngeal myiasis in common; however, anal myiasis, body cavity myiasis, aural myiasis, and intestinal myiasis are also reported. Myiasis is classified into facultative, obligatory, and accidental categories depending on their survival on dead or live tissue. Fly larvae that requires living tissue for their survival cause obligatory myiasis. In contrast, those who live on dead or necrotic tissue cause facultative myiasis. e fly larvae that are accidentally ingested or deposited on living tissue cause accidental myiasis. e first report on human myiasis in Sri Lanka was published in 1954. Only a limited number of indigenous cases were published to date. Cutaneous myiasis was the commonest form, and Chrysomya bezziana (Old world screwworm fly) and C. megacephala were documented fly larvae. Cordylobia anthropophaga (tumbu fly) was also reported in Sri Lanka as imported cases of cutaneous myiasis. Other common myiasis-causing agents such as Cochliomyia hominivorax (New world screwworm fly) and Sarcophaga spp. (flesh fly or sarcophagids) are not documented in Sri Lanka.
People who have low socioeconomic conditions and live in a poor hygienic environment are at a risk of developing myiasis. Intestinal myiasis or pseudomyiasis or accidental myiasis caused by ingestion of dipteran fly eggs via contaminated food or water is commonly seen in such communities. However, fly larval infestation is most often self-limiting and does not result in any serious complications, which is a major cause of underreporting of human myiasis throughout the world. Hitherto, a case of intestinal myiasis is not reported in Sri Lanka. Diagnosis of human myiasis is usually missed by physicians due to lack of suspicion and have little idea of the specific clinical features. erefore, this case report aims to improve physicians' knowledge of intestinal myiasis by considering intestinal myiasis in the differential diagnosis list when assessing acute and chronic abdominal pains, especially in children.
## Case presentation
A 4-year-old previously healthy boy had several (seven) episodes of colicky abdominal pain and loose stools over ten months. ese episodes were never associated with fever but have had loss of appetite and infrequent vomiting. He had perianal discomfort both in the day and night. His mother had observed the passage of live worms with faeces on all these occasions, and she emphasized that it was more than 500 in number. During this period, no one else in the family had an illness similar to food poisoning, gastroenteritis, or worm infestation.
He was first seen by a general practitioner and treated for a common intestinal helminthiasis with mebendazole 100 mg twice daily for three days without obtaining laboratory evidence. After that, the child had been seen five times by general practitioners before the first author saw him. Stool investigations were done on two occasions and were reported as normal. It was not easy to get the treatment history due to retrograde exploration. e child had been treated with various anthelmintics and antibiotics. His mother could recall that he had been given both anthelmintics and antibiotics in a combination in few occasions. He had been treated with mebendazole 100 mg twice daily for three days in two occasions, albendazole 400 mg single dose in two occasions, and once treated by two tablets of pyrantel pamoate (250 mg). Whenever those episodes were predicted as food poisoning or infective gastroenteritis, the child had received either cephalexin or metronidazole along with zinc and probiotics for 3-5 days. Usually, the child became symptom-free in 6-7 days regardless of the treatment modality, but the reappearance of the symptoms in few weeks were inevitable. e first author has seen the child in his sixth episode. e child complained of a gush of offensive loose stools with worms and abdominal cramps. A sample of stool has been sent for amoeba, ova, and cysts but became negative. Fecal culture was negative for bacteria. However, the child was treated empirically for helminthic infection using 400 mg albendazole single dose and repeated the same dose of treatment after a week. All family members were treated with the same regimen and instructed to follow hygienic measures. e child was brought to the same clinic in few weeks due to reappearance of similar symptoms. is occasion, the faeces were frothy and greenish. e parents have taken all possible hygienic measures to prevent the recurrences by introducing regular hand washing, cutting nails short etc. However, it did not prevent him getting recurrences.
He was from a semiurban area. In his garden, there were many guava trees bearing fruits. He had the habit of eating ripe guava. Upon the authors' request, the parents collected a fecal sample and guava fruit with larvae. e parents succeeded in taking video-clips of live worms on faeces and fruits. e second author examined all samples at the Department of Parasitology, Faculty of Medicine, University of Ruhuna. Live specimens were found in faeces and fruits. Faeces was examined using saline, iodine wet mounts, and Kato-Katz. It was negative for amoeba, ova, and cysts.
Live wormy specimens were submerged in hot water (∼95°C) for 30 seconds and then in 95% ethanol before examining them under a dissecting microscope. e average length of larvae was 7 mm.
## Microscopic identification of larvae.
Pictorial key of Centers for Disease Control and Prevention, USA, was referred for larval identification (http://www.cdc.gov). All specimens collected did not have a definite, hard, sclerotized head capsule. e body was smooth and had short spines but no long lateral processes. Posterior spiracles were not on peg-like tubercles. Body was tapered posteriorly but did not extend into a tail-like process. Dissected posterior spiracle showed peritreme with three distinct slits which were positioned straight. e dorsal arm of cephaloskeleton is longer than the ventral arm and posterior spiracles with incomplete peritreme.
Spiracle slits point towards opening in peritreme. Prothoracic spiracles had 15 openings. Posterior spiracle button is indistinct, and wall of slits had swellings. Tracheal trunks were not pigmented. Larvae have been identified as Cochliomyia macellaria. All larvae collected from faeces and fruit were morphologically similar. e parents were instructed to avoid possible exposure to eating fruits (guava) from their garden. e child was reviewed in 3 and 6 months. He was symptom-free, and larvae have ceased appearing in the faeces.
# Discussion
A careful evaluation of fly larvae collected from the child's faeces and the fruit revealed that the both larval groups were morphologically similar, and the key described by the Centers for Disease Control and Prevention, USA (http:// www.cdc.gov), identified the larvae as Cochliomyia macellaria (secondary screwworm). Intestinal myiasis caused by Cochliomyia macellaria (secondary screwworm) is new to world literature. Myiasis due to Cochliomyia hominivorax (New world screwworm) is well documented. Cochliomyia hominivorax is an obligatory parasite causing intestinal, auricular, oral, nasal, and woundmyiasis. Cochliomyia macellaria is a facultative parasite that infests on wound or necrotic tissue of man and livestock. Like C. hominivorax, C. macellaria does not feed on living tissues. Livestock industry experiences huge economic losses due to parasitism (myiasis) and disease transmission caused by C. macellaria maggots. Different salmonella types, swine flu, and botulism in birds are transmitted by maggots of C. macellaria.
All reported cases of intestinal myiasis have caused only by facultative or accidental fly larvae, whereas obligatory gut parasites of animals have never been encountered in human gut.
Over 50 species of fly larvae mainly belonging to families Muscidae, Calliphoridae, and Sacrophagidae have been reported from cases of intestinal myiasis. e clinical presentations of enteric myiasis reported so far and their site of infestation and species identified are summarized in. e child has had more than three episodes of abdominal pain within three months; therefore, the presentation can be considered as recurrent abdominal pain (RAP) in children. Diagnosis of RAP depends on the clear history provided by the parent and the child. e common causes of RAP such as functional abdominal pain, constipation, irritable bowel, and peptic ulcers could be diagnosed clinically by taking a careful history and through physical examination. Intestinal infections due to bacteria, protozoa, and helminths can be diagnosed by a direct stool examination and culture. RAP in Sri Lanka is most considered as functional abdominal pain (nonorganic) where associated symptoms such as fever, vomiting, and microorganism or blood in stool are not seen. Here, in this case, the child has experienced several episodes of vomiting, diarrhoea, and perianal discomfort other than the vague abdominal pain. erefore, an organic cause was considered for the RAP, and the child was investigated with stool AOC and culture. Since none of the investigation became positive, an empiric cause of medical management was considered because it has greater value than multiple exclusionary investigations.
Intestinal myiasis is also considered as an organic cause for RAP. Simple elimination of eating guava has improved the child's clinical condition. erefore, we can assume that the symptoms mentioned above were due to 'maggots' infestation, but it is an incontestable proof. Offensive greenish stools associated with colicky abdominal Dorsal arm Case Reports in Pediatrics pain and vomiting may have been triggered by the inflammation caused by fly maggots. Similar offensive breath and vomit has been reported due to gastric myiasis,. Consumption of overripe or rotten fruits such as bananaand peachhas triggered intestinal myiasis. Confirmation of transmission route by demonstrating the same species of fly larvae in guava fruit is a remarkable achievement. For the first time in Sri Lanka, such transmission route is established and for the first time in the world literature, guava as the fruit to transport fly eggs larvae. Studies conducted in Sri Lanka have documented Megaselia scalaris in ripe bananas; however, intestinal myiasis caused by them has not been reported.
ere is no specific treatment for intestinal myiasis. Anti-inflammatory and antiperistaltic agents will reduce the symptoms, whereas broad-spectrum anthelmintics and antibiotics will not be helpful. erefore, conducting continuous education and communications programmes to improve the knowledge of prevention and precautions against intestinal myiasis for physicians and public health officials is needed. Success will be based on behavioral modification such as abstain from consuming uncovered food, which has access to fly, and overripe or rotten fruits and abstain from defecating in the open. We want to emphasize that clinicians must find species and transmission route before embarking on the therapy. e only successful treatment for enteric myiasis was colonic irrigation using oral polyethylene glycol (PEG). Ivermectin was tried with no significant effect.
# Conclusion
ough the intestinal myiasis does not cause serious complication, the morbidity is substantial. erefore, a great deal of suspicion and exploring a history of passage of worms will diagnose intestinal myiasis. Identification of the species and the route of transmission is of profound importance to eliminate the disease.
## Data availability
e data used to support the finding of this study are included within the article.
## Consent
Written informed consent for the publication has been obtained from both parents of the child.
## Conflicts of interest
e authors declare that they have no conflicts of interest. |
Phase 1 study of temozolamide (TMZ) combined with procarbazine (PCB) in patients with gliomas
Temozolomide (TMZ) is an oral alkylating agent with a good safety profile and proven efficacy in the treatment of malignant glioma. Procarbazine (PCB) has been used for treating gliomas for many years and here both agents were combined in the treatment. This phase I study was designed to evaluate the efficacy and safety of TMZ alone (course 1) and TMZ in combination with PCB in subsequent courses in chemotherapy-naïve patients with malignant glioma. Patients with anaplastic astrocytoma (AA), glioblastoma multiforme (GBM) and low-grade glioma were treated with TMZ 200 mg m À2 on days 1 -5 on a 28-day cycle for course 1. Beginning with course 2, cohorts of patients received TMZ at full dose with escalating doses of PCB (50/75/100/125 mg m À2 days 1 -5 given 1 h prior to TMZ). A total of 28 patients were enrolled with three patients each at dose level 1 and 2, 16 patients at dose level 3 and six patients at dose level 4 received 182 þ cycles of treatment and were included in this analysis. In all, 16 patients had GBM, seven patients had AA, five had grade 1 or 2 glioma and the median age was 47 years. The patients had received prior surgery and radiotherapy. Responses were seen at all dose levels. Overall, there were 10 (36%) responses lasting from 2 to 17 þ months. Treatment was generally well tolerated with few grade 3 or 4 toxicities, except at dose level 4, where four patients had grade 3/4 had thrombocytopaenia at this dose and several patients had moderate-to-severe lethargy. TMZ 200 mg m À2 and PCB 100 mg m À2 were well tolerated on a daily 5 Â and four weekly cycle in patients with malignant glioma and clearly had antitumour activity.
Prognosis with patients with glioma, and in particular, high-grade (anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM)) tumours is poor. Studies have confirmed the beneficial effect of postoperative cranial irradiation, but in most series, survival beyond 2 years is 15% or less [bib_ref] Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas: a..., Walker [/bib_ref] [bib_ref] The role of chemotherapy in the treatment of gliomas in adults, Stewart [/bib_ref] [bib_ref] Meta-analysis of radiation therapy with and without adjuvant chemotherapy for malignant gliomas..., Fine [/bib_ref]. The most widely used combination in treating gliomas is procarbazine, CCNU (lomustine), vincristine (PCV). This has a limited effect on survival [bib_ref] Modified procarbazine, CCNU and vincristine brain tumours, Levin [/bib_ref] [bib_ref] A randomised study of chemotherapy with procarbazine, vincristine and lomustine with and..., Sandberg-Wollheim [/bib_ref]. The most active agents identified until recently were the nitrosoureas (BCNU, CCNU, methyl-CCNU and HECNU), and have been reported to induce responses in the range of 35 -55%. However, the majority of these responses were of short duration [bib_ref] Does chemotherapy benefit the patient with a central nervous system glioma?, Rodriguez [/bib_ref] [bib_ref] Effect of HECNU in malignant supratentorial gliomas: a phase II study, Georges [/bib_ref] [bib_ref] The role of chemotherapy in the treatment of gliomas in adults, Stewart [/bib_ref].
Temozolomide came from a synthetic programme of a number of imidazotetrazine derivatives, which exhibited broad-spectrum antitumour activity against murine models [bib_ref] Antitumour imidazotetrazines. 1. Synthesis and chemistry of 8-carbamoyl-3-(2-chloroethyl) imidazo[5,1,d]-1,2,3,5-tetrazin-4(3H)-one, a novel broad..., Stevens [/bib_ref]. TMZ was selected for further clinical development in view of its good experimental antitumour activity and low toxicity in the pre-clinical screen. In addition, its antitumour activity was also schedule-dependent [bib_ref] Antitumour activity and pharmacokinetics in mice of 8-carbamoyl-3-(2-chloroethyl) imidazo[5,1,d]-1,2,3,5-tetrazin-4(3 H)-one (CCRG 81045;..., Stevens [/bib_ref]. In the early clinical development of TMZ, administration of a single dose induced myelosupression but did not have any antitumour activity. However, when given on a daily 5 Â schedule repeated every 4 weeks, activity against malignant melanomas and gliomas was seen . TMZ spontaneously ring opens at physiological pH to produce the active intermediate MTIC, which methylates DNA at a number of sites. The main cytotoxic lesion induced by TMZ is probably at the O 6 position of guanine [bib_ref] Antitumour imidazotetrazines. XV. Role of guanine O 6 -alkylation in the mechanism..., Tisdale [/bib_ref] [bib_ref] Depletion of O 6 -alkylguanine DNA alkyltransferase correlates with potentiation of temozolomide..., Baer [/bib_ref] [bib_ref] Potentiation of temozolomide and BCNU cytotoxicity by O 6 -benzylguanine: a comparative..., Wedge [/bib_ref]. This cytotoxic lesion is repaired by the DNA-repair protein O 6 -alkylguanine DNA alkyltransferase (AGT) that accepts the methyl group onto a cysteine residue and is autoinactivated. TMZ, especially administered in repeat dosing, will deplete tumour cells of AGT [bib_ref] 6 -methylguanine and temozolomide can reverse the resistance to chloroethylnitrosoureas of a..., D'incalci [/bib_ref] [bib_ref] Effect of temozolomide and dacarbazine on O 6 -alkylguanine DNA alkytransferase activity..., Mitchell [/bib_ref].
PCB has been used as an oral agent for many years in patients with malignant lymphomas (MOPP (mustine, vincristine, procarbazine, and prednisolone)) and in PCV-treated malignant gliomas. A number of studies in the 1990s also identified that procarbazine, a DNA-alkylating agent, depletes AGT [bib_ref] O 6 -alkylguanine DNA alkytransferase and sensitivity to procarbazine in human brain..., Schold [/bib_ref] [bib_ref] Accumulation of O 6 -methylguanine in human blood leukocyte DNA during exposure..., Souliotis [/bib_ref] [bib_ref] Differential effects of procarbazine and methylnitrosoureas on the accumulation of O 6..., Valvanis [/bib_ref] [bib_ref] mutations, O 6 -alkylguanine DNA alkytransferase activity and sensitivity to procarbazine in..., Russell [/bib_ref]. This study was designed to identify whether there is potentially an increase in the therapeutic index by combining PCB and TMZ in treating patients with malignant gliomas.
# Patients and methods
Following ethics committee approval, 28 patients with malignant gliomas were enrolled in this study and their details are shown in [fig_ref] Table 1: Clinical structure [/fig_ref]. All had received prior surgery and radiotherapy, and none had received chemotherapy and all had progressive disease. The study design was such that TMZ alone was administered during course 1 to determine whether or not each patient's bone marrow was sensitive to TMZ at full dose. The second and subsequent courses of TMZ were combined with escalating doses of PCB [fig_ref] Table 2: Study design [/fig_ref]. If a patient's bone marrow was sensitive to TMZ at a dose of 200 mg m À2 daily 5 Â , this was reduced in the second course to 150 mg m À2 daily 5 Â , and then subsequent courses combined with PCB and the reduced dose of TMZ. Patients continued on 4 weekly courses of TMZ at the same dose of PCB until disease progression or evidence of major toxicity.
Responses were assessed clinically and radiologically before and after each course of treatment. Patients with gliomas had to be taking a stable dose of corticosteroid for at least 2 weeks before study entry and the baseline CT or MRI scan. Radiological shrinkage alone was not considered an acceptable measure of response in brain tumours, as there is often difficulty in forming clear margins on a CT or MRI abnormality, which includes additional areas of necrosis, oedema, and vascularity. In some cases, it may be possible to measure a clearcut reduction of 50% by CT or MRI, but in most cases it is only possible to determine a reduction in enhancement and mass effect. An objective response (OR) was defined as one that requires improvement in the Medical Research Council neurological status [fig_ref] Table 3: Medical Research Council Neurological Status [/fig_ref] by one grade as well as a clear-cut reduction in mass effect on CT or MRI assessment with a minimum duration of 4 weeks. There should also be no deterioration in any other neurological symptom or sign and no development of new neurological deficits. Responses had to be documented by two observations at least 4 weeks apart [bib_ref] Phase I trial of temozolomide using an extended continuous oral schedule, Brock [/bib_ref].
# Results
In general, the combination of TMZ and PCB was well tolerated. Dose level 1 (PCB 50 mg m À2 per day given 1 h prior to TMZ) from course 2 onwards was well tolerated without any major side effects, as was dose level 2 (PCB 75 mg m À2 daily  5). The only significant toxicity at these two dose levels was lymphocytopenia [fig_ref] Table 4: Haematological toxicities [/fig_ref]. At dose level 3 (PCB 100 mg m À2 at daily  5), two patients had falls in their white counts and four patients had thrombocytopenia all on TMZ alone. One patient had thrombocytopenia from the combination of TMZ and PCB. At dose level 4, toxicity was seen and four patients had thrombocytopenia and several patients had moderate-to-severe lethargy and malaise. The recommended dose for future studies is dose level 3. The other toxicities that were seen were skin rashes (two patients) and one patient had hepatitis A infection [fig_ref] Table 5: Other toxicities [/fig_ref].
The responses by tumour grade are shown in [fig_ref] Table 6: Response to histological grade AA ¼ anaplastic astrocytoma, GBM ¼ glioblastoma multiforme [/fig_ref]. Responses were seen in all types of glioma, with an overall response rate of 36%. [fig_ref] Table 7: Responses by dose of procarbazine [/fig_ref] shows the response by PCB dose and responses were seen at all dose levels. [fig_ref] Table 8: Response duration [/fig_ref] shows the duration of responses to 1 September 2001, the duration of responses lasting from 2 to 17 þ months. shows a response in a patient receiving treatment at dose level 3.
# Discussion
Using the criteria of response, we have previously published responses in patients with high-grade gliomas (AA and GBM) to TMZ that are well documented and approximately one in four patients achieves an objective response with clearcut neurological improvement and reduction in the area of enhancement on their MRI scan [bib_ref] Phase I trial of temozolomide using an extended continuous oral schedule, Brock [/bib_ref]. A further 25% have disease stabilisation in terms of their MRI scans and some neurological improvement when TMZ is given in the dose of 200 mg m À2 days 1 -5 schedule repeated at 4 weekly intervals. In the randomised study of TMZ against PCB, the response rate to PCB was lower, the duration of those responses shorter, and the quality of life poorer. However, PCB clearly had modest activity against high-grade gliomas [bib_ref] A Phase II study of temozolomide vs. procarbazine in patients with glioblastoma..., Yung [/bib_ref]. Neurological deficit causing moderate functional impairment (e.g. being able to move limb(s)) only with difficulty, moderate dysphasia, moderate paresis, and some visual disturbances (e.g. visual field defect) 3
Neurological deficit causing major functional impairment for example inability to move limbs, gross speech, or visual disturbance 4
No useful function; inability to make conscious responses
In this study, the combination of TMZ and PCB was generally well tolerated at doses up to dose level 3. At this dose level, there is little difference in terms of side effects between TMZ alone and the combination with PCB. We had intended to extend this cohort to 30 or 40 patients to identify whether or not the combination was superior to TMZ on its own. However, the supply of PCB was interrupted and the study had to be suspended when a total of 16 patients had been entered at dose level 3. Clearly, these results on a small number of patients are preliminary. The clinical impression is that the combination probably is a bit more active than TMZ on its own, and it may be that some of the patients who would otherwise have been classified as having stable disease would have moved into the responding group, suggesting a greater antitumour activity when the two agents are given together.
# Conclusion
TMZ and PCB, when combined at the recommended dose levels, are a reasonably effective and well-tolerated combination in treating patients with relapsed gliomas and tumour activity is seen in low-grade gliomas, AA, and GBM. Glioma grade II patient transforming to GBM. This MRI shows a patient with grade II glioma, which had transformed to GBM. This is an ongoing response to TMZ and PCB at dose level 3 at 14 þ months.
[table] Table 1: Clinical structure: temozolomide and procarbazine phase I [/table]
[table] Table 2: Study design: temozolomide and procarbazine phase I [/table]
[table] Table 3: Medical Research Council Neurological Status [/table]
[table] Table 4: Haematological toxicities. Common toxicity criteria grades 3 and 4Temozolomide alone. b Temozolomide alone, one patient. c Temozolomide alone, three patients. [/table]
[table] Table 5: Other toxicities. Common toxicity criteria grades 3 and 4 Temozolomide alone. b Hepatitis A infection, one patient. [/table]
[table] Table 6: Response to histological grade AA ¼ anaplastic astrocytoma, GBM ¼ glioblastoma multiforme. [/table]
[table] Table 7: Responses by dose of procarbazine [/table]
[table] Table 8: Response duration [/table]
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Reduction of psychological cravings and anxiety in women compulsorily isolated for detoxification using autonomous sensory meridian response (ASMR)
Objective:To explore the effects of the autonomous sensory meridian response (ASMR) on the psychological cravings and anxiety of women compulsorily isolated for detoxification.Method:Around 122 women were recruited in a female drug detoxification center.Except for the 12-week training of ASMR, the experimental conditions of the experimental group (n = 60) were the same as those of the control group (n = 62). The addiction Stroop task was used to assess the level of psychological cravings and the State-Trait Anxiety Inventory was used to assess the level of anxiety.Results:After the training, the decrease in state anxiety of the experimental group was larger than that of the control group, and the reaction time of the experimental group in the Stroop was also significantly lower than before the training.Conclusions: ASMR could thus reduce to a certain extent the state anxiety and attentional bias for drug-related clues under signaling psychological cravings among women compulsorily isolated for detoxification.HIGHLIGHTS:- Intervention effects on psychological cravings and anxiety of women isolated for detoxification - Basis for role of ASMR in regulating psychological cravings and anxiety in forced abstainers - ASMR intervention reduced forced abstainers' attentional bias to drug-related cluesK E Y W O R D Sautonomous sensory meridian responses (ASMR), detoxification, psychological cravings, forced abstainers, anxietyThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
As a global social issue, the still severe problem of drug addiction poses a serious threat to human health and social development, given the issues of extreme dependence and relapse rate. In current detoxification work, there are two commonly used models for drug addiction:
One is a physical and medical rehabilitation model based on drug substitution therapy, and the other is a social psychological rehabilitation model based on psychological intervention and cognitive behavior modification [bib_ref] The development and construction of psychological and social intervention models for drug..., Lin [/bib_ref]. In China, compulsory isolation for detoxification is the mainstay of treatment, and various compulsory, punitive, educational, and corrective technical methods are used to help drug users overcome addiction.
Among these methods, psychological cravings are particularly challenging in the context of abstinence. From the perspective of psychological research, psychological cravings refers to an addict's uncontrollable impulsive desire for the past subjective experience of psychoactive substances; and operationally, it can be defined as the psychological preference and implicit attitude derived from the longterm addictive behavior of the addict, which is difficult to control . Many factors affect psychological cravings. Among them, addicts' negative emotions are beginning to attract increasingly more attention and research interest. Susceptibility factors such as anxiety, depression, and fear play a key role in the craving for addictive substances, and to a certain extent, can positively predict or assess relapse in addicts [bib_ref] Addiction motivation reformulated: An affective processing model of negative reinforcement, Baker [/bib_ref] [bib_ref] Delineating the psychic structure of substance abuse and addictions: Should anxiety, mood..., Pani [/bib_ref] [bib_ref] The relationship between anxiety and relapse of drug addicts: the chain mediating..., Zhou [/bib_ref].
It is well known that men predominate among traditional drug abstainers, but methamphetamine (MA) abstainers are similarly divided between men and women [bib_ref] Prevalence of nonmedical methamphetamine use in the United States, Durell [/bib_ref]. Previous studies, however, have focused more on MA male abstainers [bib_ref] Association between alexithymia, social support, and duration of methamphetamine use among male..., Cui [/bib_ref] [bib_ref] Effects of acute moderate-and high-intensity aerobic exercise on oxygenation in prefrontal cortex..., Gao [/bib_ref] [bib_ref] Difference in topological organization of white matter structural connectome between methamphetamine and..., Li [/bib_ref] [bib_ref] Neurophysiological correlate of incubation of craving in individuals with methamphetamine use disorder, Zhao [/bib_ref]. It is worth noting that the mental health problems of women undergoing compulsory drug rehabilitation are more serious than those of men in China [bib_ref] Investigation and analysis on psycho-physcond of 417 case heroin addicts under compulsory..., Wen [/bib_ref] [bib_ref] Analysis on the characteristics and countermeasures of drug addicts in compulsory isolation..., Xia [/bib_ref]. Female MA abstainers exhibit more severe psychiatric symptoms than men, and they seek treatment for major depression and suicidal ideation more commonly than men [bib_ref] Patterns of psychological distress related to regular methamphetamine and opioid use, Darke [/bib_ref]. In a study, a significantly higher proportion of women with MA withdrawal met criteria for anxiety disorders than men [bib_ref] Psychopathology in methamphetamine-dependent adults 3 years after treatment, Glasner-Edwards [/bib_ref]. Notably, the intensity of craving in MA withdrawal is positively correlated with the scores obtained from the Symptoms Checklist-90 [bib_ref] Association between psychiatric symptoms and craving in methamphetamine users, Nakama [/bib_ref] , and craving in MA withdrawal is strongly correlated with anxiety, and they are often accompanied by mood disorders such as anxiety disorders, which is the starting point for developing withdrawal treatments [bib_ref] Sex differences in the association between internalizing symptoms and craving in methamphetamine..., Hartwell [/bib_ref] , which suggests the need to consider the level of psychological cravings and anxiety among women undergoing compulsory drug rehabilitation.
Further, numerous studies on the attentional bias of drug addicts have found that psychological cravings can cause addicts to skew their attention-subconscious overattention is paid to cues related to addictive substances [bib_ref] The clinical relevance of attentional bias in substance use disorders, Field [/bib_ref] [bib_ref] Depression, craving, and substance use following a randomized trial of mindfulness-based relapse..., Witkiewitz [/bib_ref]. This, in turn, causes craving and creates a vicious circle. The addiction Stroop task and dot-probe task are two commonly used research paradigms to assess attentional bias. Compared with the dot-probe task, the improved addiction Stroop task has higher internal consistency, thus being more suitable to study the attentional bias of addicts related to substances. Simultaneously, the study ofalso fully confirmed a significant association between performance in the addiction Stroop task and psychological cravings.
That is, the higher the addicts' attentional bias toward drug-related cues, the worse their Stroop task performance, and the higher their level of psychological cravings. This suggests that the Stroop interference effect can be an indirect measure of the level of psychological cravings.
Recently, media related to the autonomous sensory meridian response (ASMR) have frequently appeared on Internet platforms such as Reddit ASMR forum and the video site YouTube. ASMR is an atypical physical-psychological experience with a dynamic, wave-like, staticlike tingling sensation, triggered by a specific audio-visual stimulus. It is also called "intracranial tingling" or "intracranial orgasm." It usually originates from the back of the scalp and gradually progresses along the spine to the shoulders or limbs, accompanied by positive feelings such as relaxation, happiness, euphoria, and elevated mood. Common triggers include watching someone whisper, performing repetitive rhythmic movements, and exploring an object; however, the coverage of the tingling seems to depend on the degree to which the individual is triggered [bib_ref] Autonomous Sensory Meridian Response (ASMR): A flow-like mental state, Barratt [/bib_ref]. At present, the exploration of the physio-psychological mechanism behind ASMR is still in the early stage, and mainly focuses on the neurobasic research and case report research on the causes of positive sensory emotions, attention, improvement of pain, and social cognition [bib_ref] Autonomous sensory meridian response: Your patients already know, do you?, Reddy [/bib_ref]. But public interest in it is growing, and more researchers are actively seeking to use it as a complementary therapy for appropriate treatment.
In the first assessment of ASMR, [bib_ref] Autonomous Sensory Meridian Response (ASMR): A flow-like mental state, Barratt [/bib_ref] found that ASMR participants reported a temporary relief of chronic pain and mood improvement. Among them, 80% of the participants reported that ASMR had positive emotional effects (relaxation and euphoria).
Simultaneously, ASMR had a "placebo effect"-the first contact with ASMR media may cause a somatosensory response that meets personal expectations, while more frequent users usually experienced a sense of relaxation and satisfaction actually triggered by ASMR media [bib_ref] Expectancy effects in the autonomous sensory meridian response, Cash [/bib_ref]. Therefore, the sensitivity to ASMR may also influence its effects.
Given the positive effects of ASMR on mental health and emotional regulation (such as relieving stress, anxiety, loneliness, and insomnia), ASMR-related academic studies have gradually become popular in recent years. ASMR has been associated with other topics and technologies in exploratory research. However, there are no literature reports on the application of ASMR to forced abstainers. Therefore, this study pioneers the application ASMR to the regulation of anxiety in forced abstainers, especially the psychological cravings in the context of abstinence.
We propose that ASMR can reduce anxiety effectively and shift the addicts' attentional bias from drug-related cues progressively to eliminate the psychological cravings for drugs in the withdrawal response of forced abstainers. Besides, we also propose that ASMR sensitivity characteristics may influence the effectiveness of the emerging methods. Thus, ASMR is expected to be employed as a specialized treatment tool and ultimately help addicts to achieve the recovery of physical and mental health and return to normal social life at the earliest.
# Methods
## Research design
A randomized controlled trial was conducted, with a three-factor mixed design of 2 (ASMR training/no training) × 2 (ASMR sensitive/nonsensitive) × 2 (ASMR with semantic dialog/without semantic dialog).
This study was approved by the Ethics Committee of a medical university (Approval no. 2020-122). All participants voluntarily participated in the study and signed an informed consent form. Participants could withdraw at any time if they were unwilling or unable to continue with their participation in the study.
## Participants
According to the sample size calculation from G-Power, 128 partici- It is also controlled by international antidrug conventions and Chinese laws and regulations.
During the training period, participants were required not to participate in other psychological or behavioral treatments. A total of 193 of 650 screened women fulfilled the eligibility criteria and were not arranged to leave the drug detoxification center.
Subsequently, through a self-report survey of ASMR sensitivity, 64 sensitive participants (S) to ASMR videos were selected from the 193 eligible participants, and 64 insensitive participants (IS) were randomly selected from the remaining participants. During the ASMR training period, three participants had missing data (improper operation on the computer), two participants dropped out (isolated for 2 weeks due to fever during the COVID-19 epidemic), and one filled out invalid questionnaires multiple times. After excluding these participants, data from 122 participants were finally included in the analysis, including 60 in the ASMR group (S:IS = 30:30) and 62 in the control group (S:IS = 31:31) the balance between the groups with ASMR sensitivity characteristics. (See [fig_ref] F 1: 13 [/fig_ref].
# Materials
## Asmr videos
The foremost videos containing ASMR triggers from the ASMR video library compiled by [bib_ref] A preliminary compilation of a digital video library on triggering autonomous sensory..., Liu [/bib_ref] were selected, comprising 59 videos (i.e., 30 ASMR videos with semantic dialog and 29 ASMR videos without semantic dialog, produced by performers of different genders).
The length of each video was between one and three minutes. (See Appendix B and C for details on the material).
## Psychological cravings
The addiction task under the Stroop paradigm was used to determine the participants' level of psychological cravings. The stimulus material included 30 drug-related Chinese characters and 30 neutral Chinese characters. After randomized sorting using Excel, manual sorting was performed to make the Chinese characters of different nature appear pseudo-randomly in three colors-red, yellow, and green. This was done to avoid the continuous presence of Chinese characters of the same color or the same nature for up to three times in a row. Besides, the interference effect of Stroop was evaluated mainly through accuracy rate and reaction time. (See Appendix D for details on the material).
## State-trait anxiety
The State-Trait Anxiety Inventory (STAI) compiled bywas used, which comprises a total of 40 items divided into two subscales-State Anxiety Inventory (SAI) and Trait Anxiety Inventory (TAI)-each with 20 items. Items are scored on a 4-point Likert-type scale, and all positive emotions were scored reversely (9 items in SAI and 10 items in TAI). The scores on each subscale range from 20 to 80, and higher scores indicate higher state or trait anxiety levels. In this study, we used the Chinese version of the STAI, and its reliability and validity have been verified in previous studies [bib_ref] The Chinese version of the State-Trait Anxiety Inventory: Its relationship to different..., Shek [/bib_ref] block. The subjects were asked to select one of the three keys as quickly and correctly as possible according to the color of the Chinese characters in the picture. The different keys represented red, blue and green colors, respectively. There was a break of 2-3 min between each test group. And the procedure automatically recorded the response time and accuracy of the key to the picture within 3000 ms.
The ASMR training (watching three ASMR videos of different intensity and content) was always conducted in the same computer room in the drug detoxification center. During the training, the conditions were as follows: (1) the environment was quiet and undisturbed, and appropriate activity space was reserved for the participants;
(2) the headset functions were intact, and the volume was adjusted to 80% and the screen brightness to 50% uniformly; (3) the ASMR video was played according to the instructions.
# Statistical analysis
All statistical analyses were performed using IBM SPSS 23.0. For some raw data involving the privacy of the participants, appropriate conversions were performed first; the changes do not affect the actual statistics. All descriptive statistics were provided with 95% confidence intervals across participants. Student's t-test was used to evaluate the association between ASMR intervention and, attentional bias, or state anxiety, respectively. Three-factor repeatedmeasure ANOVA was used for the interaction effect among sensitivity, semantic dialog, and training period on attentional bias and state anxiety. For all comparisons, p < 0.05 was considered statistically significant.
# Results
## Demographic characteristics
The age range of the participants was 18-54 years, with an average age of 33.83 years (SD = 8.612); participants were mostly young.
## Psychological cravings
## Intergroup effect of asmr on attentional bias
In the intragroup analysis of differences in the addiction Stroop task performance, there was no significant difference in accuracy (Acc) comparing before and after the operation for the experimental or the control group (p = 0.525, p = 0.343). However, the reaction time (Rt) of the experimental group was significantly reduced after the training compared to before (p = 0.01), while the control group showed no significant difference (p = 0.946).
In the analysis of differences between groups, there was no significant difference in Acc or Rt between the two groups in the pretest (p > 0.05). After the training, there were no significant differences between the groups in Acc. However, the experimental group had a lower reaction time than the control group, and the difference was significant (p < 0.05). (See .
## 2.2.2
Intragroup effect of sensitivity, semantic dialog, and training period on attentional bias
The results of three-factor repeated-measure ANOVA on attentional bias showed that, for Acc and Rt, the main effect of ASMR sensitivity And none of interactions was significant either.
## Anxiety
## Intergroup effect of asmr on state anxiety
Before the training, there was no significant difference in the trait anxiety (TAI scores) between the experimental and control groups (p = 0.078). That is, the individual differences (trait anxiety, TA) were consistent in the anxiety level of the participants, which made the time dimension (state anxiety, SA) comparable between the groups.
In the analysis of differences at different points of the training, the SAI scores of the two groups were not significantly different when the training had been conducted out for 1 month. After 6 weeks of the training, the SAI score of the experimental group was significantly lower than that of pretest (8 weeks: M = −13.684±SD 2.039; 12 weeks: M = −13.577 ± SD 2.079) (p < 0.01). That is, the training effect after 6 weeks in SA reduction in the experimental group was larger than in the control group. (See .
## Intragroup effect of sensitivity, semantic dialog, and training period on state anxiety
The results of three-factor repeated-measure ANOVA on state anxiety showed that the main effect of ASMR sensitivity was not significant, and .
## F i g u r e 2
Changes in the decreased level of state anxiety (SA) between groups during the training period (M ± SE): compared with pretest, the change of the experimental group was significantly after six weeks of the training (p < 0.01)
# Discussion
To accurately explore the effects of ASMR videos on the reduction of psychological cravings and anxiety among forced abstainers, this study adopted a three-factor mixed design of 2 (ASMR training/no training) × 2 (ASMR sensitive/nonsensitive) × 2 (ASMR with semantic dialog/without semantic dialog) to conduct a controled training experiment with 122 participants depending on new drugs in a female drug detoxification center in China.
The most important result of this study was that after the training, while the Acc of the experimental group in the addiction Stroop task did not change significantly both within and between groups, the Rt was significantly lower than that of the control group. This indicates that the attentional bias of the participants of the experimental group for color-related cues reflected by the words was higher than that for drug-related cues. It has been confirmed that drug Stroop effect is prevalent in drug addiction, and this effect is positively correlated with relapse rate [bib_ref] Clinical correlates of attentional bias to drug cues associated with cocaine dependence, Kennedy [/bib_ref]. Drug
Stroop effect not only indicates poor control inhibition ability, but also attentional bias to drug-related cues. Many studies have shown that attentional bias is positively correlated with subjective craving, and its validity is even higher than that of the subjective report questionnaire [bib_ref] Assessing the severity of methamphetamine use disorder beyond the subjective craving report:..., Liang [/bib_ref] [bib_ref] Alcohol-cue exposure effects on craving and attentional bias in underage college-student drinkers, Ramirez [/bib_ref] [bib_ref] Cognition and craving during smoking cessation: an ecological momentary assessment study, Waters [/bib_ref]. Therefore, attentional bias can be used as a behavioral indicator of subjective craving.
ASMR reduces the attentional bias of drug addicts to drug-related cues, which indicates that ASMR has a benign effect on the regulation of "psychological cravings" of drug addicts in compulsory isolation in this study. In addition, in this study, the SAI score of the experimental group was significantly reduced after 4 weeks of the training period compared with that before the training, indicating that ASMR also played a benign role in regulating the SA of the people with severe abstinence. Although it was statistically shown that ASMR effectively reduced the state anxiety of the participants, considering the subjectivity of the "questionnaire report," the practice effect of the design before and after the test, as well as the influence of psychological comfort and other factors, the significant effect may be exaggerated. In general, ASMR is a unique form of relaxation that uses auditory and visual stimuli to bring about a calm state of mind and tingling sensations, like mindfulness [bib_ref] Cortical activation changes associated with autonomous sensory meridian response (asmr): Initial case..., Seifzadeh [/bib_ref]. Some studies have
shown that ASMR has the same characteristic factors as mindfulness training. It is known that mindfulness training is one of the most effective psychotherapies for addiction, and this suggests that the high correlation between ASMR and mindfulness reveals the potential for complementary psychotherapy, which may be just as effective at reducing drug cravings and anxiety as mindfulness.
Interestingly, the interaction between sensitivity, semantic dialog, and training period on state anxiety was significant. In general, the anxiety of drug addicts decreased gradually and then leveled off with the training periods; however, the changes of state anxiety in sensitive and nonsensitive participants were different in different training periods.
This suggests that the content of ASMR should be measured according to the sensitivity of the audience. In recent years, ASMR has been widely used in stress management for its ability to trigger psychologically pleasurable responses [bib_ref] Possible effect of binaural beat combined with autonomous sensory meridian response for..., Lee [/bib_ref] [bib_ref] More than a feeling: Autonomous sensory meridian response (ASMR) is characterized by..., Poerio [/bib_ref]. However, ASMR training rarely selects the type of video (for example, whether it has semantic dialog) based on the type of audience (such as sensitivity). In fact, there are differences in brain activity among spontaneous perceptual participants who are sensitive to different triggers [bib_ref] Functional connectivity associated with five different categories of Autonomous Sensory Meridian Response..., Smith [/bib_ref]. The implication for us is that in future studies, video types can be selected according to the sensitive types of the participants.
It is important to note that ASMR sensitivity/non-sensitivity were no significant main effect on state anxiety, which may reflect the universality of ASMR or may be due to the subjective reporting used in the measurement of ASMR sensitivity. Subjective report will be affected by individual judgment criteria and social credit to some extent. Apart from subjective reports, cognitive experiments and neurophysiological instruments measured anxiety and sensitivity only indirectly. In future studies, more indicators will be considered, such as attention, EEG, and respiratory rate, to assist.
In this study, both psychological cravings and anxiety were reduced in MA female abstainers through the ASMR intervention. As shown earlier, a large number of studies have shown a positive correlation between self-reported craving and anxiety in MA abstainers. It is known that weakened attentional bias to drug-related cues in withdrawal clients implies a decrease in psychological cravings, and some studies have also indicated that people's weakened attentional bias to threat reduces anxiety [bib_ref] Threat-related attentional bias in anxious and nonanxious individuals: a meta-analytic study, Bar-Haim [/bib_ref] [bib_ref] Attentional bias in youth with clinical anxiety: The moderating effect of age, Carmona [/bib_ref] [bib_ref] Attentional bias towards threatening stimuli in children with anxiety: A meta-analysis, Dudeney [/bib_ref] [bib_ref] Attentional biases toward threat: The concomitant presence of difficulty of disengagement and..., Sagliano [/bib_ref].
So what is the link between cognitive neurophysiological mechanisms of attentional bias to drug-related cues and response to threatening stimuli? We suggest that in-depth studies are warranted in order to further confirm this association.
Furthermore, in terms of participant selection, this study included a sample of female forced abstainers who were all dependent on new drugs and from a single region. Thus, it lacked representativeness in terms of region, gender, and addiction type, and the training length and number of sessions were also limited. Also, in the measurement of attentional bias, the addiction Stroop task uses words of different colors as experimental materials. Thus, there may be differences in the performance of participants according to their education level; more consideration can be given to point detection paradigms using drug-related pictures as stimulus clues. In addition, the materials in the ASMR video library used in this study were mainly created by foreign creators. Different languages, themes, and cultures may also affect the ASMR effects. In consequence, the work of creating localized ASMR materials is urgently required.
# Conclusion
This study provides a theoretical basis for the role of ASMR in regulating psychological cravings and anxiety in forced abstainers and yielded the following findings: (1) ASMR had a positive effect on the mental health of forced abstainers; (2) the ASMR training reduced forced abstainers' attentional bias to drug-related clues; and (3) after 1 month of the training, the effect of ASMR in reducing SA became significant.
# Acknowledgments
## Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
# Author contributions
MQH, HLL, SQH and QZ developed the original idea and the protocol, drafted of the manuscript. MQH, HLL and YYQ abstracted and analyzed data, improved the manuscript. SSW, YTJ, LY and XY contributed to the critical revision of the manuscript for important intellectual content.
# Data availability statement
The data that support the findings of this study are openly available in https://www.researchgate.net/profile/Qiang-Zhou-13, named "data set"
## Peer review
The peer review history for this article is available at https://publons. com/publon/10.1002/brb3.2636.
## Orcid
Xin Yu https://orcid.org/0000-0002-0830-4725
Qiang Zhou https://orcid.org/0000-0002-3045-0198
[fig] F 1: 13) = 2.263, p > 0.05, η 2 = 0.148; F (1,13) = 0.978, p > 0.05, η 2 = 0.070), semantic dialog (F (1,13) = 0.765, p > 0.05, η 2 = 0.056; F (1,13) = 0.550, p > 0.05, η 2 = 0.041), or training period (F (1,13) = 0.270, p > 0.05, η 2 = 0.020; F (1,13) = 0.472, p > 0.05, η 2 = 0.041) was not significant. [/fig]
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The Effects of Bromocriptine on Preventing Postpartum Flare in Systemic Lupus Erythematosus Patients from South China
Objective. Prolactin plays an important role on the disease flare of postpartum SLE patients. 76 pregnant SLE patients were enrolled in this study to evaluate the efficacy of bromocriptine (an inhibitor of prolactin secretion) on preventing the postpartum disease relapse. Methods. Patients were randomly divided into the treatment group (bromocriptine, 2.5 mg oral, twice a day for 14 days after delivery) and the control group. All the patients were followed up for 12 months. Clinical features were recorded every 4 weeks. Serum prolactin and estradiol levels were measured at the second week and the second month after delivery. The endpoint of the study was disease relapse and defined when SLEDAI score increased by ≥3 points from the antenatal baseline. Results. (1) Serum levels of prolactin and estradiol decreased significantly in bromocriptine treatment group at the second week ( < 0.001) and second month ( < 0.05) after delivery compared to control group. (2) The relapse rate of the treatment group was lower than the control group ( 2 = 4.68, = 0.0305). Conclusions. Two weeks of oral bromocriptine treatment in postpartum SLE patients may relieve the disease from hyperprolactinemia and hyperestrogenemia and may be beneficial in preventing the patients from disease relapse.
# Introduction
Many lupus patients experience disease flare in late trimester of pregnancy or puerperium [bib_ref] Flares of systemic lupus erythematosus during pregnancy and the puerperium: prevention, diagnosis..., Stojan [/bib_ref]. Recently, a retrospective study of pregnancies in Chinese women with systemic lupus erythematosus (SLE) demonstrated that 26 of 68 pregnancies (38%) who were stable at conception encountered disease flare during pregnancy or postpartum [bib_ref] Pregnancy in women with systemic lupus erythematosus: a retrospective study of 111..., Liu [/bib_ref]. Therefore, disease flare is a major concern of rheumatologists when facing pregnancy and puerperium SLE patients. High serum levels of sex hormones, especially prolactin and estradiol, have been considered to be associated with the lupus disease activity [bib_ref] Prolactin and autoimmune diseases, Yang [/bib_ref] [bib_ref] Hormones, immune response, and pregnancy in healthy women and SLE patients, Zen [/bib_ref]. Serum levels of prolactin and estradiol are usually increased significantly during pregnancy, puerperium, or postpartum lactation period compared to normal women [bib_ref] Effects of prolactin in stimulating disease activity in systemic lupus erythematosus, Walker [/bib_ref] , which indicated that high serum levels of prolactin and estradiol might contribute to lupus flares [bib_ref] Hormones, immune response, and pregnancy in healthy women and SLE patients, Zen [/bib_ref] [bib_ref] Sex hormones and pregnancy in rheumatoid arthritis and systemic lupus erythematosus, Østensen [/bib_ref].
Prolactin is a polypeptide hormone secreted by the anterior pituitary gland. It not only regulates the growth and differentiation of the mammary gland and ovary, but also initiates and maintains lactation. Prolactin is considered as well as a cytokine because it is secreted by immune cell and its receptor belongs to the cytokine receptors type 1 family [bib_ref] Prolactin and autoimmunity, Shelly [/bib_ref]. PRL could enhance the production of autoantibodies by influencing B-cell maturation in the early stage and promoting the survival of self-reactive B cell clones [bib_ref] Prolactin levels correlate with abnormal B cell maturation in MRL and MRL/lpr..., Legorreta-Haquet [/bib_ref]. Bromocriptine, an inhibitor of PRL secretion, is an ergot derivative that binds to dopamine (D2) receptors on normal or adenomatous lactotroph cells. Previous studies demonstrated that bromocriptine could improve the SLEDAI scores in SLE patients with mild to moderate disease activities [bib_ref] Serum free prolactin concentrations in patients with systemic lupus erythematosus are associated..., Leaños-Miranda [/bib_ref]. The same results were also found in mice [bib_ref] Bromocriptine has little direct effect on murine lymphocytes, the immunomodulatory effect being..., Neidhart [/bib_ref] [bib_ref] Differential effects of estrogen and prolactin on autoimmune disease in the NZB/NZW..., Elbourne [/bib_ref]. But for postpartum SLE patients, there were few researches on the relationship between prolactin and lupus activity. Our previous work indicated that breast-feeding after delivery had the potential of increasing the incidence of postpartum relapses in disease, and bromocriptine may eliminate this risk [bib_ref] Efficacy of oral bromocriptine in protecting the postpartum systemic lupus erythematosus patients..., Yang [/bib_ref] , but the sample size is small. So in the current study we designed a randomized controlled trial in order to get more information about the efficacy of oral bromocriptine on preventing the postpartum patients with SLE from disease relapse. The study design is presented in . Local Ethics Committee approved the protocol and written informed consents were obtained. All patients fulfilled at least 4 of the American College of Rheumatology criteria for SLE [bib_ref] Updating the American College of Rheumatology revised criteria for the classification of..., Hochberg [/bib_ref]. Pregnant lupus patients with inactive or mild-active SLE receiving no more than 10 mg of prednisone (or equivalent) per day were eligible for this study. Disease activity was measured by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [bib_ref] Derivation of the SLEDAI: a disease activity index for lupus patients, Bombardier [/bib_ref]. Patients were defined as inactive SLE when the SLEDAI scores ≤4 and mild-active with the scores between 5 to 9. Patients were excluded if they (1) had a SLEDAI score ≥10; (2) had proteinuria >500 mg/24 hours or other major organ system involvements; (3) were receiving >10 mg/day of prednisone (or equivalent) during pregnancy; or (4) had a serum level of complement 3 < 0.5 g/L.
# Patients and methods
# Methods.
Patients were randomly divided into the bromocriptine group and the control group. The bromocriptine group received 14 days of oral bromocriptine 2.5 mg twice a day within 12 hours after delivery and did not breastfeed their infants. The control group was not treated by bromocriptine or any other medicine that might influence patients' serum prolactin levels.
Demographic data and clinical characteristics were collected 1 to 4 weeks before expected date of childbirth. All patients were followed up every 4 weeks for 12 months. Clinical manifestations and laboratory findings were recorded during every visit to assess the SLE activity. Laboratory investigations included serum levels of antinuclear antibodies, anti-dsDNA antibody, complements, complete blood count, urinalysis, serum albumin, liver function, and creatinine. Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) was used to assess the disease activity. SLEDAI was performed two to four weeks before delivery and every six weeks after delivery. To minimize the influence of observation bias on outcome determination, the SLEDAI scores were calculated by two different investigators. One performed the history collection and physical examination and did not know the serologic findings, and the other one performed the serological assessment and the SLEDAI scoring.
The serum prolactin and estradiol levels were measured twice by radioimmunoassay, at the second week and the second month after delivery, respectively.
## End points of trial and definition of clinical flares.
Increasing of SLEDAI score ≥3 points from antenatal baseline data was defined as the endpoint of this trialclinical flare. Mild-to-moderate flare was defined if one or more of the following 5 features were fulfilled: (1) an increase of SLEDAI score ≥3 points but <12 points;
(2) new/worse discoid, photosensitive, cutaneous vasculitis, bullous lupus, nasopharyngeal ulcers, profundus, pleuritis, pericarditis, arthritis, and fever; (3) an increase in the prednisone dosage but <0.5 mg/kg of body weight per day; (4) initiation of therapy with either hydroxychloroquine or nonsteroidal anti-inflammatory drugs; (5) increase in the physician's global assessment score but no more than 2.5. Severe flares were defined when one or more of the following 4 features were fulfilled: (1) increasing the SLEDAI score ≥12 points; (2) new or worsening central nervous system (CNS) involvement, vasculitis, glomerulonephritis, myositis, thrombocytopenia (platelet count <60 * 10 9 cells/liter), or hemolytic anemia (Hb < 70 g/L or decreasing of Hb > 30 g/L), required doubling dose of corticosteroid or a final dosage of >0.5 mg/kg/day or hospitalization; (3) any manifestation requiring an increase in the dosage of prednisone or equivalent drug to >0.5 mg/kg/day or initiation of therapy with cyclophosphamide, azathioprine, mycophenolate mofetil, or methotrexate; (4) hospitalization because of lupus activity; or (5) increase in the physician's global assessment score >2.5 [bib_ref] The effect of combined estrogen and progesterone hormone replacement therapy on disease..., Buyon [/bib_ref].
## Statistical analyses.
Survival analysis was used to compare disease aggravation and relapse rate between two groups. Log-rank test was used to assess significant differences between two groups. Parameter data, such as SLEDAI score and sex hormone levels, were recorded as X ± SD and tested by test. Nonparameter data were analyzed by Wilcoxon rank sum test. All statistical analyses were performed on STATA 10.0 statistical software.
# Results
## The baseline demographic features and clinical
Characteristics of the Patients. Ninety-five pregnant lupus patients were screened, 76 of them qualified for the study and were enrolled into the trial. The patients were randomly divided into the treatment group ( = 38) and the control group ( = 38). All 38 patients in the treatment group were prescribed with oral bromocriptine for two weeks. There was no statistical difference on the baseline data (demographic features, laboratory findings, and SLEDAI scores) between these two groups (shown in [fig_ref] Table 1: Baseline demographic features, clinical findings, and SLEDAI scores in the pregnant SLE... [/fig_ref]. [fig_ref] Figure 2: Kaplan-Meier survival curves for relapse-free Survival between the treatment group and the... [/fig_ref]. Relative risk reduction (RRR) was 68.8% and the 95% confidence interval (CI) was 24.3%-87.1%. Absolute risk reduction (ARR) was 32.4% and the 95%CI was 11.8%-53%. The number needed to treat (NTT) was 3.1, 95%CI (1.9-8.5), which means 3.1 patients needed to be treated to protect one patient from relapse. All severe flares occurred in the control group.
## Clinical outcomes in the treatment
## Effect of bromocriptine on serum levels of prolactin and
Estradiol. The serum prolactin and estradiol levels in the treatment group were significantly lower than the levels in the control group at the second week and the second month after delivery, respectively (shown in [fig_ref] Table 2: Comparison of the serum prolactin/estradiol levels between two groups at the second... [/fig_ref]. At the end of second week, serum prolactin levels of patients in the treatment group were all in normal range, while those in the control group were higher than the upper limit of normal range (0 versus 100%). At the end of second month, 2 patients in treatment group had increased serum levels of prolactin (5.3%, 2/38), while, in control group, 17 patients experienced hyperprolactinemia (44.7%, 17/38).
## Effect of bromocriptine on disease activity of the patients after delivery.
At the 6th and 12th months after delivery, SLEDAI scores of the treatment group were significantly lower than those of the control group [fig_ref] Table 3: Change of SLEDAI within 1 years after delivery [/fig_ref].
## Cumulative doses of immunosuppressive agents.
The cumulative doses of corticosteroid and other immunosuppressive agents after 12-month follow-up were shown in [fig_ref] Table 4: Cumulative doses of corticosteroid and immunosuppressive agents for SLE in 12 months... [/fig_ref]. There were significant differences on the accumulative doses of prednisone and cyclophosphamide between two groups. In bromocriptine group, the need of immunosuppressants to keep disease-stable was significantly lower than control.
3.6. The Adverse Effects of Treatment. Three patients had mild vertigo and nausea in the treatment group and all patients could complete the 2 weeks oral bromocriptine therapy. No severe adverse event was found in this study.
# Discussion
For pregnant SLE patients, though many of them can pull through pregnancy and childbirth in the remission stage of disease, some patients still experience relapse or disease flare during the late trimester of pregnancy or puerperium period. Several studies had shown that hyperprolactinemia was associated with lupus activity in pregnancy. Prolactin can stimulate mammary growth and differentiation, and it progressively increases during these periods [bib_ref] Hormones, immune response, and pregnancy in healthy women and SLE patients, Zen [/bib_ref]. Recently, a study confirmed that prolactin levels are associated with lupus activity, lupus anticoagulant, and poor outcome in pregnancy. Significant linear correlations between prolactin, modified-systemic lupus activity measurement (m-SLAM), and lupus anticoagulant were observed [bib_ref] Prolactin levels are associated with lupus activity, lupus anticoagulant, and poor outcome..., Jara [/bib_ref]. But for postpartum SLE patients, there were few researches on the relationship between prolactin and lupus activity. The results in our study showed that the serum prolactin levels in control group were raised obviously at the second week after delivery, about 5 times the upper normal limit of prepregnancy levels. At the second month after delivery, the serum prolactin levels in the control group dropped but still much higher than those in the treatment group. In the control group, SLEDAI scores at the 6th and 12th months after delivery were much higher than those in treatment group. The results of the current study preliminarily indicated that oral bromocriptine for 2 weeks in postpartum patients with SLE may protect them from hyperprolactinemia and hyperestrogenemia and may be beneficial for preventing the patients from disease relapse.
In nonpregnancy SLE patients, some studies had been performed to determine whether there is an association between hyperprolactinemia and lupus activity, but the results were controversial. Orbach et al. [bib_ref] Prolactin and autoimmunity: hyperprolactinemia correlates with serositis and anemia in SLE patients, Orbach [/bib_ref] reported that hyperprolactinemia in lupus patients was associated with serositis and anemia but not with SLE disease activity. But most other researches indicated that hyperprolactinemia was found to be associated with SLE disease activity and conventional immunosuppressive therapy decreased PRL levels as well as SLE activity [bib_ref] Correlation of prolactin serum concentrations with clinical activity and remission in patients..., Vera-Lastra [/bib_ref] , and elevated serum bioactive prolactin concentrations in SLE patients were associated with disease activity [bib_ref] Elevated serum bioactive prolactin concentrations in patients with Systemic lupus erythematosus are..., Cárdenas-Mondragón [/bib_ref]. Serum prolactin includes several isoforms, most of them are free PRL (small prolactin, molecular weight 23 kDa) and lesser amounts are big prolactin and big big prolactin (molecular weight 45-50 and >100 kDa, resp.) [bib_ref] Biologic activity and plasma clearance of prolactin-IgG complex in patients with systemic..., Leaños-Miranda [/bib_ref]. Big big PRL or macroprolactin is a PRL variant with reduced bioactivity that may contribute to the lower disease activity and absence of symptoms in SLE [bib_ref] Correlation of prolactin serum concentrations with clinical activity and remission in patients..., Vera-Lastra [/bib_ref] [bib_ref] Elevated serum bioactive prolactin concentrations in patients with Systemic lupus erythematosus are..., Cárdenas-Mondragón [/bib_ref]. So the varied results in different studies about the relationship of prolactin and SLE disease activity may be due to the different bioassay methods that are used to access the serum level of prolactin. In the current study, what we measured was the total PRL, including free PRL, big-, and macroprolactin. Further research work should be done to address the proportion of inactive prolactin in patient serum in the future.
Bromocriptine is a dopamine receptor agonist that could inhibit prolactin secretion and reduce serum prolactin level. It could also induce humoral and cell-mediated immunosuppression, directly modulate T and B cell function through the dopamine receptor, and reduce IFN production by macrophages [bib_ref] Prolactin as a modulator of B cell function: implications for SLE, Peeva [/bib_ref] [bib_ref] Bromocriptine in rheumatic and autoimmune diseases, Mcmurray [/bib_ref]. Recently, a study showed that bromocriptine could effectively prevent maternal and fetal complications, including premature rupture of membrane, low birth weight, preterm deliveries and decrease disease activity measured by SLE Pregnancy Disease Activity Index (SLEPDAI) [bib_ref] Bromocriptine during pregnancy in systemic lupus erythematosus: a pilot clinical trial, Jara [/bib_ref].
For postpartum lupus patients, our study showed that oral bromocriptine therapy after delivery could not only reduce serum prolactin level quickly but also be helpful to decrease serum estradiol concentration to pregestation level rapidly. In bromocriptine treatment group, the serum prolactin levels were much lower than the control group at the second week after delivery. The estradiol levels in treatment group were lower than the control group as well. The results of survival analysis in this study confirmed that two weeks of oral bromocriptine therapy could reduce the SLE activity in treatment group even in one year after delivery.
Prolactin could modulate the immune function in SLE obviously but has little influence on the immune system of normal people [bib_ref] Prolactin enhances the in vitro production of IgG in peripheral blood mononuclear..., Jacobi [/bib_ref]. The adverse impacts of estradiol on SLE have been well known by rheumatologists. One study [bib_ref] Bromocriptine restores tolerance in estrogen-treated mice, Peeva [/bib_ref] indicated that hyperprolactinemia concurrence with hyperestrogenemia may have synergistic effects on SLE and estrogen could increase the production of anti-DNA autoantibody by B cell when exposed to prolactin. So, the results of our study indicated that oral bromocriptine therapy may be beneficial to pregnant SLE patients by eliminate the adverse impacts of estradiol.
An increasing body of evidences indicated that bromocriptine is a safe drug, even for the patients during pregnancy. In the current study, no severe adverse events were found in patients taking bromocriptine, and vertigo and nausea were the most common complains. Another study [bib_ref] Bromocriptine during pregnancy in systemic lupus erythematosus: a pilot clinical trial, Jara [/bib_ref] about using bromocriptine during pregnancy in SLE patients also showed that only 2 patients had mild headache and no severe adverse event or birth defect was observed. In more than 6000 pregnancies in women taking bromocriptine for hyperprolactinemia, the incidence of congenital malformations or abortions was not increased [bib_ref] Pituitary disorders during pregnancy, Molitch [/bib_ref]. The endocrine society clinical practice guideline for diagnosis and treatment of hyperprolactinemia [bib_ref] Diagnosis and treatment of hyperprolactinemia: an endocrine society clinical practice guideline, Melmed [/bib_ref] also recommends bromocriptine therapy in patients who experience symptomatic growth of a prolactinoma during pregnancy.
In conclusion, the current study preliminarily indicates that oral-taking bromocriptine for two weeks in postpartum SLE patients may eliminate the effects of hyperprolactinemia and hyperestrogenemia on disease activity. In addition to the minimal side effects and low cost of treatment, prescribing lupus patients with bromocriptine has potential benefits in preventing postpartum disease relapses in one year after delivery. However, multicenter clinical trials with large sample size should be performed to evaluate the benefit of clinical application of bromocriptine.
# Disclosure
Qiu Qian and Liang Liuqin are co-first authors.
[fig] Figure 2: Kaplan-Meier survival curves for relapse-free Survival between the treatment group and the control group. [/fig]
[table] Table 1: Baseline demographic features, clinical findings, and SLEDAI scores in the pregnant SLE patients. [/table]
[table] Table 2: Comparison of the serum prolactin/estradiol levels between two groups at the second week and the second month after delivery. [/table]
[table] Table 3: Change of SLEDAI within 1 years after delivery (mean ± SD). [/table]
[table] Table 4: Cumulative doses of corticosteroid and immunosuppressive agents for SLE in 12 months after delivery. [/table]
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Amyloid β Peptide Induces Apoptosis Through P2X7 Cell Death Receptor in Retinal Cells: Modulation by Marine Omega-3 Fatty Acid DHA and EPA
Retinal Müller glial cells have already been implicated in age-related macular degeneration (AMD). AMD is characterized by accumulation of toxic amyloid-β peptide (Aβ); the question we raise is as follows: is P2X7 receptor, known to play an important role in several degenerative diseases, involved in Aβ toxicity on Müller cells? Retinal Müller glial cells were incubated with Aβ for 48 h. Cell viability was assessed using the alamarBlue assay and cytotoxicity using the lactate dehydrogenase (LDH) release assay. P2X7 receptor expression was highlighted by immunolabeling observed on confocal microscopy and its activation was evaluated by YO-PRO-1 assay. Hoechst 33342 was used to evaluate chromatin condensation, and caspases 8 and 3 activation was assessed using AMC assays. Lipid formulation rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) used in Age-Related Eye Disease Study 2 was incubated on cells for 15 min prior to Aβ incubation. For the first time, we showed that Aβ induced caspaseindependent apoptosis through P2X7 receptor activation on our retinal model. DHA and EPA are polyunsaturated fatty acids recommended in food supplement to prevent AMD. We therefore modulated Aβ cytotoxicity using a lipid formulation rich in DHA and EPA to have a better understanding of the results observed in clinical studies. We showed that fish oil rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, represents an efficient modulator of Aβ toxicity and that P2X7 could be an interesting therapeutic target to prevent AMD.
# Introduction
Age-related macular degeneration (AMD) is a progressive degeneration of the macula, the portion of the retina used for central vision. It is the leading cause of the irreversible loss of vision in those aged over 50 years in the Western industrialized world [bib_ref] The epidemiology of age-related macular degeneration, Klein [/bib_ref]. The United Nations estimates the number of people with AMD at 20-25 million worldwide [bib_ref] Age related macular degeneration, Chopdar [/bib_ref]. As AMD progresses, it can develop into two distinct forms of late or advanced AMD: Bdry^AMD (geographic atrophy, 90 %) and Bwet^AMD (neovascular AMD, 10 %). Early stage of AMD is characterized by the formation of drusen that are deposits of extracellular material located underneath the retinal pigmented epithelium (RPE). Drusen provokes an inflammatory response and is associated with RPE atrophy. Photoreceptors overlying drusen die by apoptosis, whereas retinal Müller glial cells are activated. Under physiological conditions, Müller cells are responsible for maintaining its homeostasis, support neuronal activity, and participate in the induction, maintenance, and proper functioning of the blood-retinal barrier [bib_ref] Muller cells in the healthy and diseased retina, Bringmann [/bib_ref] [bib_ref] Glia cells of the monkey retina.2. Muller cells, Distler [/bib_ref] [bib_ref] The role of Muller cells in the formation of the blood-retinal barrier, Tout [/bib_ref]. Alterations of Müller cells under pathological conditions can contribute to retinal degeneration [bib_ref] Muller cells as players in retinal degeneration and edema. Graefes Archive for, Reichenbach [/bib_ref] [bib_ref] Treatment with 670 nm light up regulates cytochrome C oxidase expression and..., Begum [/bib_ref] [bib_ref] Extreme retinal remodeling triggered by light damage: implications for age related macular..., Marc [/bib_ref]. Especially, Müller cell dysfunction leads to photoreceptor apoptosis and blood-retinal barrier breakdown [bib_ref] Conditional Muller cell ablation causes independent neuronal and vascular pathologies in a..., Shen [/bib_ref] [bib_ref] Small interfering RNA-mediated suppression of Ccl2 in Muller cells attenuates microglial recruitment..., Rutar [/bib_ref]. There is no curative treatment against atrophic AMD, which affects 90 % of AMD patients. Indeed, consumption of micronutrients, such as zinc, β-carotene, or vitamins, has been shown to prevent AMD progression. A study reviewing the role of dietary omega-3 long chain polyunsaturated fatty acid (PUFA) in the prevention of AMD reported a 38 % reduced rate of progression to late AMD [bib_ref] Dietary omega-3 fatty acid and fish intake in the primary prevention of..., Chong [/bib_ref]. docosahexaenoic acid (DHA, C22:6 ω-3) and its precursor eicosapentaenoic acid (EPA, C20:5 ω-3) are the major structural long chain PUFAs of the membrane of photoreceptors [bib_ref] Lipid composition of photoreceptor membranes from goldfish retinas, Fliesler [/bib_ref]. DHA is essential for the biogenesis and the function of photoreceptors [bib_ref] Membrane docosahexaenoate is supplied to the developing brain and retina by the..., Scott [/bib_ref]. Moreover, EPA and DHA have antioxidant, anti-inflammatory, antiapoptotic, and antiangiogenic roles in the retina [bib_ref] Docosahexaenoic acid (DHA) has neuroprotective effects against oxidative stress in retinal ganglion..., Shimazawa [/bib_ref] [bib_ref] Docosahexaenoic acid prevents apoptosis of retina photoreceptors by activating the ERK/MAPK pathway, German [/bib_ref]. PUFA content in the retina decreases with aging and it potentially induces a dysfunction of retinal cells. Participants who reported the highest levels of EPA consumption had a reduced likelihood of AMD progression [bib_ref] The relationship of dietary omega-3 long-chain polyunsaturated fatty acid intake with incident..., Sangiovanni [/bib_ref]. Amyloid-β (Aβ) peptide is a key constituent of drusen [bib_ref] Characterization of beta amyloid assemblies in drusen: the deposits associated with aging..., Anderson [/bib_ref] [bib_ref] Amyloid-beta is found in drusen from some age-related macular degeneration retinas, but..., Dentchev [/bib_ref] [bib_ref] Soluble and mature amyloid fibrils in drusen deposits, Isas [/bib_ref]. It has been suggested that drusen could correspond to the transposition of senile plaques in Alzheimer's disease (AD). In the retina of mice models of AD, an age-dependent Aβ accumulation has been detected, possibly resulting in neurodegeneration [bib_ref] Amyloid-beta deposits lead to retinal degeneration in a mouse model of Alzheimer..., Ning [/bib_ref]. It has been found that oligomerized Aβ is more toxic than is nonoligomerized Aβ in retinal cell cultures [bib_ref] Evaluation and modulation of toxic degenerative pathways induced by beta-amyloid on retinal..., Wakx [/bib_ref]. Retinal toxicity seems to be associated with oxidative stress and pro-inflammatory response, but underlying mechanisms remain not clearly defined [bib_ref] Amyloid-beta(1-42) alters structure and function of retinal pigmented epithelial cells, Bruban [/bib_ref] [bib_ref] Microarray analysis identifies changes in inflammatory gene expression in response to amyloid-beta..., Kurji [/bib_ref]. The purinergic receptor P2X7 is an ATPgated cationic channel expressed by virtually all types of cells [bib_ref] The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor..., Surprenant [/bib_ref] [bib_ref] Lysosomal alkalinization, lipid oxidation, and reduced phagosome clearance triggered by activation of..., Guha [/bib_ref]. P2X7 is involved in oxidative stress, cell death, and inflammatory processes, all of which have been linked to AMD [bib_ref] Immune responses in age-related macular degeneration and a possible long-term therapeutic strategy..., Nussenblatt [/bib_ref] [bib_ref] Mechanism of inflammation in age-related macular degeneration: an up-to-date on genetic landmarks, Parmeggiani [/bib_ref]. A recent genetic study has demonstrated that a haplotype containing a rare genetic variant of P2X7 receptor is associated with increased susceptibility to AMD [bib_ref] A rare functional haplotype of the P2RX4 and P2RX7 genes leads to..., Gu [/bib_ref].Moreover, Notomi et al. recently proposed Brilliant Blue G (BBG), a selective P2X7 receptor antagonist, as a neuroprotective agent in retinal diseases [bib_ref] Critical involvement of extracellular ATP acting on P2RX7 purinergic receptors in photoreceptor..., Notomi [/bib_ref]. The first aim of our study was to describe the P2X7-dependent cell death pathway induced by Aβ on Müller cells. Our second aim was to modulate Aβ cytotoxicity using a lipid formulation rich in DHA and EPA, chosen for its ability to modulate toxic ocular stresses [bib_ref] New approach to modulate retinal cellular toxic effects of high glucose using..., Dutot [/bib_ref] [bib_ref] Per os administered refined olive oil and marine PUFA-rich oils reach the..., Dutot [/bib_ref].
# Methods
## Reagents
Reagents for cell culture were provided by Eurobio (Les Ulis, France), flasks and microplates from Corning (Schiphol-Rijk, The Netherlands) and chamber slides from Nunc Thermo Fisher Scientific (Rochester, NY, USA). Lipid formulation rich in DHA and EPA was provided by Yslab (Quimper, France). BBG, a specific P2X7 receptor inhibitor [bib_ref] Brilliant blue G selectively blocks ATP-gated rat P2X(7) receptors, Jiang [/bib_ref] , was purchased from Bio-Rad (Richmond, CA, USA). Hoechst 33342, YO-PRO-1, TO-PRO-3, and secondary antibodies were purchased from Invitrogen (PoortGebouw, The Netherlands). Aβ (Bachem, Weil am Rhein, Germany) was oligomerized as previously described [bib_ref] Amyloid-beta(1-42) alters structure and function of retinal pigmented epithelial cells, Bruban [/bib_ref]. Primary antibodies and IgG isotype control were provided from Santa Cruz Biotechnology (Heidelberg, Germany). All other chemicals, dyes, and kits were provided from Sigma-Aldrich (Saint Quentin Fallavier, France).
## Cell culture
The experiments were performed using the immortalized human Müller cell line MIO-M1 [bib_ref] In vitro characterization of a spontaneously immortalized human Muller cell line (MIO-M1), Limb [/bib_ref]. The MIO-M1 cell line was tested at IDEXX BioResearch (Columbia, MO, USA). The cell line was confirmed to be human and no evidence of cross-species contamination was found. The STR testing results reported for the cell line are as follows: amelogenin (X, Y), CSF1PO [bib_ref] Membrane docosahexaenoate is supplied to the developing brain and retina by the..., Scott [/bib_ref] [bib_ref] Docosahexaenoic acid (DHA) has neuroprotective effects against oxidative stress in retinal ganglion..., Shimazawa [/bib_ref] , D13S317 (13), D16S539 [bib_ref] Dietary omega-3 fatty acid and fish intake in the primary prevention of..., Chong [/bib_ref] [bib_ref] Lipid composition of photoreceptor membranes from goldfish retinas, Fliesler [/bib_ref] , D5S818 [bib_ref] Lipid composition of photoreceptor membranes from goldfish retinas, Fliesler [/bib_ref] [bib_ref] Membrane docosahexaenoate is supplied to the developing brain and retina by the..., Scott [/bib_ref] , D7S820 [bib_ref] Conditional Muller cell ablation causes independent neuronal and vascular pathologies in a..., Shen [/bib_ref] , TH01 , TPOX [bib_ref] Conditional Muller cell ablation causes independent neuronal and vascular pathologies in a..., Shen [/bib_ref] , and vWA [bib_ref] Docosahexaenoic acid prevents apoptosis of retina photoreceptors by activating the ERK/MAPK pathway, German [/bib_ref] [bib_ref] Soluble and mature amyloid fibrils in drusen deposits, Isas [/bib_ref].
MIO-M1 were cultured using Dulbecco's Modified Eagle's medium, supplemented with 10 % fetal bovine serum (FBS), 2 mM L glutamine, 50 IU/mL penicillin, and 50 IU/mL streptomycin, at 37°C in a humidified atmosphere of 5 % CO 2 , as previously described [bib_ref] Human Muller stem cell (MIO-M1) transplantation in a rat model of glaucoma:..., Bull [/bib_ref]. Confluent cultures in flasks were removed by trypsin incubation, and then the cells were seeded into 96-well (20,000 cells per well) or 24-well (38,000 cells per well) culture microplates and kept at 37°C for 24 h.
## Incubation protocols
Whenever the cells reached confluency, culture medium was removed and the cells were incubated with oligomerized Aβ 1-42 at 25 μM in 2.5 % FBS medium for 48 h at 37°C.
Excessive Aβ was used to model AMD pathology. Twenty-five micromolars was the highest Aβ concentration that did not induce any loss of cell viability in our model (data not shown).
Neat fish oil containing EPA and DHA (see composition in [fig_ref] Table 1: EPA and DHA (%) and [/fig_ref] was incubated for 15 min (100 μL/well) followed by 24 h in culture medium prior to Aβ incubation [bib_ref] New approach to modulate retinal cellular toxic effects of high glucose using..., Dutot [/bib_ref] [bib_ref] Per os administered refined olive oil and marine PUFA-rich oils reach the..., Dutot [/bib_ref] [bib_ref] Benefits and side effects of different vegetable oil vectors on apoptosis, oxidative..., Said [/bib_ref]. BBG (25 μM, according to Kawahara [bib_ref] Intracellular events in retinal glial cells exposed to ICG and BBG, Kawahara [/bib_ref] was incubated with cells for 15 min prior to Aβ incubation. BBG is a potent inhibitor of P2X7 receptor since concentrations as low as 25 μM are enough to inhibit receptor activation.
## Cell viability (necrosis assessment): alamarblue assay
The alamarBlue ® assay uses resazurin, a blue fluorogen probe, which is reduced to a red fluorescent compound (resorufin) by intracellular redox enzymes [bib_ref] Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of..., O'brien [/bib_ref]. A solution of resazurin at 0.1 mg/mL was prepared in phosphate-buffered saline (PBS) then diluted to the eleventh well in culture medium supplemented with 2.5 % FBS. The cells were exposed to resazurin solution for 6 h at 37°C, then the fluorescence signal was read (λexc=535 nm, λem=600 nm, Safire; Tecan ® , Zurich, Switzerland).
## Cytotoxicity: ldh release assay
The lactate dehydrogenase (LDH) assay measures membrane integrity as a function of the amount of cytoplasmic LDH released into the medium [bib_ref] Gentamicin as a bactericidal antibiotic in tissue-culture, Fischer [/bib_ref]. Briefly, cell supernatants were incubated with the LDH mixture containing NAD as LDH substrate and a tetrazolium dye (the mixture was prepared according to the manufacturer's instructions for Sigma kit TOX7) for 30 min. Absorbance was detected at 490 nm (Safire; Tecan ® , Zurich, Switzerland).
## P2x7 expression by immunofluorescence using confocal microscopy
After seeding in chamber slides for 24 h, the cells were fixed with 2 % paraformaldehyde and 2 mM calcium for 15 min at room temperature. The cells were then permeabilized with 0.2 % Triton X-100 for 5 min. First, the cells were incubated with primary antibody (rabbit anti-P2X7 at 5 μg/mL) or rabbit IgG (isotype control) in PBS with 1 % bovine serum albumin (BSA) for 1 h, and second, the cells were incubated with secondary antibody (Alexa Fluor 488 anti-rabbit IgG) in PBS with 1 % BSA for 1 h away from light. Third, nuclei were stained with TO-PRO-3 at 2 μg/μL for 10 min. Slides were then observed under a Leica TCS SP2 confocal microscope (Leica Microsystems) equipped with a 40× oil immersion objective. Staining specificity was carefully checked by omitting the primary antibody. Confocal imaging was performed at IFR71-IMTCE Cellular and Molecular Imaging platform (Faculté de Pharmacie, Université Paris Descartes, Paris, France).
## P2x7 activation: yo-pro-1 test
YO-PRO-1, a fluorogenic probe, enters cells through P2X7 receptor activation-induced pores and emits fluorescence when it binds DNA [bib_ref] YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with..., Idziorek [/bib_ref]. A 2-μM YO-PRO-1 solution in PBS was distributed in wells, and the microplate was placed at room temperature away from light for 10 min [bib_ref] Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor..., Dutot [/bib_ref] [bib_ref] In vitro modulation of preservative toxicity: high molecular weight hyaluronan decreases apoptosis..., Pauloin [/bib_ref] [bib_ref] Severe ocular infections with contact lens: role of multipurpose solutions, Dutot [/bib_ref] [bib_ref] Cytotoxicity of contact lens multipurpose solutions: role of oxidative stress, mitochondrial activity..., Dutot [/bib_ref] [bib_ref] Effects of toxic cellular stresses and divalent cations on the human P2X7..., Dutot [/bib_ref]. The fluorescence signal was then scanned (λexc=491 nm, λem=509 nm, Safire; Tecan ® , Zurich, Switzerland).
## Chromatin condensation: hoechst 33342 assay
Hoechst 33342 dye is used to detect chromatin condensation in cells simultaneously with propidium iodide [bib_ref] Severe ocular infections with contact lens: role of multipurpose solutions, Dutot [/bib_ref]. Hoechst 33342 enters living and apoptotic cells whereas propidium iodide enters necrotic cells faster than Hoechst 33342. A solution of Hoechst 33342 at 10 mg/ mL and propidium iodide at 0.5 mg/mL was prepared in PBS. Cells were exposed for 30 min at 37°C, then the fluorescence was read (λexc=365 nm, λem=450 nm, Safire; Tecan ® , Zurich, Switzerland).
## Caspases 3 and 8 activation: amc assays
The caspases 3 and 8 fluorometric assays were realized following the procedure for fluorometric assay of caspases 3 and 8 activity in adherent cell lines of the CASP3F and CASP8F Sigma kits. Briefly, the cells were treated with lysis buffer and incubated on ice for 20 min. Then, DEVD-AMC for caspase 3 detection or IETD-AMC for caspase 8 detection was added in each well. The samples were incubated in the dark at room temperature for 30 min. Afterward, the fluorescence signal was read (λexc=360 nm, λem=460 nm, Safire; Tecan ® , Zurich, Switzerland).
## Results exploitation and statistical analysis
All data for microtitration were obtained in fluorescence or absorbance units and expressed as a percentage of the negative control (culture medium). Each point was tested in three different wells, and experiments were reproduced in triplicate. Data are expressed as means ± standard deviation. The mean values for each test were analyzed by one-way ANOVA followed by the Dunnett test (SigmaStat 2.0; Chicago, Illinois, USA), and the level of significance was fixed at 0.05.
# Results
## Cell viability in aβ-incubated mio-m1 cells
First, we investigated cell viability in Aβ-incubated MIO-M1 cells. Aβ at 25 μM did not induce any significant decrease in cell viability according to the alamarBlue assay [fig_ref] Figure 1: Cell viability and cytotoxicity assessment after incubation of 25 μM Aβ for... [/fig_ref]. The LDH activity assay showed no significant change in the plasma membrane integrity which was detected in Aβ-incubated MIO-M1 in comparison to negative control [fig_ref] Figure 1: Cell viability and cytotoxicity assessment after incubation of 25 μM Aβ for... [/fig_ref]. shows a strong labeling of MIO-M1 cell membranes using a specific anti-P2X7 antibody compared to the isotype control antibody .
## P2x7 activation, chromatin condensation and caspase activity
Then, we analyzed P2X7 pore formation by the YO-PRO-1 assay. Fluorescence signal significantly increased (×3.16, p<0.001) when MIO-M1 were incubated with Aβ compared to negative control . When the Aβ-incubated MIO-M1 were preincubated with the P2X7 inhibitor, BBG, the fluorescence signal significantly decreased (×0.48, p<0.001) compared to Aβ-incubated MIO-M1 without preincubation, confirming activation of P2X7 by Aβ. We also observed that a specific P2X7 receptor activator, BzATP, increased P2X7 pore formation, which confirms the specificity of the YO-PRO-1 assay to evaluate P2X7 activation. We also studied chromatin condensation, an irreversible early phase of apoptosis assessed by the Hoechst 33342 assay, because even no loss of cell viability at 48 h does not mean no apoptosis at 48 h. In [fig_ref] Figure 3: Chromatin condensation after incubation of 25 μM Aβ for 48 h [/fig_ref] , Hoechst 33342 fluorescence was significantly increased (×2.11, p<0.001) in Aβ-treated MIO-M1 compared to negative control. Preincubation of Aβ-treated MIO-M1 with BBG totally inhibited chromatin condensation compared to Aβ-treated MIO-M1 without preincubation increased (×0.53, p<0.001), indicating the central role of P2X7 in the mediation of Aβ-induced condensation of Müller cell chromatin. Finally, we evaluated caspase activation. We focused our attention on caspase 8 and caspase 3. No significant difference was observed between Aβ-treated MIO-M1 compared to negative control [fig_ref] Figure 4: Caspase activation after incubation of 25 μM Aβ for 48 h [/fig_ref].
Altogether, these data indicated that Aβ first alters the chromatin state of Müller cells without inducing cell death at 48 h.
Protective Effects of EPA/DHA Associated in Fish Oil and BBG on Aβ-Cytotoxicity EPA and DHA have antioxidant and antiapoptotic roles in the retina, but their protective effects on Müller cells against Aβ remains undetermined. P2X7 pore formation was significantly decreased (×0.77, p<0.001) when Aβ-treated MIO-M1 were preincubated with EPA-DHA fish oil compared to Aβ alone, but the signal remains significantly higher than in the negative control [fig_ref] Figure 5: AβEPA-DHA oil for 15 min and incubation of 25 μM Aβ for... [/fig_ref] , suggesting that P2X7 receptor is a target for protective effects of EPA and DHA in Aβ-treated Müller cells. Preincubation of MIO-M1 with both EPA-DHA fish oil and BBG totally inhibited P2X7 pore formation (×0.33, p<0.001), meaning that BBG and EPA-DHA fish oil act synergically.
Analysis of chromatin condensation, an irreversible early phase of apoptosis assessed by the Hoechst 33342 assay, showed a significant decrease (×0.78, p<0.001) in Hoechst 33342 [fig_ref] Figure 5: AβEPA-DHA oil for 15 min and incubation of 25 μM Aβ for... [/fig_ref]. Preincubation of Aβ-treated MIO-M1 with both EPA-DHA fish oil and BBG totally inhibited chromatin condensation compared to Aβ-treated MIO-M1 with or without preincubation with EPA-DHA fish oil (×0.42, p<0.001), confirming the P2X7 receptor-mediated deleterious effects of Aβ and the potential protective role of EPA-DHA against these effects in Müller cells.
# Discussion
Müller cells have been shown to be implicated in AMD [bib_ref] Ccl2/ Cx3cr1 knockout mice have inner retinal dysfunction but are not an..., Vessey [/bib_ref] [bib_ref] Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases, Cuenca [/bib_ref] , and selective ablation of these cells led to photoreceptor apoptosis, blood-retinal barrier breakdown, and retinal neovascularization [bib_ref] Effect of glucocorticoids on neuronal and vascular pathology in a transgenic model..., Shen [/bib_ref]. We studied the Aβ effects on MIO-M1 cells to study the impact of this peptide involved in AMD pathogenesis on Müller cells. The lack of necrosis in the cells was determined by no increase in LDH levels and low levels of propidium iodide (data not shown). Apoptosis was revealed by higher levels of YO-PRO-1 and Hoechst 33342 fluorescence signals in Aβ-incubated cells than in control cells. Therefore, we report that oligomerized Aβ induces apoptosis rather than necrosis on human retinal Müller cells (MIO-M1). P2X7 receptor is involved in oxidative stress, cell death, and inflammatory processes, all of which have been linked to AMD. Aβ-induced apoptosis appears to be P2X7 cell death receptor-dependent and caspase-independent, but further investigations are needed to confirm that. We showed for the first time that P2X7 receptor activation plays a pivotal role in Aβ-induced apoptosis in Müller cells. Indeed, P2X7 receptor inhibition using a specific antagonist (BBG) drastically decreased Aβ-induced apoptosis. Our results are in accordance with previous results that showed that P2X7 receptor blockade prevents photoreceptor cell apoptosis and confers neuroprotection in the brain of a rat model of Alzheimer's disease [bib_ref] Dynamic increase in extracellular ATP accelerates photoreceptor cell apoptosis via ligation of..., Notomi [/bib_ref] [bib_ref] Block of purinergic P2X(7) receptor is neuroprotective in an animal model of..., Ryu [/bib_ref]. However, the mechanism by which BBG acts remains to be deeper studied. In our model, P2X7 activation was not associated with extrinsic caspase 8 activation, as previously described [bib_ref] P2X7 receptor-mediated apoptosis of human cervical epithelial cells, Wang [/bib_ref] [bib_ref] Activation of P2X(7)-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer..., Fu [/bib_ref]. Caspase 3, which is involved both in the extrinsic and the intrinsic pathways, was not activated in our model, meaning that Aβ peptide induces P2X7 activation leading to caspase-independent chromatin condensation in Müller cells. Apoptosisinducing factor (AIF) translocates from mitochondria to nuclei in a caspaseindependent fashion, leading to DNA fragmentation and chromatin condensation in cells undergoing apoptosis [bib_ref] Molecular characterization of mitochondrial apoptosis-inducing factor, Susin [/bib_ref]. It was previously described that Aβ-induced cell death was associated with AIF translocation [bib_ref] Carnosic acid attenuates apoptosis induced by amyloid-beta 1-42 or 1-43 in SH-SY5Y..., Meng [/bib_ref] [bib_ref] MGLuR5 activation reduces beta-amyloid-induced cell death in primary neuronal cultures and attenuates..., Movsesyan [/bib_ref].
The Age-Related Eye Disease Study 2 (AREDS2) was a multi-center, randomized trial designed to assess the effects of oral supplementation of DHA and EPA on the progression to advanced AMD. The results of this trial showed that addition of DHA and EPA as ethyl esters did not further reduce the risk of progression to advanced AMD. In our model, EPA and DHA as triglycerides in fish oil had preventive effects towards P2X7 cell death receptor-dependent apoptosis induced by Aβ. We previously demonstrated that individual synthetic DHA or EPA are not as efficient as DHA and EPA associated in fish oils in the prevention of some ocular stresses [bib_ref] New approach to modulate retinal cellular toxic effects of high glucose using..., Dutot [/bib_ref]. Moreover, appropriate proportions of DHA/EPA seem to be needed to observe the most potent effect. Fatty acids contained in the lipid formulation we selected incorporate into retinal cell membranes [bib_ref] New approach to modulate retinal cellular toxic effects of high glucose using..., Dutot [/bib_ref] , which can increase membrane fluidity and disrupt lipid rafts [bib_ref] Effects of fatty acid unsaturation numbers on membrane fluidity and alpha-secretase-dependent amyloid..., Yang [/bib_ref] [bib_ref] Membrane raft organization is more sensitive to disruption by (n-3) PUFA than..., Rockett [/bib_ref] [bib_ref] Lipid raft disruption by docosahexaenoic acid induces apoptosis in transformed human mammary..., Ravacci [/bib_ref] [bib_ref] Inhibition of cytokine signaling in human retinal endothelial cells through modification of..., Chen [/bib_ref]. The activity of the numerous receptors expressed in lipid rafts, such as P2X7 receptor [bib_ref] Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial..., Barth [/bib_ref] , may be modified. Effectively, we observed that Aβ-induced P2X7 receptor activation was reduced by our lipid formulation. This P2X7 cell death receptor blockade could occur through lipid raft disruption, as we previously showed that the EPA-DHA fish oil we used is able to modulate lipid rafts organization [bib_ref] New approach to modulate retinal cellular toxic effects of high glucose using..., Dutot [/bib_ref]. DHA (22:6 ω-3) is the precursor of EPA (20:5 ω-3), which is the omega-3 homologue of arachidonic acid (20:4 ω-6). Arachidonic acid is at the origin of pro-inflammatory mediators (prostaglandin E2); on the contrary, EPA is at the origin of anti-inflammatory mediators (prostaglandin E3) after metabolism by COX enzymes. As we observed in a previous study, an increase in EPA can lead to a decrease in arachidonic acid in cell membranes and then to a decrease in the pro-inflammatory response [bib_ref] Per os administered refined olive oil and marine PUFA-rich oils reach the..., Dutot [/bib_ref]. It is thus suggested that the high content of EPA in our fish oil formulation could help in diminishing the inflammation associated to AMD. Fish EPA-DHA and BBG exerted synergic effects in the prevention of Aβ damages in our model. As the potential application of BBG as a neuroprotective therapy has already been suggested [bib_ref] Dynamic increase in extracellular ATP accelerates photoreceptor cell apoptosis via ligation of..., Notomi [/bib_ref] , the mixture of fish EPA-DHA and BBG opens further new strategic therapeutics.
# Conclusion
For the first time, our study showed that Aβ seems to induce caspase-independent apoptosis through P2X7 receptor activation in human retinal cells. We showed that marine lipid formulation containing EPA and DHA as triglycerides, in combination with BBG, a specific P2X7 receptor inhibitor, fully prevented Aβ cytotoxic effects in our model. Therefore, marine oils rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, could represent a promising efficient modulator of Aβ toxicity.
[fig] Figure 1: Cell viability and cytotoxicity assessment after incubation of 25 μM Aβ for 48 h. a Cell viability was assessed using global redox potential. b Necrosis was assessed using extracellular LDH dosage after Aβ incubation for 48 h in MIO-M1 retinal Müller cellsFig. 2 Expression and activation of P2X7 receptor in MIO-M1 cells.a Isotype control (left) and P2X7 receptor labeling (right). Cells were observed using confocal microscopy (200×). Pictures show mergence between nuclei and P2X7 staining. b P2X7 receptor activation using the YO-PRO-1 assay was evaluated after incubation of 25 μM Aβ for 48 h. BBG at 25 μM was used as a P2X7 receptor potent inhibitor and BzATP at 300 μM was used as a positive control. ***p<0.001 compared to negative control; $$$p<0.001 compared to Aβ [/fig]
[fig] Figure 3: Chromatin condensation after incubation of 25 μM Aβ for 48 h. Chromatin condensation using Hoechst 33342 assay was evaluated after Aβ incubation for 48 h in MIO-M1 retinal Müller cells. BBG at 25 μM was used as a P2X7 receptor potent inhibitor. ***p<0.001 compared to negative control; $$$p<0.001 compared to Aβ [/fig]
[fig] Figure 4: Caspase activation after incubation of 25 μM Aβ for 48 h. Caspase 8 (a) and caspase 3 (b) activation were evaluated after Aβ incubation for 48 h in MIO-M1 retinal Müller cells signal when the Aβ-treated MIO-M1 were preincubated with EPA-DHA fish oil compared to Aβ-treated MIO-M1 without preincubation [/fig]
[fig] Figure 5: AβEPA-DHA oil for 15 min and incubation of 25 μM Aβ for 48 h in MIO-M1 retinal Müller cells. BBG at 25 μM was used as a P2X7 receptor potent inhibitor. ***p<0.001 compared to negative control; $$$p<0.001 compared to Aβ [/fig]
[table] Table 1: EPA and DHA (%) and [/table]
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Assessing knowledge of the patient bill of rights in central Saudi Arabia: a survey of primary health care providers and recipients
[bib_ref] Midwives and nurses awareness of patients' rights, Ci [/bib_ref] [bib_ref] Awareness and practice of patient's rights law in lithuania, Ducinskiene [/bib_ref] [bib_ref] An iranian perspective on patients' rights, Joolaee [/bib_ref] [bib_ref] World health organization, regional office for europe. promotion of the rights of..., Joolaee [/bib_ref]
# Patients and methods
This was a cross-sectional study conducted to explore the implementation of the PBR that was introduced re-cently in the Saudi health care system. A stratified random sampling was used to represent patients and PHC staff from public and private health care sectors. In each sector, 500 patients (18 years or older) and 500 PHC staff (physicians and nurses, who had been in the profession for the past 2 years) were selected using systematic random sampling and requested to complete the survey questionnaires. The study took place in Riyadh, the capital of Saudi Arabia, during July 2010. The survey questionnaire consisted of four parts. The first focused on personal characteristics of respondents and included questions on gender, age, educational level, and occupation. In the second part, respondents were asked whether they had knowledge about the existence of the PBR, its contents, and whether they had observed any improvement in doctor-patient relationships in the past 2 years. Responses in this section were in "yes" and "no" form. The third part asked the respondents (patients and PHC staff ) about the extent to which each aspect defined in the PBR was implemented. In this section, a 5-point Likert scale ranging from "1=strongly disagree" to "5=strongly agree" was used to assess respondents' perception about the implementation. A reliability check showed that the scale has high internal consistency (Cronbach a=0.888). The fourth part requested PHC staff to identify possible obstacles to the implementation of PBR in Saudi Arabia. In this section, PHC staff were given a list of possible obstacles, based on a review of the relevant published studies, and were instructed to mark as many obstacles as applied.
To increase the content validity of the questionnaire, a review of the relevant published studies was carried out. Two academic staff and two physicians reviewed the draft questionnaire, and it was pilot-tested. On the basis of the suggestions of the reviewers and the outcome of the pilot survey, the final questionnaire was reformulated. The respondents were assured of confidentiality and provided with an explanation regarding the purpose of the study and the importance of their contribution. The subjects gave consent to participate in the study. All questionnaires were distributed by welltrained postgraduate students. While patients completed the questionnaire during their waiting times in the PHC clinics, PHC staff were requested to complete them at times of their convenience.
The data was analyzed and presented in a descriptive fashion. In making a comparison between patients and PHC staff, the chi-square test was used to test the difference between categorical variables, and the mean values of continuous variables were compared using the t test. The level P<.05 was considered as the cutoff value for significance. The data for this study was en- ) PHC staff reported that they had observed changes in the doctor-patient relationship in the past 2 years. A further analysis indicated that, among respondents who had heard about the existence of the bill, about three-quarters (73.6%) of patients and about half (48.8%) of PHC staff had "little or very little" knowledge about the bill contents (P<.001). Similarly, a significantly higher percentage of patients than PHC staff (39.6% and 12.0%, respectively) indicated that the bill would bring "little or very little" improvement in the provision of health care (P<.001).
In this study, respondents were requested to indicate their perception about the extent to which each of the aspects (articles) listed in the bill was implemented [fig_ref] Table 2: the extent to which aspects of the patient bill of rights were... [/fig_ref]. In general, patients perceived lower mean scores for the implementation of all aspects listed in the PBR than PHC staff. In particular, patients reported significantly lower mean scores in the implementation of several aspects such as the provision of care that meets their needs, the encouragement to play roles in their health decisions, the provision of understandable information, obtaining information about treatment options and complications, and referrals to higher levels of care. Moreover, patients attending the private sector centers reported a significantly lower mean score of perception about the implementation of the aspect "obtaining information about costs of care" than PHC staff.
PHC staff reported a number of obstacles that may deter the implementation of the PBR in Saudi Arabia . The lack of knowledge about the bill among patients and PHC staff were the most cited two obstacles in implementing the PBR and were reported by about half of respondents. Obstacles such as PHC staff dissatisfaction, shortage of staff, and lack of necessary facilities were reported by more than one third of PHC staff. About a quarter of PHC staff gave "heavy workload" and "inadequate resources" as other obstacles for the implementation of the PBR. Slightly more than 15% of respondents cited that "poor coordination with other levels of care" and "insufficient managerial support" as obstacles to the implementation of the bill. PHC staff specified "other" obstacles that impede the implementation of PBR, including "time constraint," "improper staff/patient ratio," and "unsafe environment."
# Discussion
Despite the introduction of the PBR in the Saudi health care system more than 3 years ago, patients and PHC staff were not yet fully aware of the legislation. This may indicate that the process of informing health care providers and recipients about the bill has not been successfully implemented. In this study, a considerable percentage of patients and PHC staff were not aware of the PBR, its contents, and the improvements likely to occur in the delivery of health care as a result of implementing the bill. Comparing the results of the present study with those in other countries is difficult because of differences in legislation among health care systems, research methodologies used, and differences in values and norms among societies.
Interestingly, the study found that more than onethird of PHC staff were not aware of the inception of the PBR and that only half of those who were aware, knew about its contents as well. It was expected that health personnel had a comprehensive awareness about the existence of the bill and its contents; however, the results contradict this expectation. In the published studies, there is a general notion that health care pro- . obstacles to the implementation of the patient bill of rights as perceived by phC staff (only for phC staff who were aware of the existence of the bill. a respondents were instructed to select as many obstacles as applicable.
## Obstacles
viders possess higher levels of awareness about patient rights and other ethical health issues. 15 However, the results of this study imply that more efforts are needed to promote the knowledge of health care providers about the bill and its contents. In this study, the PHC staff cited several obstacles that may hinder the implementation of the PBR, such as the lack of knowledge about the bill among patients and health care providers. This is in line with other research which indicated that assuring the rights of patients are protected requires more than educating policy makers and health providers; it requires educating citizens about what they should expect from their health care providers.Other research indicated that the media plays a significant role in making people aware of their legal and social rights; however, this requires planning at a high level of health care management systems. [bib_ref] World health organization, regional office for europe. promotion of the rights of..., Joolaee [/bib_ref] PHC staff indicated that health personnel dissatisfaction, insufficient number of health staff, and lack of necessary facilities in PHC centers are among the important obstacles in implementing the PBR. These results confirm previous research studies conducted in Saudi Arabia which identified that many PHC centers lack necessary resources,have a shortage of qualified health personnel, 17 and are overcrowded. [bib_ref] Determinants of satisfaction with primary health care settings and services among patients..., Al-Qatari Gm [/bib_ref] These issues require urgent solution if the PBR is to be implemented successfully. Previous research studies indicated that where patients' rights are not protected, they look for alternative advocacy mechanisms to meet their needs and protect their rights. [bib_ref] World health organization, regional office for europe. promotion of the rights of..., Joolaee [/bib_ref] One of these mechanisms commonly used in Saudi Arabia is that many patients turn to emergency departments with primary health care problems 19 or rely on over-the-counter medication. 20 Such health-seeking behavior has been described as "inappropriate," 21 not only for patient health, but also for the health care system as a whole. Overcoming these issues might be a prerequisite to a successful implementation of the PBR. The impact of these issues on the implementation of the PBR could be examined in further research.
In summary, although patient rights are increasingly emphasized around the world, they are relatively lesser known in Saudi Arabia and are often recalled only when the health care providers make mistakes, which cause death or disability. Implementation of patient rights only seems possible when health care providers, recipients, and institutions have reached the desired levels of information and consciousness about the bill. Implementing and maintaining the PBR is the responsibility of all stakeholders of the health care system, including patients, health care providers, and policy makers.
Several limitations should be considered when interpreting the results of this study. First, the study was limited to PHC centers. Nevertheless, the findings have implications for other health care facilities as well. Second, because of time and financial constraints, the study was limited to Riyadh. Therefore, the study does not claim to be representative and the results cannot be generalized. Third, the results reported here were based on information collected by questionnaires and were subjected to the disadvantages of using such a data collection tool. Using a qualitative approach with health care stakeholders is recommended to further explore this national topic. Finally, most of the questions used in this study were nonspecific in nature and general in approach. Addressing more specific questions about the bill, its contents, and obstacles that may hinder its implementation will lead to a better understanding about the topic. Future research should attempt to address some of the concerns indicated in these limitations. Despite these limitations, it is expected that the findings are of benefit to all stakeholders of the health care system in terms of increasing awareness about the newly introduced PBR and obstacles that may hinder its implementation.
In conclusion, introducing the PBR in Saudi Arabia is a major step toward ensuring quality of health service and protection of patients' rights. However, the results that emerged from this study indicate that a considerable percentage of patients and health staff lack necessary knowledge about the bill. This suggests that health decision makers, health institutions, and the media should work together to increase the level of knowledge about the PBR and its contents. Health care policy makers should establish measures to tackle obstacles that may delay the implementation of the PBR. If appropriate strategies for increasing the level of awareness about the PBR are to be developed, more dissemination of information about patients' rights, taking into account the particularities of the Saudi population, is needed. Future research is required to establish measures that are effective in ensuring patient rights.
[table] Table 1: respondent knowledge about the existence of the patient bill of rights. ª n=All respondents, b n= only respondents who had heard about the pbr. [/table]
[table] Table 2: the extent to which aspects of the patient bill of rights were implemented as perceived by respondents by scores on a 5-point likert scale. PBR is shown inTable 1. The results indicated that only 91 (21.9%) patients and 242 (66.1%) PHC staff knew about the existence of the PBR (P<.001). Of these, only 27 (29.7%) patients and 85 (35.1% [/table]
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# Introduction
Cancer cachexia describes the cancer-related muscle wasting that contributes to the progression of many cancer types. It is defined as "a multifactorial syndrome exhibiting ongoing loss of skeletal muscle mass, with or without the loss of fat mass, leading to progressive muscle functional impairment" [bib_ref] Definition and classification of cancer cachexia: An international consensus, Fearon [/bib_ref]. Typical clinical symptoms include anorexia, involuntary weight loss, weakness, anemia, systemic inflammation, insulin resistance and increased resting energy expenditure (REE) [bib_ref] Cancer Cachexia Study, G. Definition of cancer cachexia: Effect of weight loss,..., Fearon [/bib_ref] [bib_ref] Cancer cachexia: Diagnosis, assessment, and treatment, Sadeghi [/bib_ref]. Cachexia impairs the efficacy of chemotherapy and is associated with compromised quality of life and poor survival [bib_ref] Cancer cachexia: Diagnosis, assessment, and treatment, Sadeghi [/bib_ref]. It is estimated to account for nearly one-third of cancer-related deaths and is present in more than 50% of all cancer patients at death [bib_ref] Cancer cachexia signaling pathways continue to emerge yet much still points to..., Acharyya [/bib_ref]. The distribution and severity of cachexia differs by tumor types with gastric and pancreatic cancers having the highest incidence of cachexia, and breast cancer having among the lowest incidence [bib_ref] Cancer cachexia: Diagnosis, assessment, and treatment, Sadeghi [/bib_ref] [bib_ref] Cancer cachexia: Mediators, signaling, and metabolic pathways, Fearon [/bib_ref] [bib_ref] Molecular mechanisms involved in muscle wasting in cancer and ageing: Cachexia versus..., Argiles [/bib_ref]. Despite the prevalence of cachexia and its devastating consequences, there is currently no gold standard treatment, likely because of difficulties in clinical diagnosis and the complexity of the disease. The ideal treatment for cancer cachexia is to treat the underlying cancer, but this is challenging, particularly in advanced cancers. While approximately 50 per cent of all cancer patients experience cachexia, the prevalence can be as high as 86 per cent in the last few weeks of life [bib_ref] Cancer cachexia, mechanism and treatment, Aoyagi [/bib_ref]. With such a limited therapeutic window, current management strategies focus on palliative care and improving quality of life to prolong survival. These strategies therefore aim to remove the distress of symptoms and to comfort patients and families, rather than find a cure. One of the key determinants of treatment efficacy is muscle mass because of the direct link between the preservation of lean mass and patient quality of life [bib_ref] Understanding the mechanisms and treatment options in cancer cachexia, Fearon [/bib_ref]. While the pathogenesis of cancer cachexia is multifactorial, a major contributing factor is oxidative stress. While approaches to reduce oxidative stress have therapeutic potential, few studies have investigated the merit of antioxidants to treat cancer cachexia. This review synthesizes the available information regarding antioxidants with anti-cachectic properties and identifies novel candidates with potential for attenuating cancer cachexia.
## Pathogenesis of cancer cachexia
Various factors produced by both host and tumor are thought to contribute to the development and progression of cancer cachexia [bib_ref] Disease-Induced Skeletal Muscle Atrophy and Fatigue, Powers [/bib_ref]. These multiple factors ultimately disrupt the balance between protein synthesis and protein degradation, leading to a loss of muscle mass [bib_ref] Skeletal muscle atrophy, a link between depression of protein synthesis and increase..., Eley [/bib_ref] [bib_ref] Contribution of elevated protein turnover and anorexia to cachexia in patients with..., O'keefe [/bib_ref]. Dysfunction of the membrane stabilizing dystrophin-glycoprotein complex (DGC), characterized by reduced dystrophin expression and increased glycosylation of DGC proteins, has been shown in a mouse model of gastrointestinal cancer, indicating that a loss of communication between the muscle cell membrane and the extracellular matrix could be associated with cachexia [bib_ref] Dystrophin glycoprotein complex dysfunction: A regulatory link between muscular dystrophy and cancer..., Acharyya [/bib_ref]. Increased metabolic stress, which occurs as a consequence of tumor burden, nutritional deficiency and various medical interventions such as chemotherapy, contributes to the development of cancer cachexia by elevating REE and exacerbating muscle loss [bib_ref] Understanding the mechanisms and treatment options in cancer cachexia, Fearon [/bib_ref]. Anti-cancer interventions such as chemotherapy can cause discomfort, nausea and anorexia in some patients, leading to reduced food intake, metabolic changes and muscle loss [bib_ref] Chemotherapy-induced muscle wasting: Association with NF-kappaB and cancer cachexia, Damrauer [/bib_ref]. Since oxidative stress is a major contributor to the development of cancer cachexia, factors able to mitigate excessive oxidative stress could have therapeutic potential.
therapeutic potential, few studies have investigated the merit of antioxidants to tre cer cachexia. This review synthesizes the available information regarding antiox with anti-cachectic properties and identifies novel candidates with potential for att ing cancer cachexia.
## Pathogenesis of cancer cachexia
Various factors produced by both host and tumor are thought to contribute development and progression of cancer cachexia [bib_ref] Disease-Induced Skeletal Muscle Atrophy and Fatigue, Powers [/bib_ref]. These multiple ultimately disrupt the balance between protein synthesis and protein degradation ing to a loss of muscle mass [bib_ref] Skeletal muscle atrophy, a link between depression of protein synthesis and increase..., Eley [/bib_ref] [bib_ref] Contribution of elevated protein turnover and anorexia to cachexia in patients with..., O'keefe [/bib_ref]. Dysfunction of the membrane stabilizing dystr glycoprotein complex (DGC), characterized by reduced dystrophin expression a creased glycosylation of DGC proteins, has been shown in a mouse model of gastr tinal cancer, indicating that a loss of communication between the muscle cell mem and the extracellular matrix could be associated with cachexia [bib_ref] Dystrophin glycoprotein complex dysfunction: A regulatory link between muscular dystrophy and cancer..., Acharyya [/bib_ref]. Increased me stress, which occurs as a consequence of tumor burden, nutritional deficiency and v medical interventions such as chemotherapy, contributes to the development of cachexia by elevating REE and exacerbating muscle loss [bib_ref] Understanding the mechanisms and treatment options in cancer cachexia, Fearon [/bib_ref]. Anti-cancer interve such as chemotherapy can cause discomfort, nausea and anorexia in some patients ing to reduced food intake, metabolic changes and muscle loss [bib_ref] Chemotherapy-induced muscle wasting: Association with NF-kappaB and cancer cachexia, Damrauer [/bib_ref]. Since oxidative is a major contributor to the development of cancer cachexia, factors able to mitig cessive oxidative stress could have therapeutic potential. . Pathogenesis of cancer cachexia. Numerous factors influenced by host cytokines mor-released factors result in an imbalance between protein degradation and protein synthe ultimately leads to muscle wasting and weakness [bib_ref] Skeletal muscle atrophy, a link between depression of protein synthesis and increase..., Eley [/bib_ref] [bib_ref] Contribution of elevated protein turnover and anorexia to cachexia in patients with..., O'keefe [/bib_ref]. Membrane integrity is comprom cause of potential dysfunction of the dystrophin-glycoprotein complex [bib_ref] Dystrophin glycoprotein complex dysfunction: A regulatory link between muscular dystrophy and cancer..., Acharyya [/bib_ref]. Metabolic dys tion leads to an increased resting energy expenditure (REE) and this contributes to tumor p sion and nutritional deficiencies [bib_ref] Understanding the mechanisms and treatment options in cancer cachexia, Fearon [/bib_ref]. Increased oxidative stress is a key contributor to can chexia, contributing to mechanisms that favor protein breakdown over protein synthesis t increased ubiquitin proteasome activity, mitochondrial dysfunction and dysregulation of a agy. Chemotherapy can contribute to the pathology of cancer cachexia by inducing anorex Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling is a key pathway associated with oxidative stress [bib_ref] Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in..., Li [/bib_ref]. NF-κB controls several cellular processes including inflammatory cell regulation, cell growth and apoptosis [bib_ref] The NF-kappa B and I kappa B proteins: New discoveries and insights, Baldwin [/bib_ref]. In tumor cells, NF-κB signaling has been adapted to increase cell survival and proliferation [bib_ref] Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB, Baldwin [/bib_ref]. Considering the close relationship between tumor-bearing and cancer cachexia, it is likely that NF-κB also plays a role in the progression of cancer cachexia. NF-κB signaling has an important role in skeletal muscle atrophy and fat lipolysis, including post-transcriptional suppression of myoblast determination protein 1 (MyoD) mRNA inducing protein degradation, leading to muscle wasting [bib_ref] Pyrrolidine Dithiocarbamate (PDTC) Attenuates Cancer Cachexia by Affecting Muscle Atrophy and Fat..., Miao [/bib_ref]. Host and tumor-derived factors, including proteolysis inducing factor (PIF) and TNF-α, activate NF-κB signaling, enhancing proteolysis and protein degradation [bib_ref] Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of..., Wyke [/bib_ref]. The promotor region of the proteasome C3 subunit provides a link to the binding site of NF-κB [bib_ref] Epigallocatechin-3-gallate effectively attenuates skeletal muscle atrophy caused by cancer cachexia, Wang [/bib_ref] , suggesting NF-κB's involvement in gene transcription of the ubiquitin-proteasome system. Furthermore, TNF-α treatment-induced rapid production of hydrogen peroxide and activation of NF-κB, leading to reduced total protein content and adult myosin heavy chain content in C2C12 mouse muscle myotubes in vitro [bib_ref] Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in..., Li [/bib_ref]. Inhibition of ROS with catalase suppressed the TNF-α-induced activation of NF-κB, demonstrating a link between inflammation, oxidative stress and the regulation of muscle cell size [bib_ref] Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in..., Li [/bib_ref]. NF-κB also mediates PIF-induced proteasome expression, activation of the ubiquitin-proteasome pathway and reduction of myofibrillar myosin levels [bib_ref] Reasons for low incidence of peptic ulcer in pregnancy, Schmid [/bib_ref] , with these effects attenuated by co-administration of the NF-κB inhibitor, SN50 peptide [bib_ref] Reasons for low incidence of peptic ulcer in pregnancy, Schmid [/bib_ref]. Higher levels of ROS and activated NF-κB p65 expression have been reported in the vastus lateralis muscles of cachectic patients, together with a decreased level of activated inhibitor-κB (IκB) kinases, indicating involvement of NF-κB signaling in elevating oxidative stress in cachectic cancer patients [bib_ref] Oxidative stress, redox signaling pathways, and autophagy in cachectic muscles of male..., Puig-Vilanova [/bib_ref]. These findings highlight the role of NF-κB activation in the pathogenesis of cancer cachexia and support the therapeutic potential for inhibition of ROS-induced NF-κB activation.
## Nuclear factor erythroid 2-related factor 2
Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) is a critical transcription factor that regulates the antioxidant response in cells under basal and stressed conditions [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref] [fig_ref] Figure 2: Sulforaphane [/fig_ref]. Nrf2 binds to the antioxidant response element (ARE) to regulate expression of phase II antioxidant enzymes, indicating the important role of Nrf2 in maintaining body redox homeostasis [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref]. Under basal conditions, kelch-like ECH-associated protein-1 (Keap1) represses Nrf2 signaling by facilitating poly-ubiquitination to promote proteasomal degradation of Nrf2 [bib_ref] Keap1-nrf2 signaling: A target for cancer prevention by sulforaphane, Kensler [/bib_ref]. Oxidative stress from increased ROS, inflammation and/or carcinogenic factors, disrupts the interaction between Keap1 and Nrf2 to enable nuclear translocation of Nrf2, a process that activates Nrf2 signaling [bib_ref] Nrf2 targeting by sulforaphane: A potential therapy for cancer treatment, Russo [/bib_ref] [bib_ref] Deregulation of Nrf2/ARE signaling pathway causes susceptibility of dystrophin-deficient myotubes to menadione-induced..., Choi [/bib_ref] to promote expression of antioxidant genes, including nicotinamide adenine dinucleotide phosphate (reduced form; NAD(P)H), quinone oxidoreductase-1 (NQO1), glutathione-S-transferase (GST), superoxide dismutase (SOD) and hemeoxygenase-1 (HMOX1), to attenuate the oxidative burden [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref] [bib_ref] Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in..., Son [/bib_ref].
NF-κB, demonstrating a link between inflammation, oxidative stress and the regulatio of muscle cell size [bib_ref] Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in..., Li [/bib_ref]. NF-κB also mediates PIF-induced proteasome expression, activ tion of the ubiquitin-proteasome pathway and reduction of myofibrillar myosin leve [bib_ref] Reasons for low incidence of peptic ulcer in pregnancy, Schmid [/bib_ref] , with these effects attenuated by co-administration of the NF-κB inhibitor, SN50 pep tide [bib_ref] Reasons for low incidence of peptic ulcer in pregnancy, Schmid [/bib_ref]. Higher levels of ROS and activated NF-κB p65 expression have been reported the vastus lateralis muscles of cachectic patients, together with a decreased level of act vated inhibitor-κB (IκB) kinases, indicating involvement of NF-κB signaling in elevatin oxidative stress in cachectic cancer patients [bib_ref] Oxidative stress, redox signaling pathways, and autophagy in cachectic muscles of male..., Puig-Vilanova [/bib_ref]. These findings highlight the role of NF κB activation in the pathogenesis of cancer cachexia and support the therapeutic potenti for inhibition of ROS-induced NF-κB activation.
## Nuclear factor erythroid 2-related factor 2
Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) is a critical transcription fa tor that regulates the antioxidant response in cells under basal and stressed condition [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref] [fig_ref] Figure 2: Sulforaphane [/fig_ref]. Nrf2 binds to the antioxidant response element (ARE) to regulate expre sion of phase II antioxidant enzymes, indicating the important role of Nrf2 in maintainin body redox homeostasis [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref]. Under basal conditions, kelch-like ECH-associated protein 1 (Keap1) represses Nrf2 signaling by facilitating poly-ubiquitination to promote pr teasomal degradation of Nrf2 [bib_ref] Keap1-nrf2 signaling: A target for cancer prevention by sulforaphane, Kensler [/bib_ref]. Oxidative stress from increased ROS, inflammatio and/or carcinogenic factors, disrupts the interaction between Keap1 and Nrf2 to enab nuclear translocation of Nrf2, a process that activates Nrf2 signaling [bib_ref] Nrf2 targeting by sulforaphane: A potential therapy for cancer treatment, Russo [/bib_ref] [bib_ref] Deregulation of Nrf2/ARE signaling pathway causes susceptibility of dystrophin-deficient myotubes to menadione-induced..., Choi [/bib_ref] to promo expression of antioxidant genes, including nicotinamide adenine dinucleotide phospha (reduced form; NAD(P)H), quinone oxidoreductase-1 (NQO1), glutathione-S-transferas (GST), superoxide dismutase (SOD) and hemeoxygenase-1 (HMOX1), to attenuate the o idative burden [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref] [bib_ref] Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in..., Son [/bib_ref]. localizes to the cytoplasm, where its activity is suppressed by Keap1. When cells experience extensive oxidative stress, Nrf2 is released from the Keap1 complex and translocates into the nucleus, where it promotes antioxidant and detoxifying gene expression via the antioxidant response element (ARE) complex [bib_ref] Deregulation of Nrf2/ARE signaling pathway causes susceptibility of dystrophin-deficient myotubes to menadione-induced..., Choi [/bib_ref]. SFN regulates activation of Nrf2 and increases the rate of nuclear translocation of Nrf2 [bib_ref] The natural antioxidant alpha-lipoic acid induces p27(Kip1)-dependent cell cycle arrest and apoptosis..., Dozio [/bib_ref]. SFN activates Nrf2 via direct modification of critical Keap1 cysteines, such as Cys 151 [bib_ref] Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2..., Zhang [/bib_ref]. SFN can also de-methylate the promoter region of Nrf2 and accelerate Nrf2 protein synthesis [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref] [bib_ref] Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2..., Zhang [/bib_ref]. Phosphorylated Nrf2 also inhibits phosphorylation of Smad2/3 in the transforming growth factor-β (TGF-β) signaling pathway linked to an attenuation of tissue fibrosis progression [bib_ref] Sulforaphane mitigates muscle fibrosis in mdx mice via Nrf2-mediated inhibition of TGF-beta/Smad..., Sun [/bib_ref]. In addition, SFN can increase protein synthesis and decrease protein degradation via activation of the protein kinase B (Akt)/Forkhead box O1 (FoxO1) signaling pathway [bib_ref] Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in..., Son [/bib_ref]. Activation of Nrf2 signaling also elicits a strong cytoprotective response to impair the progression of carcinogenesis [bib_ref] Keap1-nrf2 signaling: A target for cancer prevention by sulforaphane, Kensler [/bib_ref]. While Nrf2 has protective effects against various cancer types [bib_ref] Broccoli and human health: Immunomodulatory effect of sulforaphane in a model of..., Bessler [/bib_ref] [bib_ref] NRF2 and cancer: The good, the bad and the importance of context, Sporn [/bib_ref] , some studies have suggested Nrf2 activation can have negative effects by elevating the antioxidant response that contributes to a resistance to cancer treatments [bib_ref] Dual roles of NRF2 in tumor prevention and progression: Possible implications in..., Moon [/bib_ref] [bib_ref] Genetic alteration of Keap1 confers constitutive Nrf2 activation and resistance to chemotherapy..., Shibata [/bib_ref] [bib_ref] Gain of Nrf2 function in non-small-cell lung cancer cells confers radioresistance, Singh [/bib_ref]. Moreover, Nrf2 signaling has been linked to the initiation and progression of lung cancer in vivo [bib_ref] Nrf2 prevents initiation but accelerates progression through the Kras signaling pathway during..., Satoh [/bib_ref]. Nrf2-deletion in mice increased lung tumor growth but reduced the number of tumors with cancer progression [bib_ref] Nrf2 prevents initiation but accelerates progression through the Kras signaling pathway during..., Satoh [/bib_ref]. These findings suggest a dual role of Nrf2 in cancer as it prevents tumor initiation but facilitates malignant tumor progression. A delicate balance therefore exists between ROS levels and antioxidant content, indicating the need for precision when treating cancer or cancer-related muscle wasting with antioxidants.
## Polyphenols to reduce oxidative stress in cancer cachexia
Despite oxidative stress being a main driver of cancer cachexia, there is a dearth of studies investigating the therapeutic potential of antioxidants for treating cancer-related muscle wasting [fig_ref] Table 1: Cont. [/fig_ref]. Polyphenols are common phytochemicals with protective effects against oxidative stress-related diseases. They are widely distributed across plants and can be found in various plant-based foods, including fruits, vegetables and whole grains with rich antioxidant sources [bib_ref] Polyphenols and their potential role in preventing skeletal muscle atrophy, Salucci [/bib_ref]. Due to their antioxidative and anti-inflammatory properties, the potential benefits of polyphenols against different cancers have been investigated extensively [bib_ref] Natural Polyphenols for Prevention and Treatment of Cancer, Zhou [/bib_ref]. The potential for polyphenols to attenuate cancer cachexia has been attributed to a possible preservation of muscle mass through inhibition of NF-κB signaling [bib_ref] Polyphenols and their potential role in preventing skeletal muscle atrophy, Salucci [/bib_ref].
## Epigallocatechin-3-gallate
Epigallocatechin-3-gallate (EGCG) is the dominant antioxidant in green and black tea extract, having antioxidant, anti-inflammatory and anti-cancer functions [bib_ref] Epigallocatechin-3-gallate effectively attenuates skeletal muscle atrophy caused by cancer cachexia, Wang [/bib_ref]. EGCG suppresses cancer cell proliferation, inducing cell apoptosis and inhibiting cell invasion and migration to attenuate tumor progression [bib_ref] Green tea and cancer prevention, Yang [/bib_ref]. In cell culture, 48 h EGCG treatment suppressed the growth of Lewis lung carcinoma (LLC) cancer cells in a dose-dependent manner, confirming its anti-tumor effects [bib_ref] Epigallocatechin-3-gallate effectively attenuates skeletal muscle atrophy caused by cancer cachexia, Wang [/bib_ref]. In the LLC tumor-bearing mouse, 12 days of EGCG treatment reduced tumor mass and volume and attenuated the loss of body weight without altering anorexia [bib_ref] Epigallocatechin-3-gallate effectively attenuates skeletal muscle atrophy caused by cancer cachexia, Wang [/bib_ref]. The attenuation of muscle wasting was attributed to inhibition of NF-κB and the downstream E3 ligases, MuRF1 and atrogin-1. Furthermore, ECGC treatment decreased leukocytic infiltration, thus reducing inflammation in skeletal muscles of tumor-bearing mice [bib_ref] Epigallocatechin-3-gallate effectively attenuates skeletal muscle atrophy caused by cancer cachexia, Wang [/bib_ref]. In addition to the suppression of tumor growth, high dose EGCG treatment reduced the survival rate of a healthy, baby hamster kidney cells (BHK-21) by 50%, [bib_ref] Epigallocatechin-3-gallate effectively attenuates skeletal muscle atrophy caused by cancer cachexia, Wang [/bib_ref] , suggesting that higher concentrations of EGCG cause cytotoxicity in normal cells and interrupt healthy cell growth. Given that EGCG treatment in the LLC-injected mice was administrated several days prior to tumor palpability, it remains to be determined whether the protective effect of EGCG on muscle mass was due to its anti-cancer effects or direct effects on the muscle. Moreover, EGCG has a low systemic bioavailability [bib_ref] Molecular targets for the cancer preventive activity of tea polyphenols, Yang [/bib_ref] , which may reduce the efficacy of EGCG treatment in clinical studies and in vivo animal studies. EGCG treatment for cancer cachexia is a relatively preliminary concept in the field and further investigation is needed to confirm therapeutic potential.
## Resveratrol
Resveratrol is found most abundantly in the skin of grapes, peanuts and pine bark, and has been shown to exert anti-cancer effects in vitro and in vivo [bib_ref] Resveratrol, a natural product present in wine, decreases tumour growth in a..., Carbo [/bib_ref]. In Yoshida ascites hepatoma (AH-130) cells in vitro and in rats implanted with AH-130 cells, resveratrol administration reduced tumor cell number via induction of AH-130 cell apoptosis [bib_ref] Resveratrol, a natural product present in wine, decreases tumour growth in a..., Carbo [/bib_ref]. In Ehrlich ascitic carcinoma bearing mice, the combined treatment of resveratrol (10 mg/kg) with the chemotherapeutic drug, doxorubicin (5 mg/kg), twice a week via intraperitoneal injection, exerted the best reduction in tumor size and prolonged survival compared with either treatment alone [bib_ref] Resveratrol improves the anticancer effects of doxorubicin in vitro and in vivo..., Rai [/bib_ref]. These findings demonstrate the potential for resveratrol to enhance the anti-cancer effect of chemotherapies by decreasing inflammation and the oxidative stress associated with chemotherapy [bib_ref] Resveratrol improves the anticancer effects of doxorubicin in vitro and in vivo..., Rai [/bib_ref]. ↓ protein synthesis shown in LLC cells using SUnSET method; no significant changes were found with C2C12 myotubes. Resveratrol has been studied as an anti-cachectic treatment to attenuate muscle atrophy. Daily administration of resveratrol (200 mg/kg/day) via oral gavage attenuated the loss of lean body and fat mass, and gastrocnemius muscle mass in C-26 tumor-bearing mice [bib_ref] Oral resveratrol therapy inhibits cancer-induced skeletal muscle and cardiac atrophy in vivo, Shadfar [/bib_ref]. These effects were associated with inhibition of IκB kinase, the activator of NF-κB signaling, to prevent nuclear translocation and accumulation of NF-κB [bib_ref] Oral resveratrol therapy inhibits cancer-induced skeletal muscle and cardiac atrophy in vivo, Shadfar [/bib_ref] and subsequently inhibiting expression of the downstream E3 ligases, atrogin-1 and MuRF1 [bib_ref] Oral resveratrol therapy inhibits cancer-induced skeletal muscle and cardiac atrophy in vivo, Shadfar [/bib_ref]. Resveratrol inhibited PIF-induced activation of NF-κB in MAC16 tumor-bearing mice and attenuated the loss of muscle mass and whole body mass [bib_ref] Reasons for low incidence of peptic ulcer in pregnancy, Schmid [/bib_ref]. Moreover, resveratrol supplementation prevented TNF-α-induced myotube atrophy via activation of the Akt/mammalian target of rapamycin (mTOR)/FoxO1 signaling pathway, evident from increased Akt, ribosomal protein S6 kinase beta-1 (p70S6K), mTOR and eukaryotic translation initiation factor 4Ebinding protein 1 (4E-BP1) phosphorylation and decreased FoxO1 protein expression [bib_ref] Resveratrol prevents TNF-alpha-induced muscle atrophy via regulation of Akt/mTOR/FoxO1 signaling in C2C12..., Wang [/bib_ref]. These findings suggest that the mechanisms underpinning the protective effect of resveratrol for cancer cachexia likely involve a restoration of the balance between protein synthesis and protein degradation.
In mice injected with LP07 adenocarcinoma cells, the anti-cachectic effects of resveratrol (20 mg/kg/day for 15 days via intraperitoneal injection) were linked to activation of sirtuin-1 and attenuation of FoxO3 signaling [bib_ref] Curcumin and Resveratrol Improve Muscle Function and Structure through Attenuation of Proteolytic..., Penedo-Vázquez [/bib_ref]. Sirtuin-1 is a histone deacetylase enzyme that helps maintain muscle mitochondrial content and function [bib_ref] Sirtuin 1-mediated effects of exercise and resveratrol on mitochondrial biogenesis, Menzies [/bib_ref]. The resveratroldependent activation of sirtuin-1 is proposed to improve mitochondrial biogenesis to alleviate oxidative stress in cachectic LP07 tumor-bearing mice [bib_ref] Curcumin and Resveratrol Improve Muscle Function and Structure through Attenuation of Proteolytic..., Penedo-Vázquez [/bib_ref]. FoxO3 is involved in the skeletal muscle ubiquitin-proteasome, autophagy-lysosomal and mitochondrial autophagy pathways [bib_ref] FoxO transcription factors: Their roles in the maintenance of skeletal muscle homeostasis, Sanchez [/bib_ref]. However, resveratrol treatment also inhibited tumor growth, making it difficult to discern whether the attenuation of muscle wasting was due solely to a reduced tumor burden [bib_ref] Curcumin and Resveratrol Improve Muscle Function and Structure through Attenuation of Proteolytic..., Penedo-Vázquez [/bib_ref]. While these studies have shown promising effects of resveratrol, findings from other studies question the benefit of resveratrol for treating cancer cachexia. Intraperitoneal injection of resveratrol failed to attenuate the loss of muscle mass and body mass in LLC-tumor bearing mice (at both 5 mg/kg/day and 25 mg/kg/day for 15 days) and exacerbated the reduction in food intake and loss of gastrocnemius, heart and white adipose tissue mass in AH-130-tumor bearing rats (1 mg/kg/day for seven days) [bib_ref] Resveratrol does not ameliorate muscle wasting in different types of cancer cachexia..., Busquets [/bib_ref]. The discrepancies in the findings between the studies may be attributed to differences in animal models and/or tumor type [bib_ref] Oral resveratrol therapy inhibits cancer-induced skeletal muscle and cardiac atrophy in vivo, Shadfar [/bib_ref] [bib_ref] Resveratrol does not ameliorate muscle wasting in different types of cancer cachexia..., Busquets [/bib_ref]. Further studies are required to resolve these conflicts and determine the mechanisms underlying the therapeutic potential of resveratrol for cancer cachexia.
## Curcumin
Curcumin, a component of turmeric, has been investigated as a potential treatment for cancer cachexia [bib_ref] NMR-based metabolomics reveals distinct pathways mediated by curcumin in cachexia mice bearing..., Quan-Jun [/bib_ref] due to its anti-inflammatory, antioxidative and anticarcinogenic functions as a nutritional supplement [bib_ref] Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16..., Siddiqui [/bib_ref] [bib_ref] Curcumin, a natural product present in turmeric, decreases tumor growth but does..., Busquets [/bib_ref]. Curcumin is a nontoxic phytochemical with demonstrated potential to attenuate tumor growth in preclinical and clinical studies via suppression of NF-κB activity [bib_ref] Curcumin, a natural product present in turmeric, decreases tumor growth but does..., Busquets [/bib_ref] [bib_ref] Phase II trial of curcumin in patients with advanced pancreatic cancer, Dhillon [/bib_ref]. In MAC16 tumor-bearing mice, curcumin prevented muscle wasting and reversed existing muscle loss [bib_ref] Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16..., Siddiqui [/bib_ref]. Treatment with curcumin c3 complex has been used in human clinical trials [bib_ref] Phase I clinical trial of oral curcumin: Biomarkers of systemic activity and..., Sharma [/bib_ref] , where it protected skeletal muscle from wasting when orally administered a low dose (100 mg/kg/day) for 20 days, and induced weight gain relative to the control tumor-bearing group when administered at a higher dose (250 mg/kg/day) [bib_ref] Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16..., Siddiqui [/bib_ref]. In MAC16 colon tumor-bearing mice, curcumin treatment attenuated the PIF-induced increase of the 20S proteasome, and decreased expression of NF-κB, atrogin-1 and MuRF1, indicating that protection from muscle wasting was attributed to suppression of the ubiquitin-proteasome pathway and subsequent protein degradation [bib_ref] Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16..., Siddiqui [/bib_ref]. It should be noted that MAC16 colon tumor-bearing mice exhibit a gradual loss of body mass and muscle mass over 21 days, enabling the efficacy of curcumin supplementation to protect skeletal muscle mass to be assessed over a longer period [bib_ref] Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16..., Siddiqui [/bib_ref]. Curcumin was also shown to attenuate loss of body mass and improve muscle mass and limb strength gain in cachectic LP07 tumor-bearing mice without altering tumor size [bib_ref] Curcumin and Resveratrol Improve Muscle Function and Structure through Attenuation of Proteolytic..., Penedo-Vázquez [/bib_ref]. These effects were associated with increased cross-sectional area (CSA) of type I and type II muscle fibers, and a reduced proportion of muscle fibers with internal nuclei and inflammatory cell infiltration, in gastrocnemius and soleus muscles [bib_ref] Curcumin and Resveratrol Improve Muscle Function and Structure through Attenuation of Proteolytic..., Penedo-Vázquez [/bib_ref]. Compared to studies reporting beneficial outcomes of curcumin, administration (20 µg/kg/day for six days via intraperitoneal injection) failed to improve the cachectic pathology in AH-130 tumor-bearing rats despite having antitumor effects [bib_ref] Curcumin, a natural product present in turmeric, decreases tumor growth but does..., Busquets [/bib_ref]. The disparity in these reports may arise from differences in dose, route of administration and treatment duration in different animal models, as well as the low systemic bioavailability of curcumin [bib_ref] Curcumin in cancer chemoprevention: Molecular targets, pharmacokinetics, bioavailability, and clinical trials, Shehzad [/bib_ref].
While studies investigating curcumin efficacy in cancer cachexia did not measure oxidative stress directly, the role of curcumin for attenuating oxidative stress in skeletal muscle is well established. Curcumin reduced exercise-induced oxidative stress, evidenced by decreased levels of serum lactate and muscle MDA [bib_ref] Curcumin prevents muscle damage by regulating NF-kappaB and Nrf2 pathways and improves..., Sahin [/bib_ref]. Oral curcumin treatment (100 mg/kg/day) for 14 days reduced hypobaric hypoxia-induced oxidative stress and increased muscle fiber number in Sprague Dawley rats [bib_ref] High altitude mediated skeletal muscle atrophy: Protective role of curcumin, Chaudhary [/bib_ref]. The antioxidative effect of curcumin was linked with reduced activity of NF-κB and activation of Nrf2 signaling [bib_ref] Curcumin prevents muscle damage by regulating NF-kappaB and Nrf2 pathways and improves..., Sahin [/bib_ref] , effects that reflect regulation of redox balancing, protein synthesis and protein degradation. Thus, regulating redox balance may be one mechanism by which curcumin attenuates cancer cachexia.
The therapeutic potential of curcumin has also been explored in clinical trials. Based on the limited data available, curcumin reduced expression of NF-κB in some patients with pancreatic cancer [bib_ref] Phase II trial of curcumin in patients with advanced pancreatic cancer, Dhillon [/bib_ref]. Due to its poor oral absorption and weak bioavailability, curcumin supplementation had only limited benefit for these pancreatic cancer patients [bib_ref] Phase II trial of curcumin in patients with advanced pancreatic cancer, Dhillon [/bib_ref]. However, the difficulty in accurately determining redox status in skeletal muscle [bib_ref] The Janus-Faced Role of Antioxidants in Cancer Cachexia: New Insights on the..., Assi [/bib_ref] , may explain why evaluating the efficacy of antioxidant supplements like curcumin has proved challenging, especially when assessments in trials rely on measures of antioxidant levels in the blood. Improvements in the accuracy of these outcome measures are required to better evaluate the therapeutic potential of curcumin for cancer cachexia.
## Carnosol
Carnosol is a bioactive diterpene compound present in rosemary, with antioxidant, anti-inflammatory and anti-cancer properties [bib_ref] Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by..., Lu [/bib_ref]. The antioxidative function of carnosol has been well characterized and includes protection from lipid peroxidation [bib_ref] Carnosic Acid and Carnosol, Two Major Antioxidants of Rosemary, Act through Different..., Loussouarn [/bib_ref] , suppression of nitric oxide production and gene expression of inducible nitric oxide synthase [bib_ref] an antioxidant in rosemary, suppresses inducible nitric oxide synthase through down-regulating nuclear..., Lo [/bib_ref] and amelioration of the damage caused by UVB-induced ROS [bib_ref] The Mechanisms of Carnosol in Chemoprevention of Ultraviolet B-Light-Induced Non-Melanoma Skin Cancer..., Tong [/bib_ref]. Carnosol can inhibit the activities of NF-κB signaling to protect against free radical damage [bib_ref] an antioxidant in rosemary, suppresses inducible nitric oxide synthase through down-regulating nuclear..., Lo [/bib_ref] and it has been shown to reduce tumor growth in mouse models of intestinal cancer [bib_ref] Carnosol inhibits β-catenin tyrosine phosphorylation and prevents adenoma formation in the C57BL/6J/Min/+(Min/+)..., Moran [/bib_ref] , breast cancer [bib_ref] Inhibits Migration, Metastasis, and Tumor Growth of Breast Cancer via a RO.OS-Dependent..., Alsamri [/bib_ref] and skin cancer [bib_ref] The Mechanisms of Carnosol in Chemoprevention of Ultraviolet B-Light-Induced Non-Melanoma Skin Cancer..., Tong [/bib_ref].
Carnosol can protect against cancer-induced muscle wasting in in vitro and in vivo models [bib_ref] Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by..., Lu [/bib_ref]. Carnosol supplementation attenuated C2C12 myotube atrophy after exposure to C-26 cell conditioned media and ameliorated the loss of body mass in C-26 tumor-bearing mice [bib_ref] Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by..., Lu [/bib_ref]. These effects were associated with downregulation of MuRF1 expression and upregulation of Akt phosphorylation and MyoD expression, indicating suppressed protein degradation and increased protein synthesis [bib_ref] Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by..., Lu [/bib_ref]. Both in vitro and in vivo models showed reduced phosphorylation of p65, implicating a role for carnosol in suppressing NF-κB signaling in cancer cachexia [bib_ref] Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by..., Lu [/bib_ref]. While carnosol had a protective role in C-26 tumorbearing mice by maintaining body mass and adipose tissue mass, skeletal muscle mass was not improved. This suggested the increase in body mass resulted from an attenuation of fat lipolysis rather than direct effects on the regulation of skeletal muscle mass [bib_ref] Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by..., Lu [/bib_ref]. These interesting findings warrant further investigation of the therapeutic potential of carnosol for cancer cachexia. Moreover, a recent study revealed the synergistic effect of carnosol and a chemotherapeutic drug, cisplatin, where combined therapy induced the highest rates of apoptosis in MCF-7 and MDA-MB-231 breast cancer cell lines [bib_ref] Evaluate the inhibition of cytochrome P450 1A1 for enhancing breast cancer chemotherapy..., Huang [/bib_ref]. Therefore, carnosol has potential to become part of a combination therapy in the treatment of cancer and cancer-induced muscle wasting.
## Quercetin and rutin
Quercetin is an abundant flavonoid and prominent dietary antioxidant in various fruits and vegetables, such as onions, tomatoes and apples. Quercetin has antioxidative and anti-inflammatory functions, and hence therapeutic potential for treating cancer [bib_ref] Inhibition of tumor growth by quercetin with increase of survival and prevention..., Camargo [/bib_ref] [bib_ref] The Anti-Cancer Effect of Quercetin: Molecular Implications in Cancer Metabolism, Reyes-Farias [/bib_ref]. Supplementation with quercetin (0.05% (w/w) in food) for nine weeks protected against TNF-α-induced skeletal muscle atrophy via activation of Nrf-2 signaling and inactivation of the NF-κB signaling pathway to overcome oxidative stress in the C57BL/6 mouse model of high fat diet-induced obesity, confirming the antioxidant and anti-inflammatory properties of quercetin. Quercetin has significant bioavailability compared with other polyphenols, with detection in plasma 12 h after oral intake [bib_ref] Quercetin supplementation attenuates muscle wasting in cancer-associated cachexia in mice, Levolger [/bib_ref]. The high absorption in plasma was associated with whole body accumulation of quercetin since it was detected in different tissues such as the liver, skeletal muscle, heart and brain, resulting in the slow clearance of quercetin metabolites from the body [bib_ref] Quercetin supplementation attenuates muscle wasting in cancer-associated cachexia in mice, Levolger [/bib_ref].
Quercetin has been shown to protect against cancer-induced muscle wasting in vivo [bib_ref] Quercetin Supplementation Attenuates the Progression of Cancer Cachexia in ApcMin/+Mice, Velázquez [/bib_ref] [bib_ref] Quercetin supplementation attenuates muscle wasting in cancer-associated cachexia in mice, Levolger [/bib_ref]. Oral administration of quercetin (25 mg/kg/day) to Apc Min/+ mice for three weeks attenuated the loss of whole body mass and increased gastrocnemius and quadriceps muscle mass [bib_ref] Quercetin Supplementation Attenuates the Progression of Cancer Cachexia in ApcMin/+Mice, Velázquez [/bib_ref]. These effects may have resulted from reduced inflammation, based on the decrease in plasma IL-6 levels [bib_ref] Quercetin Supplementation Attenuates the Progression of Cancer Cachexia in ApcMin/+Mice, Velázquez [/bib_ref]. However, treatment failed to improve muscle function in the Apc Min/+ mouse model of cancer cachexia [bib_ref] Quercetin Supplementation Attenuates the Progression of Cancer Cachexia in ApcMin/+Mice, Velázquez [/bib_ref]. Supplementation of 250 mg/kg quercetin to a daily chow diet for 20 days attenuated both the loss of body mass and the reduction in gastrocnemius and tibialis anterior muscle mass in C-26 tumor bearing mice, but did not improve grip strength [bib_ref] Quercetin supplementation attenuates muscle wasting in cancer-associated cachexia in mice, Levolger [/bib_ref]. Micro-CT analysis of the hindlimb revealed that quercetin supplementation completely prevented the tumor-induced reduction in muscle volume [bib_ref] Quercetin supplementation attenuates muscle wasting in cancer-associated cachexia in mice, Levolger [/bib_ref]. A substantial (albeit a non-statistically significant) decrease was detected in the expression of E3 ubiquitin ligases, atrogin-1 and MuRF1, in treated mice, indicating an attenuation of protein degradation. While these studies suggest a positive outcome of quercetin with a potential anti-cachectic function, the underlying mechanism remains undetermined. Interestingly, tumor mass was not significantly decreased with quercetin. Furthermore, the experiment did not control for the increase in food intake in the quercetin supplemented group [bib_ref] Quercetin supplementation attenuates muscle wasting in cancer-associated cachexia in mice, Levolger [/bib_ref]. While quercetin has proposed anti-cachectic benefits, a mechanism for these effects has not been established at the cellular level, and this diminishes the significance of these outcomes. Moreover, oral quercetin (50 mg/kg/day) for 1 h prior and during 15-day doxorubicin exposure, reduced chemotherapy-induced oxidative stress in the spleen via suppression of apoptosis, reducing inflammation and increasing the antioxidant response in Sprague-Dawley rats. The protective effect of quercetin against chemotherapy-induced cytotoxicity indicates its potential for attenuating chemotherapy associated muscle atrophy in cancer cachexia. Future studies using pair-fed groups to control for potential treatment-related changes in food intake, as well as additional clinically relevant end-point analyses such as survival, are warranted in order to fully investigate the therapeutic potential of quercetin in cancer cachexia.
Similar to quercetin, rutin, a quercetin glycoside more commonly seen in edible plants, has similar high bioavailability, making it a suitable candidate for nutritional therapy [bib_ref] Bioavailability of rutin and quercetin in rats, Manach [/bib_ref]. Supplementation with rutin (413 mg/kg/day) for 24 weeks increased survival in HPV16-tumor bearing mice and increased gastrocnemius muscle mass, which was associated with inhibition of NF-κB signaling [bib_ref] HPV16 induces a wasting syndrome in transgenic mice: Amelioration by dietary polyphenols..., Gil Da Costa [/bib_ref]. Rutin also alleviated carcinogenesis via suppression of cyclo-oxygenase-2 in K14-HPV16 mice [bib_ref] Curcumin and Rutin Down-regulate Cyclooxygenase-2 and Reduce Tumor-associated Inflammation in HPV16-Transgenic Mice, Moutinho [/bib_ref]. Studies have demonstrated the synergistic benefit of rutin in combination with chemotherapeutic drugs to further reduce cell proliferation in different cancer cell lines via activation of apoptosis, thereby enhancing the anti-cancer effect of chemotherapy. Thus, rutin has significant potential in combination therapies to attenuate cancer cachexia.
## Genistein, daidzein and morin
Genistein and daidzein are isoflavones abundant in soy products, with anti-inflammatory and antioxidative properties [bib_ref] Dietary Supplementation with Isoflavones Prevents Muscle Wasting in Tumor-Bearing Mice, Hirasaka [/bib_ref]. Supplementation with soy isoflavones (mainly genistein and daidzein) for three weeks attenuated LLC tumor-induced muscle wasting by increasing both the overall muscle mass and size of individual muscle fibers within the gastrocnemius. These effects were associated with decreased expression of the ubiquitin-related E3 ligases, atrogin-1 and MuRF1, and likely mediated by ERK signaling to exert muscle-protecting function against cancer cachexia [bib_ref] Dietary Supplementation with Isoflavones Prevents Muscle Wasting in Tumor-Bearing Mice, Hirasaka [/bib_ref]. Soy isoflavones have also been widely discussed in the context of breast cancer, since they are plant-derived substances that activate signaling via estrogen receptors [bib_ref] Genistein modulates the anti-tumor activity of cisplatin in MCF-7 breast and HT-29..., Hu [/bib_ref]. However, the benefit of soy isoflavones for breast cancer patients has been controversial [bib_ref] Soy and isoflavones consumption and breast cancer survival and recurrence: A systematic..., Qiu [/bib_ref]. In the MCF-7 breast cancer cell line, high dose (100 µM) of genistein combined with the chemotherapeutic agent, cisplatin, suppressed breast cancer cell growth and proliferation, whereas 10 µM of genistein antagonized the action of cisplatin to induce cancer cell apoptosis [bib_ref] Genistein modulates the anti-tumor activity of cisplatin in MCF-7 breast and HT-29..., Hu [/bib_ref]. Further investigation confirmed that oral genistein supplementation (5 mg/kg/day) for three weeks counteracted cisplatin chemotherapy in breast cancer-bearing mice [bib_ref] Genistein interferes with antitumor effects of cisplatin in an ovariectomized breast cancer..., Ma [/bib_ref]. The anti-cancer effect of these soy isoflavones (genistein in particular) therefore remains controversial due to insufficient evidence. Such conflict raises doubt about the potential of these soy isoflavones to attenuate cancer-induced muscle atrophy. Future investigation is warranted to address these concerns.
Morin is a type of flavonoid, found in plants including Moraceae, Malpighiaceae, Myrtaceae, almond hulls and seaweeds [bib_ref] Morin suppresses cachexia-induced muscle wasti.ing by binding to ribosomal protein S10 in..., Yoshimura [/bib_ref]. Supplementation with a morin-rich diet for three weeks reduced tumor weight and progression in LLC-tumor bearing mice, demonstrating a powerful anti-cancer effect [bib_ref] Morin suppresses cachexia-induced muscle wasti.ing by binding to ribosomal protein S10 in..., Yoshimura [/bib_ref]. In addition, morin has been shown to have anticachectic potential, by attenuating cancer-induced muscle wasting in these same mice [bib_ref] Morin suppresses cachexia-induced muscle wasti.ing by binding to ribosomal protein S10 in..., Yoshimura [/bib_ref]. In vitro studies revealed that morin suppressed cancer cell growth via decreasing protein synthesis [bib_ref] Morin suppresses cachexia-induced muscle wasti.ing by binding to ribosomal protein S10 in..., Yoshimura [/bib_ref]. In contrast, morin (10 µM) increased protein synthesis in C2C12 myotubes, and this was associated with increased cell viability [bib_ref] Morin suppresses cachexia-induced muscle wasti.ing by binding to ribosomal protein S10 in..., Yoshimura [/bib_ref]. Moreover, oral morin treatment (50 mg/kg/day) for 30 days reduced the damage caused by a single injection of cisplatin in Sprague-Dawley rats by ameliorating chemotherapeutic drug-induced oxidative stress and activating antioxidant signaling cascades in isolated renal mitochondria [bib_ref] Ameliorative effects of morin on cisplatin-induced toxicity in renal mitochondria isolated from..., Ijaz [/bib_ref]. These findings suggest a benefit for morin supplementation in reducing chemotherapy-induced cytotoxicity, which may also have positive effect on ameliorating chemotherapy-related oxidative stress in skeletal muscle. Further investigation is needed to evaluate the potential for morin to attenuate cancer cachexia.
## Other antioxidants with therapeutic potential for cancer cachexia
Given the etiology of cancer cachexia is multifactorial, it is likely that only targeting a single factor may be inadequate and that a multimodal approach is needed to confer benefits. Identifying the most effective strategies that improve food intake, enhance protein synthesis, reduce protein degradation and decrease inflammation, are needed before testing the safety and efficacy of their different combinations. Since oxidative stress is one of the main contributing factors favoring protein breakdown over synthesis, through mitochondrial dysfunction and dysregulation of autophagy, including a safe and effective antioxidant could be an important for combination therapies. To date, all of the antioxidants examined for treating cancer cachexia are pharmacologically similar in their anti-inflammatory, anti-cancer and antioxidative properties. Thus, it is important to also investigate other antioxidants with similar pharmacological properties to increase the feasibility and efficacy of combination approaches.
## Ursolic acid
Ursolic acid (UA) is a natural pentacyclic triterpenoid carboxylic acid commonly found in some herbs and fruit wax, such as apples, pears, prunes and other fruits [bib_ref] Ursolic acid in cancer prevention and treatment: Molecular targets, pharmacokinetics and clinical..., Shanmugam [/bib_ref] , which has antioxidant, anti-inflammatory and anticancer properties [bib_ref] Ursolic acid supplementation decreases markers of skeletal muscle damage during resistance training..., Bang [/bib_ref]. As a plantderived antioxidant, the anti-cancer effect of UA has been well characterized, with antiinflammatory and chemoprotective effects demonstrated in various cancer types in cell (in vitro) and rodent (in vivo) models [bib_ref] Ursolic acid in cancer prevention and treatment: Molecular targets, pharmacokinetics and clinical..., Shanmugam [/bib_ref]. The anti-cancer and anti-inflammatory effects of UA are mediated via various signaling cascades, including the signal transducer and activator of transcription 3 (STAT3) and NF-κB signaling pathways [bib_ref] Ursolic acid inhibits multiple cell survival pathways leading to suppression of growth..., Shanmugam [/bib_ref] and the Akt/p70S6K signaling pathways [bib_ref] Ursolic acid inhibits colorectal cancer angiogenesis through suppression of multiple signaling pathways, Lin [/bib_ref] to suppress tumor growth, survival and metastases [bib_ref] Ursolic acid in cancer prevention and treatment: Molecular targets, pharmacokinetics and clinical..., Shanmugam [/bib_ref].
In addition to promising anti-cancer properties, UA was shown to improve skeletal muscle health in various pathologies [fig_ref] Table 2: Effects of Ursolic acid on skeletal muscle in vivo and in vitro [/fig_ref]. Acute (12 h) and short-term (three days) supplementation with UA attenuated muscle wasting induced by fasting [bib_ref] mRNA expression signatures of human skeletal muscle atrophy identify a natural compound..., Kunkel [/bib_ref] and hypobaric hypoxia, by promoting protein synthesis [bib_ref] Ursolic acid ameliorates hypobaric hypoxia-induced skeletal muscle protein loss via upregulating Akt..., Rathor [/bib_ref] , attenuating protein degradation [bib_ref] mRNA expression signatures of human skeletal muscle atrophy identify a natural compound..., Kunkel [/bib_ref] and reducing oxidative stress [bib_ref] Ursolic acid ameliorates hypobaric hypoxia-induced skeletal muscle protein loss via upregulating Akt..., Rathor [/bib_ref]. Longer UA treatment (5-6 weeks) improved muscle mass and function and increased slow and fast muscle fiber size without altering fiber type composition [bib_ref] mRNA expression signatures of human skeletal muscle atrophy identify a natural compound..., Kunkel [/bib_ref] [bib_ref] Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity,..., Kunkel [/bib_ref]. Moreover, oral UA supplementation (0.27% in food) for five weeks caused muscle hypertrophy and improved muscle function in healthy mice [bib_ref] mRNA expression signatures of human skeletal muscle atrophy identify a natural compound..., Kunkel [/bib_ref]. ↓-decreased and ↑-increased.
Oral UA supplementation (20 mg/kg/day) for three days to healthy rats increased body mass and muscle protein content, in the absence of changes in pro-inflammatory cytokines, ROS levels or oxidation markers [bib_ref] Ursolic acid ameliorates hypobaric hypoxia-induced skeletal muscle protein loss via upregulating Akt..., Rathor [/bib_ref]. While the beneficial effects of UA on muscle have been established, higher doses of UA (200 mg/kg, twice daily via intraperitoneal injection) for seven days reduced body mass and cellular energy stores in aging, despite improved muscle regeneration and a fast-to-oxidative fiber type shift [bib_ref] Short-term ursolic acid promotes skeletal muscle rejuvenation through enhancing of SIRT1 expression..., Bakhtiari [/bib_ref]. These discrepancies may be due to differences in methodology across studies, including treatment duration and route of administration and highlight the need for clarifying studies [fig_ref] Table 2: Effects of Ursolic acid on skeletal muscle in vivo and in vitro [/fig_ref].
The attenuation of atrophy by UA is proposed to occur via induction of insulin-like growth factor-1 (IGF-1) and activation of Akt/mTOR1 signaling [bib_ref] mRNA expression signatures of human skeletal muscle atrophy identify a natural compound..., Kunkel [/bib_ref] [bib_ref] Ursolic acid stimulates mTORC1 signaling after resistance exercise in rat skeletal muscle, Ogasawara [/bib_ref]. UA-mediated IGF-1 induction is localized to skeletal muscle, increasing muscle mass via Akt/mTOR signaling [bib_ref] Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity,..., Kunkel [/bib_ref] , evident from enhanced Akt and p70S6K phosphorylation and reduced glycogen synthase kinase 3β (GSK3β) expression [bib_ref] Ursolic acid ameliorates hypobaric hypoxia-induced skeletal muscle protein loss via upregulating Akt..., Rathor [/bib_ref] [bib_ref] Ursolic acid stimulates mTORC1 signaling after resistance exercise in rat skeletal muscle, Ogasawara [/bib_ref]. While short-term UA treatment was insufficient to increase muscle IGF-1 content, it was able to enhance resistance exercise-induced activation of Akt/mTOR signaling [bib_ref] Ursolic acid stimulates mTORC1 signaling after resistance exercise in rat skeletal muscle, Ogasawara [/bib_ref]. Longer-term UA treatment would be expected to increase IGF-1 levels and Akt phosphorylation, whereas acute UA treatment would likely to enhance Akt/mTOR signaling cascades. In clinical studies, UA demonstrated potential for attenuating resistance-exercise-induced muscle damage in a pilot study [bib_ref] Ursolic acid supplementation decreases markers of skeletal muscle damage during resistance training..., Bang [/bib_ref] , therefore supporting the therapeutic potential for UA to attenuate muscle wasting in cancer. The high bioavailability of UA [bib_ref] Ursolic acid in cancer prevention and treatment: Molecular targets, pharmacokinetics and clinical..., Shanmugam [/bib_ref] and its ability to sustain upregulated protein synthesis, make it a promising candidate for combination therapies.
## Sulforaphane
Sulforaphane (SFN) is a natural dietary isothiocyanate that was first identified and isolated from broccoli in 1992 [bib_ref] A major inducer of anticarcinogenic protective enzymes from broccoli: Isolation and elucidation..., Zhang [/bib_ref]. It is produced by the enzymatic action of myrosinase on glucopharanin, a relatively stable precursor found in cruciferous vegetables such as broccoli, Brussels sprouts and cabbage [bib_ref] Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect..., Fahey [/bib_ref] [bib_ref] Protective effect of sulforaphane against oxidative stress: Recent advances, Guerrero-Beltran [/bib_ref]. SFN is a potent inducer of detoxification enzymes with both chemoprotective and anti-carcinogenic potential [bib_ref] A major inducer of anticarcinogenic protective enzymes from broccoli: Isolation and elucidation..., Zhang [/bib_ref]. It induces several phase II enzymes including GST and glucuronosyltransferases, defining its antioxidative action [bib_ref] Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect..., Fahey [/bib_ref]. The expression of these enzymes continues throughout the duration of SFN administration and has been reported in various organs, indicating its systemic antioxidant response [bib_ref] The complexity of the Nrf2 pathway: Beyond the antioxidant response, Huang [/bib_ref]. In addition, SFN has an absolute bioavailability of around 80%, which is much higher than that of other polyphenols commonly used in dietary supplements [bib_ref] Absolute bioavailability and dosedependent pharmacokinetic behaviour of dietary doses of the chemopreventive..., Hanlon [/bib_ref] [bib_ref] Sulforaphane: Its "Coming of Age" as a Clinically Relevant Nutraceutical in the..., Houghton [/bib_ref]. Similar to curcumin, SFN is a major regulator of Nrf2 signaling [bib_ref] Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide..., Thimmulappa [/bib_ref] , enhancing nuclear translocation of Nrf2, where it binds the ARE to produce phase II antioxidant enzymes [bib_ref] Nrf2 targeting by sulforaphane: A potential therapy for cancer treatment, Russo [/bib_ref] [bib_ref] Deregulation of Nrf2/ARE signaling pathway causes susceptibility of dystrophin-deficient myotubes to menadione-induced..., Choi [/bib_ref]. SFN therefore plays an important role in the survival signaling pathway in the face of extensive oxidative stress resulting from cancer, muscle atrophy and chemotherapy [bib_ref] Keap1-nrf2 signaling: A target for cancer prevention by sulforaphane, Kensler [/bib_ref] [bib_ref] Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in..., Son [/bib_ref] [bib_ref] Sulforaphane alleviates muscular dystrophy in mdx mice by activation of Nrf2, Sun [/bib_ref]. Overall, SFN shows promising potential as a bioactive compound for treating toxicity related to oxidative stress, although further studies need to confirm its therapeutic potential.
SFN's ability to combat carcinogenesis was first proposed in the 1990s [bib_ref] Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect..., Fahey [/bib_ref] and it has more recently gained attention as a potential dietary supplement for treating cancer [bib_ref] Absolute bioavailability and dosedependent pharmacokinetic behaviour of dietary doses of the chemopreventive..., Hanlon [/bib_ref]. The chemoprotective effects of SFN are well described, with potential to treat cancers of the breast, prostate and lungs [bib_ref] Nrf2 targeting by sulforaphane: A potential therapy for cancer treatment, Russo [/bib_ref] [bib_ref] Sulforaphane inhibits the growth of KPL-1 human breast cancer cells in vitro..., Kanematsu [/bib_ref] [bib_ref] Sulforaphane suppresses EMT and metastasis in human lung cancer through miR-616-5p-mediated GSK3beta/beta-catenin..., Wang [/bib_ref] [bib_ref] Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic..., Zhang [/bib_ref]. SFN suppressed KPL-1 human breast cancer cell proliferation and induced apoptosis in a dose-dependent manner in in vitro and in vivo studies [bib_ref] Sulforaphane inhibits the growth of KPL-1 human breast cancer cells in vitro..., Kanematsu [/bib_ref] , and restored Nrf2 expression and downstream antioxidative enzymes in TRAMP prostate tumor-bearing mice [bib_ref] Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic..., Zhang [/bib_ref] [bib_ref] Gamma-tocopherol-enriched mixed tocopherol diet inhibits prostate carcinogenesis in TRAMP mice, Barve [/bib_ref]. While preliminary clinical data support the potential for SFN to treat prostate cancer [bib_ref] Transcriptional changes in prostate of men on active surveillance after a 12-mo..., Traka [/bib_ref] , further clinical trials with larger cohorts are required to more comprehensively determine SFN's therapeutic efficacy.
SFN has reported benefits for skeletal muscle atrophy [fig_ref] Table 3: Effects of SFN on skeletal muscle in vivo and in vitro [/fig_ref]. SFN attenuated dexamethasone-induced C2C12 myotube atrophy in vitro by inhibiting the increase in myostatin and atrogin-1 expression [bib_ref] Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in..., Son [/bib_ref]. Similarly, in porcine muscle stem cells (satellite cells), SFN inhibited myostatin gene expression while promoting an increase in mRNA expression of myostatin inhibitors, Smad7 and Smad-specific E3 ubiquitin protein ligase 1 (Smurf1) [bib_ref] Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells, Fan [/bib_ref]. These findings reveal a role for SFN in decreasing the rate of protein degradation via reducing E3 ligase-mediated ubiquitination in muscle stem cells and in differentiated myotubes. Furthermore, acute SFN pre-treatment increased muscle cell survival rate and myotube diameter and reduced intracellular ROS levels under menadioneinduced and starvation-induced oxidative stress [bib_ref] Deregulation of Nrf2/ARE signaling pathway causes susceptibility of dystrophin-deficient myotubes to menadione-induced..., Choi [/bib_ref] [bib_ref] Sulforaphane ameliorates serum starvation-induced muscle atrophy via activation of the Nrf2 pathway..., Moon [/bib_ref] , suggesting SFN can attenuate oxidative stress in C2C12 myotubes. SFN administration has also been shown to influence the regulation of skeletal muscle growth and regeneration [bib_ref] Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in..., Son [/bib_ref] [bib_ref] Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells, Fan [/bib_ref] [bib_ref] Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic..., Whitman [/bib_ref]. In C2C12 myoblasts, 4 h exposure to SFN induced Nrf2 activation, but inhibited or delayed myotube differentiation, evidenced by a decrease in MyoD, myogenin and myosin expression [bib_ref] Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic..., Whitman [/bib_ref]. In addition, siRNA-mediated reduction of Nrf2 revealed an inverse association between Nrf2 activation and the progression of myotube differentiation in C2C12 muscle cells [bib_ref] Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic..., Whitman [/bib_ref]. These findings are consistent with the alternate role of Nrf2 by inhibiting the initiation of cell growth [bib_ref] NRF2 and cancer: The good, the bad and the importance of context, Sporn [/bib_ref]. Similarly, administration of SFN to isolated muscle stem cells reduced MyoD mRNA expression and proliferation [bib_ref] Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells, Fan [/bib_ref]. There is considerable variability between studies investigating the potential benefits of SFN treatment on skeletal muscle in vitro because of methodological differences, including different SFN concentrations and treatment durations. While SFN has been shown to attenuate chemical-induced muscle damage [bib_ref] Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in..., Son [/bib_ref] [bib_ref] Sulforaphane ameliorates serum starvation-induced muscle atrophy via activation of the Nrf2 pathway..., Moon [/bib_ref] [bib_ref] Sulforaphane modulates CX3CL1/CX3CR1 axis and inflammation in palmitic acid-induced cell injury in..., Faridvand [/bib_ref] , the activation of Nrf2 after SFN treatment in healthy myoblasts [bib_ref] Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic..., Whitman [/bib_ref] and muscle stem cells [bib_ref] Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells, Fan [/bib_ref] is somewhat contradictory, as SFN treatment may delay myotube differentiation. This discrepancy may be attributed to the different cell types and growth stages being studied but identifying the effects of SFN on muscle regeneration and muscle stem cell proliferation is worthy of more comprehensive investigation. In animal models in vivo, SFN pre-treatment (25 mg/kg/day for three days) reduced muscle damage in rats caused by exhaustive exercise, evidenced by decreased levels of CK and LDH [bib_ref] Sulforaphane treatment protects skeletal muscle against damage induced by exhaustive exercise in..., Malaguti [/bib_ref] , and increased expression and activity of GST, GR and NQO1 in vastus lateralis muscles, linked to activation of Nrf2 signaling [bib_ref] Sulforaphane treatment protects skeletal muscle against damage induced by exhaustive exercise in..., Malaguti [/bib_ref]. Similarly, mice treated with SFN (25 mg/kg) four times prior to exercise had improved exercise endurance capacity by overcoming oxidative stress-induced muscle damage. Pre-treatment with SFN enhanced gene expression of antioxidants such as HMOX1, NQO1, γ-GCS and catalase in mouse gastrocnemius muscles, and protected against oxidative stress damage by reducing oxidative biomarkers including CK, LDH, GSH/GSSG ratio and TBARS. Whether SFN exerts its effects directly on skeletal muscle, or via systematic protection against oxidative stress, remains unresolved.
SFN has been reported to improve skeletal muscle pathology in mouse models of Duchenne muscular dystrophy (DMD) [bib_ref] Sulforaphane alleviates muscular dystrophy in mdx mice by activation of Nrf2, Sun [/bib_ref] [bib_ref] Sulforaphane Attenuates Muscle Inflammation in Dystrophin-deficient mdx Mice via NF-E2-related Factor 2..., Sun [/bib_ref] , type 2 diabetes [bib_ref] Sulforaphane protects against skeletal muscle dysfunction in spontaneous type 2 diabetic db/db..., Wang [/bib_ref] and sarcopenia [bib_ref] Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling, Bose [/bib_ref]. Both short-term (four weeks) and long-term (three months) treatment with SFN improved the dystrophic muscle pathology by reducing oxidative stress and inflammation [bib_ref] Sulforaphane mitigates muscle fibrosis in mdx mice via Nrf2-mediated inhibition of TGF-beta/Smad..., Sun [/bib_ref] [bib_ref] Sulforaphane alleviates muscular dystrophy in mdx mice by activation of Nrf2, Sun [/bib_ref]. SFN (0.5 mg/kg/day for one month) also attenuated muscle dysfunction related to type 2 diabetes, by increasing muscle mass and improving muscle function and structure in vivo [bib_ref] Sulforaphane protects against skeletal muscle dysfunction in spontaneous type 2 diabetic db/db..., Wang [/bib_ref]. A SFN-supplemented diet for 12 weeks, prolonged survival, increased muscle function and improved muscle regeneration in aged mice [bib_ref] Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling, Bose [/bib_ref]. While it is difficult to compare the effects of SFN across studies due to differences in routes of administration, the dose and treatment duration [fig_ref] Table 3: Effects of SFN on skeletal muscle in vivo and in vitro [/fig_ref] , taken together, these data support SFN as a potential treatment for cancer cachexia because of its anti-cancer effects and properties that confer muscle protection.
## Honokiol and magnolol
Honokiol and its isomer magnolol are two major components in Magnolia officinalis, a traditional Chinese herb [bib_ref] Phenolic constituents from the stem bark of Magnolia officinalis, Shen [/bib_ref]. Honokiol and magnolol are polyphenolic compounds with antioxidant and anti-inflammatory properties [bib_ref] Protective effect of magnolol against hydrogen peroxide-induced oxidative stress in human lens..., Yao [/bib_ref] [bib_ref] Anti-inflammatory effects of magnolol and honokiol are mediated through inhibition of the..., Lee [/bib_ref] shown to reduce LPS-induced inflammation and nitric oxide expression via inhibition of NF-κB signaling [bib_ref] Anti-inflammatory bioactivities of honokiol through inhibition of protein kinase C, mitogen-activated protein..., Chao [/bib_ref]. Magnolol has been shown to overcome hydrogen peroxide-induced oxidative stress in the human lens epithelial cell culture [bib_ref] Protective effect of magnolol against hydrogen peroxide-induced oxidative stress in human lens..., Yao [/bib_ref]. Magnolol can also activate detoxifying and antioxidative enzymes, including GST, in healthy ICR mice, further confirming magnolol's antioxidative actions [bib_ref] Anti-clastogenic effect of magnolol on benzo(a)pyrene-induced clastogenicity in mice, Saito [/bib_ref]. While honokiol and magnolol are less well studied antioxidants, they have been proposed to have anti-cancer functions, with in vitro and in vivo studies revealing inhibition of tumor-cell proliferation and growth, and induction of apoptosis [bib_ref] Therapeutic applications of compounds in the Magnolia family, Lee [/bib_ref].
In addition to these proposed anti-cancer functions, both honokiol and magnolol have reported beneficial functions in skeletal muscle. Oral administration of honokiol (1 mg/kg/day) for five consecutive days reduced muscle damage induced by eccentric exercise in adult male rats, reducing the production of proinflammatory cytokines, cyclooxygenase-2 and inducible nitric oxide synthase, via inhibition of NF-κB signaling [bib_ref] Honokiol protects rats against eccentric exercise-induced skeletal muscle damage by inhibiting NF-kappaB..., Chiang [/bib_ref]. Magnolol attenuated cancer-induced muscle wasting of C2C12 myotubes in vitro after exposure to C-26 cancer cell conditioned media. Myotube atrophy was attenuated through increased protein synthesis and reduced protein degradation in a dose-dependent manner, decreasing cancer-induced myostatin expression and subsequent downstream signaling [bib_ref] Magnolol inhibits myotube atrophy induced by cancer cachexia through myostatin signaling pathway..., Ge [/bib_ref]. In that study, magnolol was administered along with C-26 cancer cell conditioned media, at the beginning of differentiation, making it difficult to discern whether the treatment acted during differentiation or maturation. Magnolol has been shown to have beneficial effects on skeletal muscle in a mouse model of bladder cancer [bib_ref] Supplementation of Magnolol Attenuates Skeletal Muscle Atrophy in Bladder Cancer-Bearing Mice Undergoing..., Chen [/bib_ref] , with magnolol supplementation (1 mg/kg/day for three weeks via intraperitoneal injection) and chemotherapy significantly reducing cancer-induced weight loss and chemotherapyinduced anorexia [bib_ref] Supplementation of Magnolol Attenuates Skeletal Muscle Atrophy in Bladder Cancer-Bearing Mice Undergoing..., Chen [/bib_ref]. While the mass of the gastrocnemius muscle was not increased compared with the untreated tumor-bearing group, magnolol supplementation significantly reduced proteasomal activity, as well as myostatin and MuRF1 expression via IGF-1-regulated signaling cascades [bib_ref] Supplementation of Magnolol Attenuates Skeletal Muscle Atrophy in Bladder Cancer-Bearing Mice Undergoing..., Chen [/bib_ref]. Compared with the chemotherapy-only group, magnolol supplementation reduced production of proinflammatory cytokines, including TNF-α, IL-6 and IL-1β, via suppression of NF-κB activation [bib_ref] Supplementation of Magnolol Attenuates Skeletal Muscle Atrophy in Bladder Cancer-Bearing Mice Undergoing..., Chen [/bib_ref]. Therefore, magnolol may complement chemotherapy to achieve a greater benefit, highlighting the need for combination approaches to treat cancer cachexia. While there is evidence that honokiol and magnolol have anti-cancer and muscle protecting properties, these studies are preliminary and require confirmation.
## Pomegranate extract
Pomegranate extract (PE) contains high polyphenol contents derived from pomegranate juice, peel and seed oil of Punica granatum fruit, including pomegranate ellagitannins (punicalagin), ellagic acid and gallic acid [bib_ref] Pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-kappaB-dependent mechanism, Rettig [/bib_ref] [bib_ref] Cancer Chemoprevention by Pomegranate: Laboratory and Clinical Evidence, Adhami [/bib_ref] [bib_ref] Effect of pomegranate peel polyphenols on human prostate cancer PC-3 cells in..., Ma [/bib_ref]. Among these polyphenols, punicalagin is the most abundant bioactive ellagitannin of pomegranate fruit [bib_ref] Ellagic acid, pomegranate and prostate cancer-A mini review, Bell [/bib_ref]. PE has been widely discussed in cancer research as a potential nontoxic chemo-preventive dietary agent [bib_ref] Pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-kappaB-dependent mechanism, Rettig [/bib_ref] with its anti-cancer properties demonstrated through inhibiting proliferation of MCF-7 breast cancer cells in vitro [bib_ref] Antiproliferative effects of pomegranate extract in MCF-7 breast cancer cells are associated..., Shirode [/bib_ref]. In addition, PE has been suggested as a promising dietary treatment for the prevention of androgen-independent prostate cancer both in vitro and in vivo [bib_ref] Pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-kappaB-dependent mechanism, Rettig [/bib_ref]. However, clinical trials applying long-term PE supplementation for localized prostate cancer patents failed to demonstrate detectable improvement after the 18-month treatment [bib_ref] A randomized phase II study of pomegranate extract for men with rising..., Paller [/bib_ref]. While PE has anti-cancer and anti-inflammatory effects in lung, skin and colon cancer in preclinical in vitro and in vivo studies, there are limited clinical data and optimal dose and treatment duration have yet to be determined.
In addition to the potential anti-cancer effects of PE, studies have also suggested its ability to protect skeletal muscle. Oral supplementation with a commercial punicalagin-rich PE (Pomanox-P30) for six weeks alleviated TNF-α-induced activation of NF-κB signaling cascade, reduced cytokine induction and protected against skeletal muscle mass loss in mice with acute TNF-α-induced inflammation [bib_ref] Pomegranate extract prevents skeletal muscle of mice against wasting induced by acute..., Rodriguez [/bib_ref]. PE supplementation also reduced TNF-α-induced oxidative stress, as evidenced by reduced mRNA expression of nox2 and nox4, indicating reduced oxidation with PE treatment [bib_ref] Pomegranate extract prevents skeletal muscle of mice against wasting induced by acute..., Rodriguez [/bib_ref]. The study also suggested that PE pre-treatment maintained activity of the Akt/mTORC1 pathway, and limited activation of the ubiquitin proteasome pathway, indicating favoring of protein synthesis over degradation [bib_ref] Pomegranate extract prevents skeletal muscle of mice against wasting induced by acute..., Rodriguez [/bib_ref]. Therefore, while limited data are currently available, PE is proposed to have protective effects against the loss of muscle mass. Further studies need to confirm these proposed benefits of PE on skeletal muscle. PE has gained attention as a food supplement in sport science, with high-polyphenol containing pomegranate juice given twice daily to participants for seven days prior to intense eccentric exercise, found to enhance strength and delay muscle soreness [bib_ref] The effect of pomegranate juice supplementation on strength and soreness after eccentric..., Trombold [/bib_ref]. Several human trials have shown promising outcomes for PE in different exercise modalities by improving performance, enhancing power output and vascular oxygen content and accelerating muscle recovery. However, the mechanisms underlying these benefits of PE on skeletal muscle remain to be determined and studies should investigate these effects at the cellular level to confirm potential therapeutic benefit. Lastly, PE has also been shown to alleviate oxidative stress and inflammation induced by the chemotherapeutic 5-Fluorouracil (5-FU) in human immortal keratinocyte cells, suggesting the potential for PE to protect against the off-target effects of 5-FU chemotherapy [bib_ref] Protective Effect of Pomegranate on Oxidative Stress and Inflammatory Response Induced by..., Rapa [/bib_ref].
## Ellagic acid and urolithin a, b
Ellagic acid (EA) is a naturally occurring phenolic constituent that is a subgroup of ellagitannins found in many fruits and plants, including grapes, nuts, berries, green tea and pomegranates [bib_ref] Research progress on the anticarcinogenic actions and mechanisms of ellagic acid, Zhang [/bib_ref]. EA has significant chemo-preventive effects against different types of cancer, including breast, prostate, colon and skin cancer, and elicits anti-inflammatory and antioxidative effects in vitro and in vivo [bib_ref] Research progress on the anticarcinogenic actions and mechanisms of ellagic acid, Zhang [/bib_ref]. Pre-treatment with EA (100 mg/kg) daily for one week significantly reduced skeletal muscle damage induced by ischemia/reperfusion (I/R) and ameliorated I/R-induced oxidative stress by decreasing the level of MDA and inducing antioxidative enzymes, including SOD, catalase and GSH-Px in gastrocnemius muscles of Sprague-Dawley rats [bib_ref] A comparative study on the antioxidant effects of hesperidin and ellagic acid..., Ekinci Akdemir [/bib_ref]. Similarly, EA attenuated the loss of muscle mass in carbon tetrachloride-induced muscle wasting [bib_ref] The effect of ellagic acid on caspase-3/bcl-2/Nrf-2/NF-kB/TNF-α /COX-2 gene expression product apoptosis..., Aslan [/bib_ref]. In Wistar rats, intraperitoneal injection of EA (10 mg/kg, five times per week for eight weeks) decreased carbon tetrachlorideinduced muscle damage and oxidative stress by increasing expression of caspase-3 and Nrf2 while decreasing B-cell lymphoma 2 (bcl-2), NF-κB, TNF-α, MDA and cyclooxygenase-2 (COX-2) expression [bib_ref] The effect of ellagic acid on caspase-3/bcl-2/Nrf-2/NF-kB/TNF-α /COX-2 gene expression product apoptosis..., Aslan [/bib_ref]. Based on these preliminary findings, EA may have potential to attenuate cancer cachexia. However, discrepancies in methodology, including variations in dose, route of administration and duration of treatment, limit rigorous comparisons between these studies, and highlight the need for better controlled studies.
Urolithin A and B are the metabolites of EA or ellagitannin, with slight variations in phenolic hydroxylation patterns [bib_ref] Identification of urolithin a as a metabolite produced by human colon microflora..., Cerdá [/bib_ref]. Urolithins are normally produced by the microflora in the gastrointestinal tract after digestion of fruits containing high level of EA or ellagitannin [bib_ref] Identification of urolithin a as a metabolite produced by human colon microflora..., Cerdá [/bib_ref] [bib_ref] Urolithin B, a newly identified regulator of skeletal muscle mass, Rodriguez [/bib_ref]. Due to the low bioavailability and aqueous solubility of EA, it is likely that these colonic metabolites are the bioactive compounds responsible for the reported anti-inflammatory, antioxidative and anti-cancer effects of EA [bib_ref] Cacciotti, I. Strategies to improve ellagic acid bioavailability: From natural or semisynthetic..., Ceci [/bib_ref]. Urolithin A and B have reported anti-cancer effectswith additional benefits in skeletal muscle. Pre-treatment with urolithin A (15 µM) for 24 h attenuated TNF-α-induced inflammation and prevented activation of NF-κB signaling to protect wasting of C2C12 myotubes in vitro [bib_ref] Pomegranate extract prevents skeletal muscle of mice against wasting induced by acute..., Rodriguez [/bib_ref]. Urolithin A is a first-in-class activator of mitophagy [bib_ref] Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy, Luan [/bib_ref]. Since reduced mitophagy is one of the hallmarks of the pathology in DMD, urolithin A has been trialed in studies using the wellcharacterized mdx mouse model of DMD [bib_ref] Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy, Luan [/bib_ref]. Dietary supplementation with urolithin A (50 mg/kg) for 10 weeks restored disrupted mitophagy and mitochondrial respiratory capacity in mdx mice [bib_ref] Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy, Luan [/bib_ref]. In addition, urolithin A treatment enhanced myocellular quality by increasing expression of α-dystrobrevin and β-dystroglycan, the structural proteins required to restore muscle morphology in mdx mice, and it reduced the percentage of centrally nucleated fibers (a hallmark of regeneration), and increased muscle cross sectional area [bib_ref] Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy, Luan [/bib_ref]. Furthermore, 24 h treatment of urolithin B (15 µM) enhanced the growth and differentiation of C2C12 myotubes by increasing protein synthesis and attenuating protein degradation [bib_ref] Urolithin B, a newly identified regulator of skeletal muscle mass, Rodriguez [/bib_ref]. Administration of urolithin B (10 µg/day) via mini-osmotic pumps in C57BL/6 mice for three weeks, increased body mass and increased the mass of tibialis anterior, soleus and quadriceps muscles [bib_ref] Urolithin B, a newly identified regulator of skeletal muscle mass, Rodriguez [/bib_ref]. Furthermore, urolithin B (10 µg/day for seven days) attenuated denervation-induced muscle atrophy through suppression of FoxO1 and FoxO3, MAFbx, MuRF1 and myostatin expression [bib_ref] Urolithin B, a newly identified regulator of skeletal muscle mass, Rodriguez [/bib_ref]. Overall, ellagic acid, urolithin A and urolithin B are potent antioxidants with anti-cancer and muscle-protective effects. Their therapeutic potential for cancer cachexia requires further investigation.
## Other polyphenols
In addition to the polyphenols already discussed, evidence has emerged over the past 5-10 years of other polyphenol compounds with the potential to protect against cancer cachexia. Hesperidin is a natural occurring flavonoid with significant antioxidative properties, most abundant in citrus fruits, such as oranges, tangerines and lemons [bib_ref] Therapeutic potential of hesperidin and its aglycone hesperetin: Cell cycle regulation and..., Ferreira De Oliveira [/bib_ref]. It has demonstrated anti-cancer properties through attenuating the growth of breast, lung and colon cancer cells in vitro and in vivo, via induction of apoptosis, regulating cell cycle and reducing oxidative stress [bib_ref] Therapeutic potential of hesperidin and its aglycone hesperetin: Cell cycle regulation and..., Ferreira De Oliveira [/bib_ref]. The combination of hesperidin (50 mg/kg/day, orally) and doxorubicin for 16 days significantly reduced tumor size and mass, while overcoming chemo-resistance to doxorubicin in Ehrlich ascitic tumor-bearing mice [bib_ref] Effect of hesperidin on mice bearing Ehrlich solid carcinoma maintained on doxorubicin, Khedr [/bib_ref]. This highlights a potential role for hesperidin as a combination therapy for treating cancer. In addition to its anti-cancer functions, pre-treatment with hesperidin (100 mg/kg/day, orally), one week prior to ischemia/reperfusion (I/R) injury, protected skeletal muscle from subsequent I/R injury-induced oxidative stress through reductions in MDA and increased SOD expression in gastrocnemius muscles of Sprague-Dawley rats [bib_ref] A comparative study on the antioxidant effects of hesperidin and ellagic acid..., Ekinci Akdemir [/bib_ref]. Further investigation is required to determine its mechanism of action in skeletal muscle and the potential to ameliorate cancer-induced muscle wasting.
Pterostilbene is a bioactive polyphenol, found in grapes, wine and berries, which shares similar chemical properties with resveratrol but has greater in vivo bioavailability, suggesting better translational potential [bib_ref] SIRT1 activation by pterostilbene attenuates the skeletal muscle oxidative stress injury and..., Cheng [/bib_ref]. Similar to resveratrol, pterostilbene has chemo-protective and chemo-preventive properties, with demonstrated effects in various cancers, including breast, colon, prostate and liver, through activation of Nrf2 signaling and antioxidant activities, inhibition of STAT3 activity and induction of apoptosis [bib_ref] Pterostilbene in Cancer Therapy, Obrador [/bib_ref]. In vitro, pterostilbene (100 µM) inhibited cancer cell growth over time in chemo-resistant T24 cells, indicating pterostilbene may have potential in combination therapies to increase sensitivity of chemotherapy in cancer [bib_ref] Pterostilbene induces autophagy and apoptosis in sensitive and chemoresistant human bladder cancer..., Chen [/bib_ref]. In addition, oral pterostilbene supplementation (50 mg/kg/day) to Sprague-Dawley rats for four weeks promoted muscle adaptation and enhanced endurance capacity during exercise training by promoting slow-twitch fiber formation, increasing angiogenic factor expression and improving mitochondrial function [bib_ref] Pterostilbene Enhances Endurance Capacity via Promoting Skeletal Muscle Adaptations to Exercise Training..., Zheng [/bib_ref]. In a mouse model of collagen-VI-deficient of myopathy, pterostilbene (90 mg/kg/day, oral gavage for five days) restored autophagy in autophagy-deficient muscle, counteracting the major pathogenic marker of collagen-VI-related myopathies [bib_ref] The Polyphenol Pterostilbene Ameliorates the Myopathic Phenotype of Collagen VI Deficient Mice..., Metti [/bib_ref]. Lastly, pre-treatment with pterostilbene (2.5, 5 or 10 mg/kg/day, intraperitoneal injection) to Sprague-Dawley rats one week prior to I/R injury inhibited subsequent I/R injury-induced oxidative stress in a dose-dependent manner by reducing apoptosis and activating SIRT1-FoxO1/p53 signaling in skeletal muscle [bib_ref] SIRT1 activation by pterostilbene attenuates the skeletal muscle oxidative stress injury and..., Cheng [/bib_ref]. Based on the beneficial effects of pterostilbene on skeletal muscle and its anti-cancer properties, future studies should investigate the potential for pterostilbene to attenuate cancer-induced muscle wasting.
## Dual-function of antioxidants in cancer cachexia
Targeting oxidative stress has significant promise to treat cancer cachexia but conflicting findings question the safety of antioxidant therapy in some cancer populations. Administration of an antioxidant cocktail enriched in EGCG, curcumin and vitamin C, had no benefit in cancer cachexia and instead enhanced weight loss in tumor-bearing mice, resulting in premature death [bib_ref] Antioxidant supplementation accelerates cachexia development by promoting tumor growth in C26 tumor-bearing..., Assi [/bib_ref]. Individually, EGCG [bib_ref] Epigallocatechin-3-gallate effectively attenuates skeletal muscle atrophy caused by cancer cachexia, Wang [/bib_ref] and curcumin [bib_ref] Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16..., Siddiqui [/bib_ref] have been shown to counteract tumor growth and ameliorate cancer cachexia in vivo, but their combination failed to improve skeletal muscle atrophy and survival in the C-26 mouse model of cancer cachexia, likely due to the failure to reduce plasma TNF-α levels, systematic oxidative stress and ubiquitin-proteasome system activation [bib_ref] Antioxidant supplementation accelerates cachexia development by promoting tumor growth in C26 tumor-bearing..., Assi [/bib_ref]. In addition to the controversy surrounding the efficacy of antioxidants for cancer prevention, the potential for antioxidants to treat cancer cachexia remains equivocal. The dual effect of ROS may be responsible for this ambiguity since they can both promote proliferation and induce cancer cell death [bib_ref] ROS function in redox signaling and oxidative stress, Schieber [/bib_ref]. Cancer cells can influence levels of ROS and antioxidants to achieve a steady ROS state, avoiding oxidative-stress-induced cell apoptosis [bib_ref] ROS function in redox signaling and oxidative stress, Schieber [/bib_ref] [bib_ref] Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes..., Nogueira [/bib_ref]. Therefore, antioxidant supplements could be either beneficial or deleterious depending on the cancer conditions. Reducing oxidative stress might accelerate or attenuate tumor growth depending on the tumor type and location [bib_ref] Antioxidant supplementation accelerates cachexia development by promoting tumor growth in C26 tumor-bearing..., Assi [/bib_ref].
The benefits of antioxidant supplements for treating cancer cachexia in humans remains inconclusive. Among different types of cancer, the benefits of antioxidants were limited to cachectic patients with reduced antioxidant activity and high level of ROS in the blood [bib_ref] Cachexia and oxidative stress in cancer: An innovative therapeutic management, Mantovani [/bib_ref]. These findings suggest that antioxidant therapies may be beneficial for attenuating cachexia in patients with antioxidant deficiencies [bib_ref] The Janus-Faced Role of Antioxidants in Cancer Cachexia: New Insights on the..., Assi [/bib_ref] but potentially harmful for patients with a normal redox balance [bib_ref] The Janus-Faced Role of Antioxidants in Cancer Cachexia: New Insights on the..., Assi [/bib_ref] [bib_ref] Cachexia and oxidative stress in cancer: An innovative therapeutic management, Mantovani [/bib_ref]. Therefore, the therapeutic application of antioxidants for cancer cachexia remains questionable because of these competing effects on ROS regulation. Given the contradictory data reported to date, it is important to better understand the mechanisms underlying the action of antioxidants and their regulation of tumor growth and muscle mass, particularly for different cancer types and disease stages. Furthermore, it is crucial that optimal models for specific cancers be identified to facilitate the most appropriate pre-clinical studies that can inform clinical translation.
# Conclusions
This review highlighted the potential for dietary antioxidants to attenuate cancerinduced muscle wasting, identifying those able to attenuate cancer-induced muscle wasting, some with specific anti-cancer effects and others with potential muscle protective functions that have yet to be tested in the context of cancer cachexia. Considering the balance between the positive and negative effects of antioxidants that have been identified so far, therapeutic application of antioxidants for cancer cachexia requires closer examination. Since cancer cachexia is a multifactorial syndrome, combinatorial treatments will likely be necessary. As antioxidants are commonly used as dietary supplements, their role and application in these multimodal treatments is worthy of further investigation.
[fig] Figure 2: Sulforaphane (SFN) activates the Nrf2 signaling pathway. Under normal conditions, Nr localizes to the cytoplasm, where its activity is suppressed by Keap1. When cells experience exte sive oxidative stress, Nrf2 is released from the Keap1 complex and translocates into the nucleu where it promotes antioxidant and detoxifying gene expression via the antioxidant respon [/fig]
[fig] Author: Contributions: Conceptualization, W.L., K.S., K.T.M. and G.S.L.; investigation, W.L.; resources, G.S.L.; writing-original draft preparation, W.L.; writing-review and editing, K.S., K.T.M. and G.S.L.; visualization, W.L., K.S., K.T.M. and G.S.L.; supervision, K.S., K.T.M., G.S.L. All authors have read and agreed to the published version of the manuscript. [/fig]
[table] Table 1 Table 1: Effect of treatment with polyphenols for cancer cachexia. Cont. [/table]
[table] Table 1: Cont. [/table]
[table] Table 2: Effects of Ursolic acid on skeletal muscle in vivo and in vitro. [/table]
[table] Table 3: Effects of SFN on skeletal muscle in vivo and in vitro. [/table]
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External validation of the NeuroImaging Radiological Interpretation System and Helsinki computed tomography score for mortality prediction in patients with traumatic brain injury treated in the intensive care unit: a Finnish intensive care consortium study
Background Admission computed tomography (CT) scoring systems can be used to objectively quantify the severity of traumatic brain injury (TBI) and aid in outcome prediction. We aimed to externally validate the NeuroImaging Radiological Interpretation System (NIRIS) and the Helsinki CT score. In addition, we compared the prognostic performance of the NIRIS and the Helsinki CT score to the Marshall CT classification and to a clinical model. Methods We conducted a retrospective multicenter observational study using the Finnish Intensive Care Consortium database. We included adult TBI patients admitted in four university hospital ICUs during 2003-2013. We analyzed the CT scans using the NIRIS and the Helsinki CT score and compared the results to 6-month mortality as the primary outcome. In addition, we created a clinical model (age, Glasgow Coma Scale score, Simplified Acute Physiology Score II, presence of severe comorbidity) and combined clinical and CT models to see the added predictive impact of radiological data to conventional clinical information. We measured model performance using area under curve (AUC), Nagelkerke's R 2 statistics, and the integrated discrimination improvement (IDI). Results A total of 3031 patients were included in the analysis. The 6-month mortality was 710 patients (23.4%). Of the CT models, the Helsinki CT displayed best discrimination (AUC 0.73 vs. 0.70 for NIRIS) and explanatory variation (Nagelkerke's R 2 0.20 vs. 0.15). The clinical model displayed an AUC of 0.86 (95% CI 0.84-0.87). All CT models increased the AUC of the clinical model by + 0.01 to 0.87 (95% CI 0.85-0.88) and the IDI by 0.01-0.03. Conclusion In patients with TBI treated in the ICU, the Helsinki CT score outperformed the NIRIS for 6-month mortality prediction. In isolation, CT models offered only moderate accuracy for outcome prediction and clinical variables outweighing the CT-based predictors in terms of predictive performance.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
# Introduction
Traumatic brain injury (TBI) is one of the most common causes of mortality among young persons [bib_ref] The impact of traumatic brain injuries: a global perspective, Hyder [/bib_ref] [bib_ref] Epidemiology of head injury, Jennett [/bib_ref]. In recent years, it has been identified as an increasing risk of mortality and morbidity among elderly as well [bib_ref] Changing patterns in the epidemiology of traumatic brain injury, Roozenbeek [/bib_ref]. Glasgow Coma Scale (GCS) has been traditionally used as a measure of TBI severity upon admission to hospital. GCS is easy and fast to assess; however, it does not give information on structural information on potential intracranial lesions.
As computed tomography (CT) has become widely available, several classifications and scoring systems have been developed for additional information on TBI prognosis. These include, e.g., Marshall CT classification [bib_ref] A new classification of head injury based on computerized tomography, Marshall [/bib_ref] , Rotterdam CT-based score [bib_ref] Prediction of outcome in traumatic brain injury with computed tomographic characteristics: a..., Maas [/bib_ref] , Helsinki CT score [bib_ref] Predicting outcome in traumatic brain injury: development of a novel computerized tomography..., Raj [/bib_ref] , and Stockholm CT score [bib_ref] Extended analysis of early computed tomography scans of traumatic brain injured patients..., Nelson [/bib_ref].
The practical use of CT scores is to give clinicians more quantitative and comparable tools to assess the severity of TBI and estimate need for operative treatment and prognosis. For research purposes, CT scores are used for injury severity standardization and comparison. Recently, a new CT score, NeuroImaging Radiological Interpretation System (NIRIS), was introduced [bib_ref] Neuroimaging radiological interpretation system for acute traumatic brain injury, Wintermark [/bib_ref]. The NIRIS consists of five categories ranging from 0 to 5, with an increasing intracranial injury load with an increasing number. NIRIS was developed to consolidate imaging findings into different categories of ordinal severity to inform specific patient management actions [bib_ref] Neuroimaging radiological interpretation system for acute traumatic brain injury, Wintermark [/bib_ref]. The NIRIS has been earlier validated against Marshall CT classification and Rotterdam CT score [bib_ref] Validation of the revised Neu-roImaging Radiological Interpretation System for acute traumatic brain..., Dewangan [/bib_ref] [bib_ref] Neuroimaging radiological interpretation system for acute traumatic brain injury, Wintermark [/bib_ref] [bib_ref] Validation of the Neu-roImaging Radiological Interpretation System for acute traumatic brain injury, Zhou [/bib_ref]. To our knowledge, NIRIS has not been validated nor compared to more granular Helsinki CT score.
In this study, we aimed to perform an external validation study of the NIRIS and to compare it with the Helsinki CT score and a clinical model for predicting 6-month mortality. We hypothesized that both CT scores would add predictive performance when compared just with clinical data and that the Helsinki CT score would outperform the NIRIS, as it is more granular. We also report performance statistics of the widely used Marshall CT classification system.
# Methods and materials
The ethics committee of Helsinki University Hospital
## Study design and population
We performed a multicenter retrospective observational study using data that were prospectively collected from the Finnish Intensive Care Consortium (FICC) database. The FICC database is a nationwide prospectively data-collecting database including all ICU-treated patients from the majority of all ICUs in Finland [bib_ref] Association of automated data collection and data completeness with outcomes of intensive..., Reinikainen [/bib_ref]. In Finland, all specialized tertiary intensive care of TBI patients is centralized to five tertiary ICUs. Four of these ICUs participate in the FICC covering approximately two-thirds of the population in Finland. From these four tertiary ICUs, we included all adult TBI patients (age ≥ 18 years) admitted from January 1, 2003, to December 31, 2013 (readmissions excluded). Patients were excluded if no primary CT scan was available, and if Glasgow Coma Scale (GCS) or pre-admission functional status was missing.
Six-month case fatality was used as endpoint for analysis (available for all Finnish citizens through the Finnish population registry).
## Ct assessment
All patients in the study had non-contrast CT scan taken at the admission to hospital. Patients with only post-operative CT scans, CT angiography, or MRI scans were excluded. All available CT images were classified according to the Marshall CT classification system, the Helsinki CT score, and the updated version of NIRIS [bib_ref] Validation of the Neu-roImaging Radiological Interpretation System for acute traumatic brain injury, Zhou [/bib_ref] by two authors (JV, RR). The CT classification systems are described in [fig_ref] Table 1: Description of Marshall CT classification, Helsinki CT score, and NIRIS methods Abbreviations [/fig_ref].
# Statistical analysis
The Marshall CT classification [bib_ref] A new classification of head injury based on computerized tomography, Marshall [/bib_ref] and the NIRIS [bib_ref] Neuroimaging radiological interpretation system for acute traumatic brain injury, Wintermark [/bib_ref] [bib_ref] Validation of the Neu-roImaging Radiological Interpretation System for acute traumatic brain injury, Zhou [/bib_ref] were treated as categorical variables, NIRIS being ordinal. The Helsinki CT score was originally constructed as an ordinal scale, but due to its many levels and numeric distribution, it can be treated as a numeric variable [bib_ref] Predicting outcome in traumatic brain injury: development of a novel computerized tomography..., Raj [/bib_ref].
We performed first-level customization of the CT scores by fitting a new logit function to the respective CT score [bib_ref] Impact of different customization strategies in the performance of a general severity..., Moreno [/bib_ref]. The Helsinki CT-based score (hereafter referred to as Helsinki CT score), Marshall CT-based classification (hereafter referred to as Marshall CT class), and NIRIS-based (hereafter referred to as NIRIS).
We created a clinical "base model" that included age, GCS score (worst measured GCS score during the first ICU-day or as the last reliable GCS for intubated and/or sedated patients), a modified Simplified Acute Physiology Score II (SAPS II, without the age, GCS score, and chronic comorbidity component), and the presence of a chronic comorbidity (according to the SAPS II and Acute Physiology and Chronic Evaluation [APACHE] II definitions). The SAPS II and APACHE scores were assessed during the first 24 h of ICU treatment. We separately added age, GCS score, and chronic comorbidities to give them more weight in our base model [bib_ref] Predicting six-month mortality of patients with traumatic brain injury: usefulness of common..., Raj [/bib_ref]. To the base model, the three different CT scores (NIRIS, Marshall CT classification, and Helsinki CT score) were separately added.
We assessed the individual CT scores and the combined base + CT scores by calculating the Nagelkerke's R 2 and the area under the receiver operating characteristics curve [bib_ref] A note on a general definition of the coefficient of determination, Nagelkerke [/bib_ref]. Nagelkerke's R 2 gives a value between 0 and 1 resembling explained variance, where the value 1 indicates a model that fully explains the outcome. The AUC values range from 0.5 to 1 with 0.5 indicating at the level of chance and 1 indicating a perfect model. We assessed the calibration by using the Hosmer-Lemeshow test for all models apart from NIRIS and Marshall, as these consist of less than 10 groups.
We compared AUCs between models using a DeLong test. We considered p-values under 0.05 statistically significant.
In addition to the AUC analysis, we calculated the integrated discrimination improvement (IDI) as the AUC might be more insensitive in model comparisons in which the baseline model has performed well [bib_ref] Interpreting incremental value of markers added to risk prediction models, Pencina [/bib_ref]. The IDI is a category free measure of the discrimination ability between two logistic regression prediction models. The IDI can be defined as the difference in discrimination slopes between two models one with, and the other without, the added variable. It can be estimated with the following equation: Î DI = p new,events −p old,events − p new,nonevents −p old,nonevents , where p new,events is the mean of the new model-based predicted probabilities of an event for those who develop events, p old,events is the corresponding quantity based on the old model, p new,nonevents is the mean of the new model-based predicted probabilities of an event for those who do not develop events, and p old,nonevents is the corresponding quantity based on the old model [bib_ref] Evaluating the added predictive ability of a new marker: from area under..., Pencina [/bib_ref]. Another representation of the IDI can be formulated with the following equation:
[formula] IDI = IS new − IS old −(IP new − IP old ) , [/formula]
where IS = ∫ sensitivity and IP = ∫ 1 − specificity and the subscripts "new" and "old" correspond to the new model and the old model, respectively [bib_ref] Evaluating the added predictive ability of a new marker: from area under..., Pencina [/bib_ref]. The IDI reflects the mean magnitude of the change in outcome probability with the addition of the new variable in the prediction model. It is the area between the curves (the old model and the new model) in a plot where on the y-axis are both sensitivity and 1-specificity, and on the x-axis is the calculated risk. The model performance is improved when the new model moves the reference curve of sensitivity toward the top-right corner and the reference curve of 1-specificity toward lower-left corner. Conversely, model performance is reduced when the new model moves the reference curve of sensitivity toward lower-left corner and the reference curve of 1-specificity toward top-right corner [bib_ref] New metrics for assessing diagnostic potential of candidate biomarkers, Pickering [/bib_ref] [bib_ref] Comparing risk scoring systems beyond the ROC paradigm in survival analysis, Uno [/bib_ref]. The IDI ranges theoretically from − 2 to 2 representing the overall risk discrimination improvement [bib_ref] Evaluating the added predictive ability of a new marker: from area under..., Pencina [/bib_ref] [bib_ref] Novel metrics for evaluating improvement in discrimination: net reclassification and integrated discrimination..., Pencina [/bib_ref] [bib_ref] New metrics for assessing diagnostic potential of candidate biomarkers, Pickering [/bib_ref] [bib_ref] Risk reclassification analysis investigating the added value of fatigue to sickness absence..., Roelen [/bib_ref].
# Results
Altogether, 3031 patients met the inclusion criteria [fig_ref] Figure 1: Study's patient flow chart [/fig_ref]. Patient characteristics are listed in [fig_ref] Table 2: Patient characteristics Abbreviations [/fig_ref]. The median patient age was 55 years, 78% were male, 90% were functionally independent prior to admission, and 8.3% suffered from significant comorbidity. Almost half (47%) had a GCS between 3 and 8 in the first 24 h or before intubation. Approximately one-third (33%) of the patients required operative treatment, 24% were ICP monitored, and majority of the patients (66%) were intubated and mechanically ventilated. Seven percent of patients died during ICU treatment, 13% died in hospital, and 23% in 6 months after the TBI.
The distribution of NIRIS categories, Marshall CT classes, and median Helsinki CT scores is shown in . NIRIS category 2 (43%) was the most frequent, followed by NIRIS category 4 (22%) and 3 (17%). The most frequent Marshall classes were EML/NEML (44%) and II (43%). The median Helsinki CT score was 2.0 (IQR 2.0-4.0). NIRIS categories 0-1 correlated well with Marshall classes I and II. Most patients with a NIRIS category of II had a Marshall class of II. The majority of patients with a NIRIS category of 3 and 4 had a Marshall class indicating mass lesion (EML/NEML).
Non-survivors and patients with a low GCS score 3-8 in the first 24 h, or before intubation, more often belonged to a higher NIRIS category than survivors. The median age, frequency of mechanical ventilation, operative treatment, and ICP monitoring increased with a rising NIRIS category. When divided further into groups of NIRIS categories and respective GCS groups , the correlation between ICP monitoring and increasing NIRIS category was not as clear (Supplemental [fig_ref] Table 1: Description of Marshall CT classification, Helsinki CT score, and NIRIS methods Abbreviations [/fig_ref]. The 6-month mortality in patients with NIRIS categories 0, 1, 2, 3, and 4 was 8.4%, 5.3%, 17.0%, 27.0%, and 46.6%, respectively.
## Performance of the models
Of the CT models, Helsinki CT displayed best discrimination (AUC 0.73 vs. 0.70 for NIRIS vs. 0.68 for Marshall, and explained variance (Nagelkerke's R 2 0.20 vs. 0.15 for NIRIS vs. 0.14 for Marshall) [fig_ref] Table 5: Performance measures of the CT scores, base model, and combined models for... [/fig_ref].
The base model displayed an AUC of 0.86 (95% CI 0.84-0.87) with an explained variance of 0.43. The addition of all CT models increased the AUC by + 0.01 to 0.87 (p < 0.001, [fig_ref] Table 5: Performance measures of the CT scores, base model, and combined models for... [/fig_ref] , Note that some CT images had more than one exclusion criteria
# Discussion
## Key findings
In this large multicenter observational study, including 3031 patients from four academic centers in Finland, we compared three different CT scores to their ability to predict 6-month mortality in patients with TBI treated in the ICU. Of the three CT models in isolation, the Helsinki CT score displayed the best performance for 6-month mortality prediction. However, after adding the individual CT models to a clinical base model, only a moderate improvement in predictive performance predicting the 6-month mortality could be seen: AUC by + 0.01 to 0.87 (95% CI 0.85-0.88) and the IDI by 0.01-0.03, which is small, but statistically significant, and makes the AUC analysis more robust. Our results suggest that the choice of CT model when adjusting for case-mix in patients with TBI is of less importance than the adjustment of clinical variables such as age, GCS score, comorbidities, and acute physiological derangements (e.g., SAPS II).
## Comparison to previous studies
The NIRIS has been validated in a more general TBI population and compared to the Marshall CT classification and the Rotterdam CT score [bib_ref] Validation of the revised Neu-roImaging Radiological Interpretation System for acute traumatic brain..., Dewangan [/bib_ref] [bib_ref] Validation of the Neu-roImaging Radiological Interpretation System for acute traumatic brain injury, Zhou [/bib_ref]. Their results showed that the NIRIS performed similarly to the Marshall CT classification and the Rotterdam CT score in terms of predicting mortality, but markedly better in terms of discriminating the needed interventions and intensity of patient care. This is in line with the original aim of the NIRIS to predict TBI patient care based on initial imaging.
The study populations in both original article introducing the NIRIS [bib_ref] Neuroimaging radiological interpretation system for acute traumatic brain injury, Wintermark [/bib_ref] and in the validation article by Zhou et al., [bib_ref] Validation of the Neu-roImaging Radiological Interpretation System for acute traumatic brain injury, Zhou [/bib_ref] done in Stanford Hospital, USA, had a more general TBI population compared to our already ICU-admitted study population. Hence, our study population had more severe injuries and higher mortality: approx. 2% in earlier studies with the NIRIS compared to almost 13% during hospitalization in our study. This is also seen in the NIRIS categories as category 2 is the most common (43%) in our study population compared to category 0 in Zhou et al. [bib_ref] Validation of the Neu-roImaging Radiological Interpretation System for acute traumatic brain injury, Zhou [/bib_ref] (77%). Furthermore, categories 3 and 4 include 39% of our study population that leaves less than 20% for categories 0 and 1. A validation study [bib_ref] Validation of the revised Neu-roImaging Radiological Interpretation System for acute traumatic brain..., Dewangan [/bib_ref] of NIRIS conducted in India had a similar mortality rate in hospital (14%) than in our study; however, the patients were general TBI patients, not only ICUadmitted TBI patients.
In contrast to the NIRIS, the Helsinki CT score was developed for outcome prediction in patients with TBI treated in the ICU [bib_ref] Predicting outcome in traumatic brain injury: development of a novel computerized tomography..., Raj [/bib_ref]. The Helsinki CT score has been validated in pediatric TBI patients (AUC 0.84) [bib_ref] Validation of prognostic models in intensive care unit-treated pediatric traumatic brain injury..., Mikkonen [/bib_ref] , penetrating TBI patients (AUC 0.90) [bib_ref] Prognostic performance of computerized tomography scoring systems in civilian penetrating traumatic brain..., Lindfors [/bib_ref] , and adult TBI patients (AUC 0.70-0.81) [bib_ref] Predicting mortality after traumatic brain injury: prognostic model based on admission characteristics, Charry [/bib_ref] [bib_ref] Selection of CT variables and prognostic models for outcome prediction in patients..., Khaki [/bib_ref] [bib_ref] Evaluation of computed tomography scoring systems in the prediction of short-term mortality..., Rodrigues De Souza [/bib_ref] [bib_ref] Evaluation of novel computerized tomography scoring systems in human traumatic brain injury:..., Thelin [/bib_ref] [bib_ref] Helsinki computed tomography scoring system can independently predict long-term outcome in traumatic..., Yao [/bib_ref] with good performance. We found similar performance measures in the present cohort (AUC 0.73, Nagelkerke's R 2 0.20). Thus, due to differences in design and granularity, it is not surprising that the Helsinki CT score outperformed the NIRIS in terms of outcome prediction. Furthermore, in line with previous results, clinical variables seem to be more important predictors that CT predictors [bib_ref] Predicting six-month mortality of patients with traumatic brain injury: usefulness of common..., Raj [/bib_ref].
The clinical importance of early CT imaging in patients with significant TBI is undisputed. However, current CT models seem to be of limited additional prognostic value compared to clinical variables in terms of mortality prediction. There is some obvious selection bias to this, as all patients were admitted to the ICU. Thus, the association between variables such as midline shift and mass lesions is diluted since these may be, at least partly, reversible due to surgical treatment [bib_ref] Evaluation of novel computerized tomography scoring systems in human traumatic brain injury:..., Thelin [/bib_ref]. It is possible that the predictive performance of the current CT models could be improved by including spatial and volumetric parameters.
The SAPS II and APACHE scores are measured during the first 24 h of ICU treatment and thus contain more information than the CT models based upon admission characteristics. This will be in favor of the clinical model in terms of prognostic accuracy.
# Strengths and limitations
Some strengths should be highlighted. We used a large multicenter high-quality database collecting data prospectively. Thus, we were able to include more than three thousand patients in our study. In addition, there was a small number of missing data, and we had a complete 6-month follow-up. Our patient cohort also represents well the general ICUtreated TBI population in Finland as the referral population of the four neurointensive ICUs is approximately 3.5 million people, encompassing two-thirds of the Finnish population. Some limitations should be acknowledged. The FICC is a general ICU database and lacks some TBI-specific parameters, like specific neurosurgical procedures, admission GCS score, pupillary light reactivity, and thus information on IMPACT or CRASH models. The FICC database used in this study did not include data on such devastating injuries that the more aggressive treatment was withheld. Information regarding admission due to organ donation has been added later. Still, for case-mix adjustment, our base model displayed good statistical performance. Second, we only had all-cause mortality as an outcome measure. Predicting functional outcome would be desirable as well. Noteworthy, the Helsinki CT score was designed to predict outcome while Marshall CT and NIRIS were not. Thus, this might skew the results in favor of the Helsinki CT score when predicting outcome. Third, we highlight that we only included patients treated in a university hospital ICU and did not include milder TBIs. In this study, we did not include in comparisons the two other significant CT scores, namely, the Stockholm CT score and the Rotterdam CT score. Earlier, both of these scores have been validated against the Helsinki CT score [bib_ref] Evaluation of novel computerized tomography scoring systems in human traumatic brain injury:..., Thelin [/bib_ref] and the Rotterdam CT score against the NIRIS [bib_ref] Validation of the revised Neu-roImaging Radiological Interpretation System for acute traumatic brain..., Dewangan [/bib_ref] [bib_ref] Validation of the Neu-roImaging Radiological Interpretation System for acute traumatic brain injury, Zhou [/bib_ref]. The Stockholm CT score has shown to have superior predictive power, to some extent, over the Helsinki CT score and the Rotterdam CT score [bib_ref] Evaluation of novel computerized tomography scoring systems in human traumatic brain injury:..., Thelin [/bib_ref]. Further work should be done to compare the Stockholm CT score to the NIRIS.
# Conclusion
In patients with TBI treated in the ICU, the Helsinki CT score outperformed the NIRIS for 6-month mortality. However, clinical variables outweighed the current CT-based models in terms of predictive performance. Thus, accounting for clinical variables when adjusting for TBI injury severity is imperative.
## Supplementary information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00701-022-05353-0.
Author contribution Juho Vehviläinen: study design, manuscript preparation, data analysis, data interpretation. Markus Skrifvars: data acquisition, review and editing, data interpretation. Matti Reinikainen: data acquisition, review and editing, data interpretation. Stepani Bendel: data acquisition, review and editing, data interpretation. Tero Ala-Kokko: data acquisition, review and editing, data interpretation. Sanna Hoppu: data acquisition, review and editing, data interpretation. Ruut Laitio: data acquisition, review and editing, data interpretation. Jari Siironen: data acquisition, review and editing, data interpretation. Rahul Raj: study design, manuscript preparation, data analysis, review and editing, data interpretation.
Funding Open Access funding provided by University of Helsinki including Helsinki University Central Hospital. Independent funding support has been received from Helsinki University Hospital (State funding, Finland VTR TYH2018227); Finska Läkaresällskapet; Medicinska Understödsföreningen Liv & Hälsa; and Svenska Kulturfonden. The funders had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. The first and last author had full access to all the data in the study and had final responsibility for the decision to submit for publication.
# Declarations
## Consent to participate
There was no need for patient consent.
[fig] Figure 1: Study's patient flow chart. Abbreviations: CT = computed tomography; MRI = magnetic resonance imaging; GCS = Glasgow Coma Scale; FICC = Finnish Intensive Care Consortium; TBI = traumatic brain injury. [/fig]
[fig] 0. 0: (0.0, 0.0) 2.0 (0.0, 2.0) 2.0 (2.0, 4.0) 4.0 (2.0, 4.0) 5.0 (4.0, 9.0) positive (ranging from 0.011 to 0.028) for all CT models when they were added to the base model. [/fig]
[table] Table 1: Description of Marshall CT classification, Helsinki CT score, and NIRIS methods Abbreviations: EML, evacuated mass lesion; NEML, non-evacuated mass lesion; EDH, epidural haematoma; NIRIS, NeuroImaging Radiological Interpretation System; SDH, subdural haematoma; ICH, intracerebral haematoma (parenchymal); IVH, intraventricular hemorrhage; *The revised NIRIS definitions9 [/table]
[table] Table 2: Patient characteristics Abbreviations: IQR, interquartile range; LOS, length of stay; NIRIS, NeuroImaging Radiological Interpretation System. 1 A modified World Health Organization/Eastern Cooperative Oncology Group classification system implemented by the Finnish Intensive Care Consortium. 2 Any chronic comorbidity according to Acute Physiology and Chronic Health Evaluation II or Simplified Acute Physiology Score II. [/table]
[table] Table 5: Performance measures of the CT scores, base model, and combined models for predicting 6-month mortality * Based on age, GCS, chronic comorbidities, and modified SAPS II. ǂ AUC comparison using DeLong test to NIRIS for CT models and to Base model for Clinical + CT models. Abbreviations: AUC , area under curve; CT, computed tomography; GCS, Glasgow Coma Scale; IDI, integrated discrimination improvement; NIRIS, NeuroImaging Radiological Interpretation; SAPS, Simplified Acute Physiology Score. The Hosmer-Lemeshow test was not applicable for NIRIS and Marshall CT as these consist of less than 10 categories [/table]
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Perampanel in Brain Tumor-Related Epilepsy: A Systematic Review
Citation: Tabaee Damavandi, P.; Pasini, F.; Fanella, G.; Cereda, G.S.; Mainini, G.; DiFrancesco, J.C.; Trinka, E.; Lattanzi, S. Perampanel in Brain Tumor-Related Epilepsy: A Systematic Review. Brain Sci. 2023, 13, 326. https://doi.Abstract: Brain tumor-related epilepsy (BTRE) is a common comorbidity in patients with brain neoplasms and it may be either the first symptom or develop after the tumor diagnosis. Increasing evidence suggests that brain tumors and BTRE share common pathophysiological mechanisms. Glutamatergic mechanisms can play a central role in promoting both primary brain tumor growth and epileptogenesis. Perampanel (PER), which acts as a selective antagonist of glutamate α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, may play a role both in the reduction in tumor growth and the control of epileptiform activity. This systematic review aimed to summarize the pre-clinical and clinical evidence about the antitumor properties, antiseizure effects and tolerability of PER in BTRE. Eight pre-clinical and eight clinical studies were identified. The currently available evidence suggests that PER can be an effective and generally well-tolerated therapeutic option in patients with BTRE. In vitro studies demonstrated promising antitumor activity of PER, while no role in slowing tumor progression has been demonstrated in rat models; clinical data on the potential antitumor activity of PER are scarce. Additional studies are needed to explore further the effects of PER on tumor progression and fully characterize its potentialities in patients with BTRE.
# Introduction
Brain tumor-related epilepsy (BTRE) is common in patients with brain neoplasms, being the onset symptom in 15-30% of cases, and presenting later in the course of the disease in a further 20-45% of patients [bib_ref] Epilepsy in Patients with Brain Tumours: Epidemiology, Mechanisms, and Management, Van Breemen [/bib_ref] [bib_ref] Optimizing Antiepileptic Drug Treatment in Tumoral Epilepsy, Perucca [/bib_ref]. Brain tumor-related epilepsy severely impacts the morbidity and quality of life of affected people, yet its pathogenesis remains poorly understood. Of note, BTRE is drug-resistant in up to 15 to 25% of cases in high-grade gliomas, and choosing the best antiseizure medication (ASM) might be challenging in this more vulnerable population due to drug toxicity and interactions with antineoplastic agents [bib_ref] Tumor-Related Epilepsy: Epidemiology, Pathogenesis and Management, Chen [/bib_ref].
Brain neoplasms such as high-grade gliomas have few therapeutic options. The standard medical treatment for high-grade gliomas is concomitant chemotherapy with temozolomide and radiotherapy. Even if current therapeutic approaches can improve progression-free survival, the prognosis of patients with high-grade gliomas still remains poor with the 5-year survival rate rarely reaching 5%; innovative therapies are, hence, needed [bib_ref] Effects of Radiotherapy with Concomitant and Adjuvant Temozolomide versus Radiotherapy Alone on..., Stupp [/bib_ref]. There is evidence suggesting that brain tumors and tumor-associated epilepsy can share pathophysiological mechanisms: tumor growth can promote the generation of seizures which, in turn, can drive tumor progression. Glutamatergic mechanisms including the altered expression of glutamate transporters, excessive glutamate release and glutamate receptors activation, and increased extracellular glutamate concentrations can play a key role in promoting both primary brain tumor growth and epileptogenesis [bib_ref] Electrical and Synaptic Integration of Glioma into Neural Circuits, Venkatesh [/bib_ref].
In glioma cells, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for glutamate are often overexpressed. By activating oncogenic signaling cascades and cytoskeletal remodeling, glutamate stimulates autocrine and paracrine responses in glioma cells, which lead to tumor growth and invasion [bib_ref] Glutamate Release Promotes Growth of Malignant Gliomas, Takano [/bib_ref] [bib_ref] Glutamine Metabolism in Brain Tumors, Natarajan [/bib_ref]. While the pathophysiology of gliomas and cancer-associated epilepsy remains unclear, it may be speculated that AMPA receptor antagonists may have both anticonvulsant activity and antitumor effects. Furthermore, several cancer cell lines, including lung carcinoma, astrocytoma, neuroblastoma, and rhabdomyosarcoma/medulloblastoma, were observed to be more sensitive to conventional cytotoxic chemotherapy when AMPA receptors were blocked.
This insight into the role of glutamate in brain tumor growth has pointed to promising therapeutic options to treat tumor-related seizures and exert additional antitumor effects. In a phase II trial in patients with newly diagnosed glioblastoma, talampanel, an AMPA receptor antagonist, has been associated with longer survival when combined with temozolomide and radiotherapy compared to treatment with temozolomide and radiotherapy alone [bib_ref] Talampanel with Standard Radiation and Temozolomide in Patients with Newly Diagnosed Glioblastoma:..., Grossman [/bib_ref] ; these findings, however, have not been confirmed in a second phase II trial [bib_ref] Phase 2 Trial of Talampanel, a Glutamate Receptor Inhibitor, for Adults with..., Iwamoto [/bib_ref].
Perampanel (PER) is a non-competitive AMPA receptor antagonist with a five-fold longer half-life than talampanel and good blood-brain barrier penetration. The drug has been approved in Europe and the USA as an adjunctive treatment of focal seizures in patients aged ≥ 4 years (and as monotherapy in the USA), and as an adjunctive treatment of primary generalized tonic-clonic seizures associated with idiopathic generalized epilepsy in patients aged ≥ 12 years (and ≥7 years in Europe). Given its action as a selective antagonist of glutamate AMPA receptors and the role of altered glutamate homeostasis in the migration and invasion of brain tumor cells, PER may have a role both in the reduction in tumor growth and the control of epileptiform activity.
Here, we perform a systematic review of the pre-clinical and clinical evidence about the antitumor properties, the antiseizure effects, and the tolerability of PER in BTRE.
# Materials and methods
We performed a systematic literature search using MEDLINE (accessed by Pubmed), Embase, the Cochrane Central Register of Controlled Trials (CENTRAL), and the US National Institutes of Health Clinical Trials Registry (http://www.clinicaltrials.gov (accessed on 22 December 2022)) from inception to week one of October 2022 (update on week one of February 2023). The search strategy included "perampanel", "brain tumor" and "brain neoplasm" as keywords in different combinations using Boolean operators; no filters were applied. Duplicates, reviews, and articles in languages other than English were excluded. The risk of bias in any included clinical trial was assessed using the RoB 2 tool [bib_ref] RoB 2: A Revised Tool for Assessing Risk of Bias in Randomised..., Sterne [/bib_ref] , whilst it was not assessed individually for other study types (observational cohort studies and case series/case reports) that, instead, were considered at high risk of bias [bib_ref] Exploring the Evidence for Broad-Spectrum Effectiveness of Perampanel: A Systematic Review of..., Trinka [/bib_ref]. This systematic review is reported according to the recommendations of the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement [bib_ref] Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement, Moher [/bib_ref].
# Results
We identified 123 records through database and trial registers searching. After the removal of duplicates, we screened 97 records. Of these, 78 were excluded as they were considered ineligible (reviews, not related to the topic) and three were excluded because the full texts were not in any of the languages reported in the inclusion criteria. We assessed 16 reports for eligibility, which were subsequently included in the review [fig_ref] Figure 1: Flow diagram of study selection process [/fig_ref] : eight were pre-clinical and eight were clinical studies. The clinical studies included were obser-vational studies (four prospective, two retrospective) and two case series and were hence, considered at high risk of bias; no clinical trials were identified.
# Results
We identified 123 records through database and trial registers searching. After the removal of duplicates, we screened 97 records. Of these, 78 were excluded as they were considered ineligible (reviews, not related to the topic) and three were excluded because the full texts were not in any of the languages reported in the inclusion criteria. We assessed 16 reports for eligibility, which were subsequently included in the review [fig_ref] Figure 1: Flow diagram of study selection process [/fig_ref] : eight were pre-clinical and eight were clinical studies. The clinical studies included were observational studies (four prospective, two retrospective) and two case series and were hence, considered at high risk of bias; no clinical trials were identified.
## Pre-clinical studies
One of the first experimental studies available in the literature was performed by Cunningham and colleagues [bib_ref] Targeting Elevated Glutamate in Brain Tumour Related Epilepsy, Cunningham [/bib_ref] , and it showed that PER was able to block inter-ictal discharges in ex vivo human peritumoral brain slices.
Lange and colleagues [bib_ref] AMPA Receptor Antagonist Perampanel Affects Glioblastoma Cell Growth and Glutamate Release In..., Lange [/bib_ref] used patient-derived low-passage cell lines of glioblastoma and metastasis cells to study the biological and molecular effects of PER, levetiracetam, valproate, and carbamazepine on brain tumor cells. Perampanel was the only drug that showed antiproliferative effects on glioblastoma cell lines, whereas carbamazepine, valproate, and levetiracetam failed to decrease proliferation. The antiproliferative effects
## Pre-clinical studies
One of the first experimental studies available in the literature was performed by Cunningham and colleagues [bib_ref] Targeting Elevated Glutamate in Brain Tumour Related Epilepsy, Cunningham [/bib_ref] , and it showed that PER was able to block inter-ictal discharges in ex vivo human peritumoral brain slices.
Lange and colleagues [bib_ref] AMPA Receptor Antagonist Perampanel Affects Glioblastoma Cell Growth and Glutamate Release In..., Lange [/bib_ref] used patient-derived low-passage cell lines of glioblastoma and metastasis cells to study the biological and molecular effects of PER, levetiracetam, valproate, and carbamazepine on brain tumor cells. Perampanel was the only drug that showed antiproliferative effects on glioblastoma cell lines, whereas carbamazepine, valproate, and levetiracetam failed to decrease proliferation. The antiproliferative effects of PER were not explained by enhanced apoptosis, as shown by cell cycle analysis and caspase activity assay, but rather by its effect on cell metabolism, as evidenced by decreased glucose uptake in glioblastoma cells. In addition, PER decreased glutamate levels in all cell lines. To determine how PER could affect glutamate release and metabolism, a real-time PCR analysis was performed. Among the genes studied, PER showed a transcriptional effect in glioblastoma cells by increasing the expression of BCAT1 and GLUL. While BCAT1 increases intracellular glutamate levels, promoting glioma proliferation [bib_ref] BCAT1 Promotes Cell Proliferation through Amino Acid Catabolism in Gliomas Carrying Wild-Type..., Tönjes [/bib_ref] , GLUL converts glutamate to glutamine, decreasing cytosolic glutamate levels. Conversely, there were no transcriptional effects on gene expression in metastasis cell cultures.
Lai and colleaguesexamined the effects of PER on sodium currents in various cell types, including U87 glioma cells. According to this study, the peak and late components of voltage-gated Na+ currents (I Na ) in glioma cells decreased after exposure to 1 and 3 µM PER.
Salmaggi et al. [bib_ref] Synergistic Effect of Perampanel and Temozolomide in Human Glioma Cell Lines, Salmaggi [/bib_ref] evaluated the impact of PER alone or in combination with temozolomide on the growth of glioblastoma cell lines U87, U138, A172, and the grade III astrocytoma cell line SW1783. Consistent with the results of Lange [bib_ref] AMPA Receptor Antagonist Perampanel Affects Glioblastoma Cell Growth and Glutamate Release In..., Lange [/bib_ref] , PER showed antitumor activity in all cell lines, and the combination of PER and temozolomide had a significant synergistic effect, with the A172 line being the most sensitive. The antitumor activity was related to a pro-apoptotic effect which occurred to varying degrees in all four glioma cell lines and was more striking at higher doses of the drug. Perampanel has been also shown to upregulate the expression of several GluR subunits in both U87 and U138 cells.
Tatsuoka et al. [bib_ref] Anti-tumor Effects of Perampanel in Malignant Glioma Cells, Tatsuoka [/bib_ref] used six malignant glioma cell lines (A-172, AM-38, T98G, U-138MG, U-251MG, and YH-13) to assess the antitumor effect of PER. The drug showed inhibitory effects on cell viability in a dose-dependent manner on all cell lines at 72 h. Five of the six cell lines exhibited significantly reduced cell viability with 1.0 µM PER treatment. At this dosage, no significant change was observed in the proportions of cells in the G0/G1, S, and G2/M phases in the T98G and U-251MG lines, which indicated that the inhibitory effect may not be due to the accumulation of cells at specific cell cycle phases. On the other hand, the protein expression level of cleaved caspase-3 was markedly greater in the PER group compared with the control, suggesting a pro-apoptotic role of the drug. Of note, in the A-172, T98G, and U-251MG cell lines, the combination of temozolomide and PER demonstrated significant additive inhibitory effects on cell viability. The same research team investigated the antitumor effects of PER, carbamazepine, valproate, and levetiracetam at therapeutic blood concentrations on six cell lines [bib_ref] Anti-tumor effects of anti-epileptic drugs in malignant glioma cells, Yagi [/bib_ref]. Perampanel demonstrated significant inhibition of cell proliferation in all cell lines; the same effect was observed in three cell lines treated with carbamazepine, four cell lines treated with valproate, and two cell lines treated with levetiracetam [bib_ref] Anti-tumor effects of anti-epileptic drugs in malignant glioma cells, Yagi [/bib_ref]. The combined antitumor effects of temozolomide were further explored using T98G and U-251MG cell lines. The results confirmed the presence of such effects in both cell lines treated with PER, as well as in T98G cells treated with levetiracetam; no such effects were observed in cells treated with carbamazepine and valproate [bib_ref] Anti-tumor effects of anti-epileptic drugs in malignant glioma cells, Yagi [/bib_ref]. Additionally, PER suppressed cell migration in T98G and U-251MG cells acting on the expression of different genes involved in cell migration, adhesion, and infiltration [bib_ref] Anti-tumor effects of anti-epileptic drugs in malignant glioma cells, Yagi [/bib_ref].
Mayer and colleagues [bib_ref] Perampanel Attenuates Epileptiform Phenotype in C6 Glioma, Mayer [/bib_ref] were the first to study the effects of PER on glioma C6 cells both in vitro and in vivo. In their experiment, PER reduced glucose uptake in vitro without affecting extracellular glutamate levels. Additionally, PER prevented recurrent epileptiform discharges in brain slices from animals bearing C6 glioma, and these effects were shown to be AMPA-receptor dependent. In vivo, daily therapy with PER was associated with a trend toward reduction in tumor size, although without reaching statistical significance or affecting animal survival.
Lange et al. [bib_ref] Perampanel Add-on to Standard Radiochemotherapy in Vivo Promotes Neuroprotection in a Rodent..., Lange [/bib_ref] also assessed the neuroprotective potential of PER as an add-on treatment to standard radiochemotherapy in a rodent glioma model. First, they used orthotopically cultured F98 glioma cells in Fischer rats as a model of glioma progression with an epileptiform phenotype. While radiochemotherapy reduced tumor size, the addition of PER had no effect on tumor progression. However, when PER was co-administered with standard radiochemotherapy, glutamatergic network activity was maintained in healthy peritumoral tissue significantly more efficiently than standard radiochemotherapy or PER alone in rats bearing F98 glioma. Furthermore, PER showed a significant anticonvulsant effect regardless of whether it was co-administered with radiochemotherapy.
The main findings of the pre-clinical studies are summarized in [fig_ref] Table 1: Main characteristics and findings of pre-clinical studies [/fig_ref].
## Clinical studies
## Anti-seizure effects of perampanel
In all the included clinical studies, PER was given as an add-on ASM in adults with BTRE and uncontrolled seizures.
In the first study investigating the efficacy of PER in BTRE patients, Vecht and colleagues [bib_ref] Seizure Response to Perampanel in Drug-Resistant Epilepsy with Gliomas: Early Observations, Vecht [/bib_ref] reported an objective seizure response in nine out of 12 patients (75%), with a seizure frequency reduction greater than 50% in three patients and seizure freedom in six patients.
In a small case series of 12 patients, Izumoto et al. [bib_ref] Seizures and Tumor Progression in Glioma Patients with Uncontrollable Epilepsy Treated with..., Izumoto [/bib_ref] reported a ≥50% responder rate, defined as a reduction in seizure frequency ≥ 50%, in all the patients included, with six patients (60%) reaching seizure freedom; two patients discontinued PER treatment.
Dunn-Pirio and colleagues [bib_ref] Adjunctive Perampanel for Glioma-Associated Epilepsy, Dunn-Pirio [/bib_ref] conducted a single-arm prospective study on PER as an add-on treatment in patients with focal-onset glioma-associated seizures. Six out of eight subjects observed a benefit from adjunctive PER in terms of seizure reduction, even though no details about the entity of seizure reduction were provided and an unspecified improvement in seizure control was reported. The authors highlighted how most patients who had a decrease in seizure activity had IDH1 mutant gliomas. However, sampling error cannot be excluded because of the high rate of IDH1 mutant tumors in the study population.
Chonan et al. [bib_ref] Experience of Low Dose Perampanel to Add-on in Glioma Patients with Levetiracetam-Uncontrollable..., Chonan [/bib_ref] evaluated the efficacy of low-dose PER as the first add-on treatment in glioma patients who had uncontrolled seizures while receiving levetiracetam. Seventeen out of 18 patients became seizure-free at a median follow-up of 10.6 (range 1-21) months and a dose of 2-4 mg PER added to levetiracetam. The median time to achieve seizure freedom was 11 days (range 0-2 months). No significant difference was observed in PER efficacy according to the presence of IDH1 mutation.
In a small retrospective study, Maschio and colleagues [bib_ref] Perampanel in Patients with Brain Tumor-Related Epilepsy in Real-Life Clinical Practice: A..., Maschio [/bib_ref] demonstrated a ≥50% responder rate of 81.8% after 12 months of treatment with PER in BTRE patients with uncontrolled seizures. A higher responder rate was observed in IDH1-mutated compared to IDH1-wild-type patients.
Subsequently, the same group of researchconducted a small prospective pilot study in 26 patients with uncontrolled BTRE and found a ≥50% responder rate of 95.2% at Brain Sci. 2023, 13, 326 6 of 13 6 months. As opposed to previous results, no significant differences in seizure control were shown in patients with/without IDH1 mutation and with/without MGMT methylation.
The multicenter, observational PERADET studyreported a ≥50% responder rate of 90.4% at 12 months, with 33.3% of patients being seizure-free in the per-protocol population; in the intent-to-treat analysis, the responder rate was 66.6% at 12 months, with 25% of patients being seizure-free. Patients with IDH1 mutation and patients with MGMT methylation seemed to respond better to PER treatment; the molecular analysis, however, was not available in all included patients.
In a small case series about the efficacy of PER in BTRE patients by Heugenhauser et al. [bib_ref] Unterberger, I. Perampanel in Brain Tumor and SMART-Syndrome Related Epilepsy-A Single Institutional..., Heugenhauser [/bib_ref] , PER was effective to reduce seizure frequency in four of the five patients with uncontrolled BTRE; a ≥50% seizure frequency reduction was observed in two patients (40%). Interestingly, PER improved seizure control also in two patients with SMART (stroke-like migraine attacks after radiation therapy) syndrome.
The main findings about the antiseizure activity of PER in clinical studies are reported in [fig_ref] Table 2: Main characteristics of clinical studies and efficacy findings of perampanel in patients... [/fig_ref]. The responder rate was 40%, with 2 patients experiencing a ≥50% reduction in seizure frequency after add-on treatment with PER. One patient was seizure-free after 1 year Abbreviation: PER = perampanel.
## Antitumor effects of perampanel
Only one clinical study evaluated the potential antitumor effect of PER through MRI volume analysis of tumor lesions [bib_ref] Seizures and Tumor Progression in Glioma Patients with Uncontrollable Epilepsy Treated with..., Izumoto [/bib_ref]. Izumoto and colleagues assessed the efficacy of PER on seizures and tumor progression in 12 glioma patients with uncontrolled epilepsy. All the patients had a glial tumor type, presented recurrent seizure activity at the time of initiating PER, and had used at least one ASM before the treatment with PER. All patients with malignant glioma had been treated with the Stupp regimen before PER initiation. Tumor progression was assessed within 6 months with analysis of tumor volume and peritumoral edema on FLAIR MRI. Volumetric imaging analysis showed a decreased volume of hyperintense lesions in eight patients, suggesting an inhibitory effect of PER on tumor progression and/or peritumoral brain edema. Furthermore, volume reduction showed a correlation with the plasma concentration of PER. The results of this small study might have been affected by concurrent antitumor treatment: of note, six patients received temozolomide and three patients received bevacizumab along with PER.
## Tolerability and safety of perampanel
Across the clinical studies, PER was generally titrated slowly by starting at the dose of 2 mg once a day for 2-4 weeks and then increasing the dose by 2 mg every 2-4 weeks to reach seizure control and/or follow the study protocol. Dunn-Pirio et al. [bib_ref] Adjunctive Perampanel for Glioma-Associated Epilepsy, Dunn-Pirio [/bib_ref] originally proposed a fast-titration approach up-titrating PER by 2 mg every week; the study, however, required a protocol amendment and a 2-week up-titration schedule was considered after side effects (fatigue and dizziness) were experienced by four patients.
In the included studies, the overall prevalence of adverse effects in BTRE patients treated with PER was quite variable, ranging from 11% [bib_ref] Experience of Low Dose Perampanel to Add-on in Glioma Patients with Levetiracetam-Uncontrollable..., Chonan [/bib_ref] to 50% [bib_ref] Seizure Response to Perampanel in Drug-Resistant Epilepsy with Gliomas: Early Observations, Vecht [/bib_ref]. The most experienced adverse effects were dizziness/vertigo (18% of all patients treated with PER), fatigue (9%), aggressiveness/irritability/agitation (9%), and anxiety (5%). Other reported adverse effects were confusion, insomnia, nausea, and somnolence/drowsiness. Adverse effects of PER are generally reported to increase with increasing doses. A total of eight patients [bib_ref] Seizures and Tumor Progression in Glioma Patients with Uncontrollable Epilepsy Treated with..., Izumoto [/bib_ref] [bib_ref] Adjunctive Perampanel for Glioma-Associated Epilepsy, Dunn-Pirio [/bib_ref] [bib_ref] Perampanel in Patients with Brain Tumor-Related Epilepsy in Real-Life Clinical Practice: A..., Maschio [/bib_ref] required a down-titration of PER because of tolerability issues: adverse effects improved following the decrease in PER dosage, and all patients continued the treatment. In the included studies, the maximum daily dose of PER reached 12 mg, reported in four patients [bib_ref] Seizure Response to Perampanel in Drug-Resistant Epilepsy with Gliomas: Early Observations, Vecht [/bib_ref] : two patients experienced no adverse effects while for the other two, no data were available. Regarding the known risk of hepatotoxicity associated with PER, only Heugenhauser et al. [bib_ref] Unterberger, I. Perampanel in Brain Tumor and SMART-Syndrome Related Epilepsy-A Single Institutional..., Heugenhauser [/bib_ref] reported measuring laboratory hepatic parameters in their cohort, showing no relevant changes.
The main findings about the tolerability of PER in clinical studies are summarized in . . Main tolerability findings of perampanel in patients with brain tumor-related epilepsy.
## Study (year) patients with adverse effects (%) adverse effects (% of patients) discontinuation of per due to adverse effects (% of patients)
Vecht et al.
# Discussion
## Pre-clinical evidence
Pre-clinical studies suggest that epileptogenesis and tumor growth in gliomas share common pathophysiological mechanisms through glutamatergic pathways and the activa-tion of AMPA receptors, supporting the potential beneficial effects of glutamate receptor antagonists [bib_ref] Perampanel Add-on to Standard Radiochemotherapy in Vivo Promotes Neuroprotection in a Rodent..., Lange [/bib_ref] [fig_ref] Figure 2: Mechanism of action of perampanel and its potential role in antitumor growth [/fig_ref].
# Discussion
## Pre-clinical evidence
Pre-clinical studies suggest that epileptogenesis and tumor growth in gliomas share common pathophysiological mechanisms through glutamatergic pathways and the activation of AMPA receptors, supporting the potential beneficial effects of glutamate receptor antagonists [bib_ref] Perampanel Add-on to Standard Radiochemotherapy in Vivo Promotes Neuroprotection in a Rodent..., Lange [/bib_ref] [fig_ref] Figure 2: Mechanism of action of perampanel and its potential role in antitumor growth [/fig_ref]. Through the upregulation of glutamate receptors-including AMPA-and post-synaptic structural genes, glioma cells establish synaptic connections. The neuronal electrochemical signaling results in the release of glutamate in the synaptic cleft. Glutamate molecules bind the AMPA receptors and depolarize glioma cells, thus promoting tumor proliferation, invasiveness, and metastatic colonization. Perampanel non-competitively binds AMPA receptors, inhibiting the sodium and potassium cytosolic influx and blocking the depolarization of both neuronal and glioma cells.
The pre-clinical studies provided preliminary evidence that PER may have anti-tumor potentialities. Several in vitro studies demonstrated that PER exhibits antitumor activity, which is increased in the presence of temozolomide in some of the malignant glioma cell lines. The mechanisms by which this activity is mediated are not clear yet. According to Lange's study [bib_ref] AMPA Receptor Antagonist Perampanel Affects Glioblastoma Cell Growth and Glutamate Release In..., Lange [/bib_ref] , PER acts on cell metabolism by decreasing glucose uptake in glioblastoma cells, without causing apoptosis. Conversely, a pro-apoptotic effect has been demonstrated in the studies by Salmaggi and Tatsuoka [bib_ref] Synergistic Effect of Perampanel and Temozolomide in Human Glioma Cell Lines, Salmaggi [/bib_ref] [bib_ref] Anti-tumor Effects of Perampanel in Malignant Glioma Cells, Tatsuoka [/bib_ref]. The discrepancy between these studies regarding the presence of pro-apoptotic activity can be explained by the different methods used to detect it. In the study by Salmaggi et al., PER upregulated the expression of several GluR subunits in two different glioma cell lines. The modulation of GluR subunits could decrease the permeability of glioma cells to calcium, leading to the inhibition of cell migration and the induction of apoptotic cell death [bib_ref] Blockage of Ca(2+)-Permeable AMPA Receptors Suppresses Migration and Induces Apoptosis in Human..., Ishiuchi [/bib_ref]. Of note, in the study by Tatsuoka et al., the pro-apoptotic effect was obtained with concentrations lower than those employed by Salmaggi et al. Further research is needed to better understand how apoptosis is affected; interestingly, the role of PER on the expression of caspase-8, caspase-9, and poly(ADP-ribose) polymerase (PARP) or in the presence of caspase inhibitors has not been investigated yet [bib_ref] Anti-tumor Effects of Perampanel in Malignant Glioma Cells, Tatsuoka [/bib_ref]. Tatsuoka et al. also demonstrated that the combination of PER with tiplaxtinin further decreased cell viability in cells with high expression levels of SERPINE1 that were resistant to PER. Tiplaxtinin inhibits SERPINE1, a serine protease inhibitor that has been proposed as a factor for tumor migration and invasion in several types of cancer, including gliomas [bib_ref] Identification of SERPINE1 as a Regulator of Glioblastoma Cell Dispersal with Transcriptome..., Seker [/bib_ref]. These findings overall suggest that the antitumor effect of PER may be diminished in malignant gliomas with higher expression levels of SERPINE1. Interestingly, showed that the antitumor effect of PER can result not only from the inhibition of cell proliferation but also from the inhibition of cell migration: PER reduced the expression of Rac1, RhoA, and N-cadherin, which are involved in promoting cell motility, and increased the expression of E-cadherin, which has opposing effects. [bib_ref] Anti-tumor effects of anti-epileptic drugs in malignant glioma cells, Yagi [/bib_ref]. The mechanisms underlying the inhibitory effect on cell growth exerted by other ASMs were not examined in detail [bib_ref] Anti-tumor effects of anti-epileptic drugs in malignant glioma cells, Yagi [/bib_ref].
In addition, in the study by Lai and colleagues, PER suppressed voltage-gated Na+ currents in glioma cells. It is known that gliomas are characterized by the differential expression of voltage-gated sodium channel subtypes, which determine the expressed I Na phenotype [bib_ref] Molecular Characterization of Voltage-Gated Sodium Channels in Human Gliomas, Schrey [/bib_ref]. A few studies have shown that some agents acting as inhibitors of voltagegated sodium channels can prolong the survival of patients with malignant gliomas [bib_ref] Sodium Ion Channel Mutations in Glioblastoma Patients Correlate with Shorter Survival, Joshi [/bib_ref] [bib_ref] Impact of Particular Antiepileptic Drugs on the Survival of Patients with Glioblastoma..., Guthrie [/bib_ref]. Further studies are needed to determine whether the blocking effect of PER on the transient and persistent components of I Na can have a synergistic and beneficial role in patients with BTRE.
While several studies have demonstrated the antitumor activity of PER in vitro, no role of the drug in slowing tumor progression has been demonstrated in rat models so far. However, in the study by Lange et al. [bib_ref] Perampanel Add-on to Standard Radiochemotherapy in Vivo Promotes Neuroprotection in a Rodent..., Lange [/bib_ref] PER showed a potential neuroprotective role when co-administered with standard radiochemotherapy since it preserved the glutamatergic network activity in healthy peritumoral tissue. Additionally, in the study conducted by Mayer et al. [bib_ref] Perampanel Attenuates Epileptiform Phenotype in C6 Glioma, Mayer [/bib_ref] on a C6 glioma rodent model, the lack of tumor inhibitory effect of PER could be related to a too-low administered dose. Moreover, while C6 cells exhibit moderate amounts of AMPA receptors in vitro [bib_ref] Ca 2+ -Permeable AMPA Receptors Regulate Growth of Human Glioblastoma via Akt..., Ishiuchi [/bib_ref] , it is not yet known whether the glutamatergic pathway plays a role in tumor progression in vivo. Given the limited available evidence, more research and in vivo experiments with rodents are needed to investigate the possible antitumoral properties of PER. Similarly, more studies are warranted to investigate how PER works as an anticonvulsant agent in the long-term in rodents and the side effects at different therapeutic doses.
## Clinical evidence
Since the publication of an anecdotal case report in 2015 [bib_ref] Perampanel in the Treatment of a Patient with Glioblastoma Multiforme without IDH1..., Rösche [/bib_ref] in a patient with IDH-1 wild-type glioblastoma-associated epilepsy, an increasing number of observational small studies exploring the efficacy of PER in BTRE have been published.
In all the clinical studies where the seizure baseline was calculated, PER given as add-on treatment in BTRE patients has been shown to be highly efficacious in reducing seizure frequency, with a cumulative ≥50% responder rate of 86.4% (89 out of 103 patients). With the limits of data interpretation due to the differences in the lengths of follow-up ranging from 1 to 21 months, seizure freedom was achieved by 45% (50 out of 111) of all patients.
Interestingly, recent studies have shed light on the potential role of tumor molecular markers, such as IDH1 mutation and MGMT methylation in seizure occurrence in patients with gliomas [bib_ref] Potential Influence of IDH1 Mutation and MGMT Gene Promoter Methylation on Glioma-Related..., Feyissa [/bib_ref]. More specifically, the mutated form of IDH1 generates 2-hydroxyglutarate (2-HG), which is an oncometabolite and structurally resembles glutamate; it has been hypothesized that 2-HG may promote seizures by functioning as a glutamate receptor agonist. However, although promising, clinical evidence on this topic is contrasting, with three studies [bib_ref] Adjunctive Perampanel for Glioma-Associated Epilepsy, Dunn-Pirio [/bib_ref] [bib_ref] Perampanel in Patients with Brain Tumor-Related Epilepsy in Real-Life Clinical Practice: A..., Maschio [/bib_ref] suggesting better seizure control with PER in BTRE patients with IDH1 mutation and MGMT methylation, and two studies pointing out no significant difference between the two molecular signatures. Additionally, the PER antitumor effect, hypothesized in several in vitro studies, has been studied in only one clinical study [bib_ref] Seizures and Tumor Progression in Glioma Patients with Uncontrollable Epilepsy Treated with..., Izumoto [/bib_ref]. Future clinical trials and observational studies involving larger cohorts of patients are strongly warranted to better explore these interesting and promising relationships.
Add-on therapy with PER in patients with BTRE was generally well tolerated, and the rate of treatment discontinuation due to adverse events across the studies was 6.3% (eight out of 128 patients). Regarding the titration scheme, the overall low incidence of adverse effects reported with a 2-week up-titration suggests that a slow titration may represent a good strategy to minimize the risk of side effects. Shortcomings in the interpretation of findings related to tolerability included the variable and sometimes short lengths of the follow-up, and the ascertainment of adverse events occurrence through patients' subjective reports in almost all studies; the study by Maschio et al.was the only one to propose a more accurate self-report multi-item questionnaire to collect adverse events. Importantly, the PERADET study-the only multicentric and prospective study available so far-evaluated the quality of life of patients with BTRE before and during the treatment with PER, and showed no changes; these findings further supported the overall good tolerability of PER. Additional issues need to be acknowledged for evaluating the tolerability of ASMs in BTRE. First, the adverse effects of ASMs are known to be more frequent in patients with BTRE, and the adverse effects of anticancer therapies could heavily influence their tolerability, exacerbating or even triggering their unwanted effects (e.g., nausea) [bib_ref] Optimizing Antiepileptic Drug Treatment in Tumoral Epilepsy, Perucca [/bib_ref]. Moreover, tumor progression may act as an important confounding factor, potentially being the physical and/or psychological contributor to some of the adverse effects attributed to ASMs (e.g., headache, dizziness, anxiety). Reasonably, an ASM can be considered responsible for the adverse effects when they occur at the initiation of the therapy, after an increase in drug dosage, or when they disappear after discontinuing treatment.
Selecting the appropriate ASM in patients with BTRE is challenging and several issues need to be considered, such as the risk of drug-drug interactions, concurrent antitumor treatments, the cognitive status of patients and other neurological symptoms. Levetiracetam has been suggested to be the preferred choice as first-line treatment in patients with BTRE [bib_ref] First-line antiepileptic drug treatment in glioma patients with epilepsy: Levetiracetam vs valproic..., Van Der Meer [/bib_ref] [bib_ref] Management of epilepsy in brain tumor patients, Van Der Meer [/bib_ref] ; in case of poor seizure control with monotherapy, potential add-on ASMs include lacosamide, perampanel, and valproic acid [bib_ref] Management of epilepsy in brain tumor patients, Van Der Meer [/bib_ref]. First-generation ASMs (e.g., carbamazepine, phenobarbital, phenytoin and valproic acid) are well known to cause drug-drug interactions: chemotherapeutic agents can reduce the concentration of firstgeneration ASMs and these ASMs can reduce the activity of chemotherapeutic agents. Moreover, phenytoin and phenobarbital shorten the half-life and increase the total body clearance of dexamethasone and prednisone [bib_ref] Interactions between antiepileptic and chemotherapeutic drugs, Vecht [/bib_ref]. Valproic acid, an enzyme inhibitor, can increase the toxicity of cisplatin, etoposide and nitrosoureas, and it has been also associated with coagulopathy, especially thrombocytopenia, which may worsen in combination with chemotherapy [bib_ref] Assessment of need for hemostatic evaluation in patients taking valproic acid: A..., Post [/bib_ref]. Because of extensive hepatic metabolism, PER could be prone to interactions with cytochrome P450 substrates. In fact, it is known that the concomitant use of known moderate or strong CYP3A4 inducers, such as carbamazepine, phenytoin, and oxcarbazepine, can decrease the plasma levels of PER by approximately 50% to 67%. In contrast, PER itself does not seem to show significant enzyme inhibition or induction [bib_ref] Impact of perampanel on pharmacokinetics of concomitant antiepileptics in patients with partial-onset..., Majid [/bib_ref]. So far, drug interactions leading to a worsening of the tolerability of PER in BTRE have not been reported.
# Conclusions
Currently available evidence, despite being based on a few non-randomized, noncontrolled studies characterized by small sample sizes and open to high risk of bias, suggests the role of PER as a viable and effective therapeutic option to improve seizure control in patients with BTRE. Although present and not negligible, adverse effects are generally of modest impact and do not greatly impact treatment retention. In vitro studies indicated promising antitumor properties of PER, while clinical data on its potential antitumor activity are still scarce. Additional controlled trials and observational studies in larger cohorts are needed to further explore the effects of PER on tumor progression and fully characterize its potentialities in patients with BTRE. To date, one phase I-II, interventional, open-label, pilot trial is ongoing to measure the effect of PER on peritumoral hyperexcitability using intraoperative electrocorticography at the time of initial glioma resection as well as seizure control in patients with newly diagnosed high-grade gliomas compared to standard of care treatment. A phase IV clinical trial in adult patients with biopsy-proven highgrade glioma and focal epilepsy is also ongoing to evaluate the efficacy and safety of PER compared with alternate ASMs, assess the change in neurocognitive function and brain magnetic resonance imaging progression over the course of PER treatment, and identify a biomarker-specific response to seizure-reduction. Data Availability Statement: Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.
[fig] Figure 1: Flow diagram of study selection process. [/fig]
[fig] Figure 2: Mechanism of action of perampanel and its potential role in antitumor growth. Through the upregulation of glutamate receptors-including AMPA-and post-synaptic structural genes, glioma cells establish synaptic connections. The neuronal electrochemical signaling results in the release of glutamate in the synaptic cleft. Glutamate [/fig]
[fig] Author: Contributions: Conceptualization, G.S.C.; methodology, P.T.D. and F.P.; data curation, P.T.D. and F.P.; writing-original draft preparation, P.T.D., F.P., G.F., G.S.C., G.M. and S.L.; writing-review and editing, P.T.D., F.P., G.F., J.C.D., E.T. and S.L.; supervision, S.L. All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors. Informed Consent Statement: Not applicable. This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors. [/fig]
[table] Table 1: Main characteristics and findings of pre-clinical studies.PER showed systematic inhibitory effects on cell proliferation in patient-derived low-passage cell lines of glioblastoma. Metastasis cells were more resistant to PER.Glucose uptake was attenuated in all glioblastoma cells after exposure to PER, whereas apoptosis was not inducedLai et al. (2019) In vitro PER suppressed voltage-gated Na+ currents in U87 glioma cells PER reduced glucose uptake in vitro without affecting extracellular glutamate levels. PER prevented recurrent epileptiform discharges in brain slices from rats bearing C6 glioma. PER did not reduce tumor size PER showed anticonvulsant properties in rodent F98 glioma model. PER as an add-on treatment to radiochemotherapy had no effect on tumor progression, but preserved the glutamatergic network activity on healthy peritumoral tissuePER showed antitumor activity in glioblastoma cell lines U87, U138, A172 and the grade III astrocytoma cell line SW1783, via a pro-apoptotic effect. The combination of PER and temozolomide had a significant synergistic effect PER showed inhibitory effects on cell viability in a dose-dependent manner on malignant glioma cell lines A-172, AM-38, T98G, U-138MG, U-251MG and YH-13, via a pro-apoptotic effect Inhibitory effect on cell proliferation of PER confirmed on A-172, AM-38, T98G, U-138MG, U-251MG and YH-13 cell lines. PER and temozolomide showed synergistic effect on T98G and U-251MG lines. PER suppressed migration of T98G and U-251MG cells. Abbreviation: PER = perampanel. [/table]
[table] Table 2: Main characteristics of clinical studies and efficacy findings of perampanel in patients with brain tumor-related epilepsy. [/table]
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MicroRNA Target Site Identification by Integrating Sequence and Binding Information
High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute protein members can pinpoint microRNA target sites within tens of bases, but leaves the identity of the microRNA unresolved. A flexible computational framework that integrates sequence with cross-linking features reliably identifies the microRNA family involved in each binding event, considerably outperforms sequence-only approaches, and quantifies the prevalence of noncanonical binding modes.
The resolution of CLIP experiments has been generally insufficient to unambiguously identify the acting miRNA via sequence matches. Recent protocols, including iCLIP and PAR-CLIP, increased resolution by providing additional signals diagnostic of cross-linked locations. In particular, PAR-CLIP obtains high cross-linking efficiency due to the presence of the nucleoside analog 4-thiouridine, which leads to characteristic thymine-to-cytosine transitions in the library reads in the vicinity of protein-mRNA cross-linking. To define a narrow region (typically ~30nt) most likely bound by the protein, our previously published PARalyzer softwarequantifies this T-to-C transition profile relative to background read density via a kernel density estimator [fig_ref] Figure 1: Figure 1. [/fig_ref].
MiRNA target sites are characterized by one of several types of seed sequence matches unique to each miRNA family 7 (canonically, a perfect 7nt complement to positions 2-8 of the miRNA). In AGO-immunoprecipitated libraries, the cross-linking site indicates a radius for RISC-mediated miRNA heteroduplex formation on the mRNA substrate, and restricts the search space for functional seed matches. Furthermore, the PAR-CLIP transition signal is preferentially located directly 5′ of the miRNA seed match on the mRNA, likely due to the biophysical properties of the heteroduplex within functional miRNP complexes [fig_ref] Figure 1: Figure 1. [/fig_ref]. mRNA conservation levels immediately 3′ of peaks indicate that many of these seed matches are selectively maintained [fig_ref] Figure 1: Figure 1. [/fig_ref].
Despite continuous improvements in de novo computational predictions of target sites, efforts have been fundamentally limited by the brevity of sequence motifs describing functional miRNA-target relationships. The quantitative information in AGO PAR-CLIP datasets motivated us to develop an integrative computational method to identify miRNA target sites at the nucleotide level with high accuracy. It combines the T-to-C transition signal with miRNA seed-pairing and its evolutionary conservation, as well as the spatial relation between binding and sequence features. We implemented the model within a general-purpose hidden Markov model (HMM) frameworkcalled MUMMIE (Multivariate Markov Modeling Inference Engine). We model the shape of PAR-CLIP signals with a 6state topology [fig_ref] Figure 1: Figure 1. [/fig_ref] , in which state 5 expands into a 41-state submodel for detection of seed matches of various types [fig_ref] Figure 1: Figure 1. [/fig_ref]. The model is parameterized to preferentially predict seed matches that are long, are located closely 3′ to a PAR-CLIP peak, and are highly conserved. Tradeoffs between these features are naturally accounted for during prediction so that suboptimal sites can still be detected, albeit with lower scores (posterior probabilities; [fig_ref] Figure 1: Figure 1. [/fig_ref].
Most current target predictors identify candidate sites within whole transcripts, usually parameterized on the indirect cumulative effect on mRNA or protein steady-state levels. The availability of in vivo direct binding data changes the problem of target prediction into one of assigning the most likely miRNA(s) to each of the observed AGO binding locations. We applied suitable site-level target predictors to cross-linked regions from multiple datasets, and evaluated the improvement obtained by the explicit joint model in the new target predictor. Analyses were limited to regions covered by aligned PAR-CLIP reads (termed groups); this dramatically increases the accuracy of methods compared to their application without the benefit of CLIP information [fig_ref] Figure 2: Figure 2. [/fig_ref]. Because the identity of the miRNA in bound AGO complexes is unknown, we assessed the relative prediction accuracy via signal-to-noise ratio (SNR) based on randomized shuffling of miRNA sequences (i.e., enrichment of complementary miRNA seeds over decoy sequences among the predictions; see Online Methods).
Specifically, we compared MUMMIE to the following alternatives: The latest release of the state-of-the-art de novo miRNA target site predictor, TargetScan 10 , utilizes conservation evidence as well as various sequence context scores, but makes no explicit use of CLIP information. MIRZA 11 implements a new biophysical model of target recognition, based on context-specific RNA duplex formation estimated on high-quality PAR-CLIP data. We contrasted these model-based approaches to two baseline strategies for identifying miRNA target sites from PAR-CLIP data: choosing the nearest seed match to each PARalyzer peak, or predicting all seed matches within each PARalyzer cluster (the maximal interval in which T-to-C transition rate exceeds background expectation 6 ). All approaches used the 100 most highly-expressed miRNAs in the cell line, which delivered the best trade-off between SNR and sensitivity .
On a PAR-CLIP dataset from a lymphoblastoid cell line infected with Epstein-Barr virus (LCL-BAC) 12 , our model achieves higher sensitivity, specificity, and SNR than other approaches [fig_ref] Figure 1: Figure 1. [/fig_ref]. Thus, the joint model has a greater ability to discriminate real from decoy target sites (higher SNR), and it does so by predicting fewer randomized decoy sites within those CLIP groups (higher specificity). TargetScan limits predictions to the three "canonical" seed matches (7mer-A1, 7mer-m8, 8mer-A1), while our model predicts seven types (6mer3-8, 6mer2-7, 7mer-m8, 7mer-m1, 7mer-A1, 8mer-A1, 8mer-m1; see also . MIRZA's energy-based model scores the whole duplex and is therefore agnostic to specific seed matches. While it reaches a higher sensitivity than MUMMIE, by far the largest contribution of "noncanonical" targets appear to involve the 6mer seeds included in MUMMIE. The baseline models exhibited competitive sensitivity, but at very low SNR/specificity levels. Results on additional PAR-CLIP data sets prepared by MNase digestion .
To make results as comparable as possible, we used TargetScan's branch length score as conservation evidence 10 , which is unavailable for 6mer seed matches. This effectively biases our model against predicting 6mer sites when parameterized to weight conservation highly, and helps improve specificity and SNR at the low-sensitivity end of the spectrum [fig_ref] Figure 2: Figure 2. [/fig_ref]. Increasing the number of miRNAs represented in our model beyond the 100 most highly expressed ones progressively degrades performance , illustrating the complementary benefit of miRNA expression measurements in addition to RISC binding information. Examples of several previously or newly experimentally validated MUMMIE predictions 12 involving different seed types are given in Supplementary Figs. 6 and 7.
To directly address the question of whether predicted targets were due to the presence of the specified miRNA, we contrasted the LCL-BAC predictions with those from three additional Epstein-Barr infected cell lines, each of which has one viral miRNA deleted (BHRF1-1, BHRF1-2, and BHRF1-3, respectively). The PAR-CLIP signal in the deletion lines was nearly absent at the locations of predictions involving a BHRF miRNA in the original cell line [fig_ref] Figure 2: Figure 2. [/fig_ref]. For two miRNAs, the loss of sites was accompanied by consistent upregulation in steady-state target mRNA levels as measured by Illumina sequencing. MUMMIE-based observations agreed with MIRZA-predicted targets .
As shown in previous studies, the presence of a perfect seed match cannot explain all CLIP groups. In the LCL-BAC dataset, the sensitivity of MUMMIE does not exceed 80% at the most lenient settings; a naive scan for 6-mer seed matches to the top 100 expressed miRNAs does not exceed 84% [fig_ref] Figure 1: Figure 1. [/fig_ref]. This motivated us to investigate the extent to which binding with an imperfect seed match might explain remaining CLIP groups. We focused on a recently described class of imperfect targets, namely "bulge" sites, which have been suggested to explain as many as 25% of all the HITS-CLIP clusters for miR-124. We adapted our model to sites in which one of the mRNA residues pairs with the miRNA only during an initial annealing step but becomes unpaired in the final duplex, forming a bulge in the mRNA. In addition to the previously proposed pivot pairing between mRNA positions 5-6, we investigated potential bulges between positions 4-5 and positions 3-4. Bulge site predictions showed distinctly lower SNR profiles, and when combining all three pivot locations, predictions covered only ~15-20% of orphan groups (~1-2% of total groups; Supplementary Table 2, Supplementary Figs. 11, 12, and 13). Even MIRZA, which does not make any seed type assumptions, assigns miRNAs to only a small fraction of additional clusters.
Our findings illustrate the superiority of transcriptomic assays over de novo sequence analysis for identifying functional sites active in a specific condition, as well as of principled modeling techniques over ad hoc strategies. By jointly modeling multiple relevant variables -including evolutionary conservation, residue transition rates, spatial positioning, and sequence composition -MUMMIE provides for finer discrimination between true and decoy sites. There is room for further improvement, via incorporation of additional evidence including structural accessibility, and relative or absolute production or steady-state levels of miRNAs or mRNAs. The framework provides flexibility in the ability not only to incorporate additional covariates, but also to choose the desired compromise between sensitivity and specificity, and to define additional classes of seed matches (e.g., centered sites 15 or compensatory 3′ binding. However, in our comparative evaluation, genuinely imperfect seed sites appeared to account for a small fraction of total targets only. The remaining 20% of AGO clusters may reflect low-affinity transitory binding, experimental noise, or AGO targets that do not involve miRNAs.
Due to the exploitation of complementary information sources, we have arrived at a performance level where the question of miRNA targeting is no longer a binary one, but one of quantitative impact of a miRNA on a transcript (cf. ; this idea has specifically been pursued in the concurrently developed MIRZA). To this aim, however, multiple genomic data sets have to be generated; recent sequence-only target predictors remain more generally applicable, albeit at the cost of significant reductions in accuracy. Yet, distinct transition patterns are also observed for other post-transcriptional regulators such as Quaking and Pumilio 4 , suggesting the utility of such models for RNA-binding protein target identification in general.
# Online methods
## Par-clip data generation
Multiple Argonaute 2 PAR-CLIP libraries from two different human cell line sources (LCLs and HEK293) were used in this study. Established lymphoblastoid cell lines (LCL-BAC, LCL-BAC-D1, LCL-BAC-D2, LCL-BAC-D3) from the same donor were infected with variations of EBV B95-8 Bacmid. LCL-BAC-D1, LCL-BAC-D2, and LCL-BAC-D3 (the "deletion lines") were infected with EBV miRNA-deficient viruses lacking miR-BHRF1-1, miR-BHRF1-2, and miR-BHRF1-3 expression, respectively 20 . All LCLs were maintained in RPMI 1640 medium supplemented with 15% FBS and 10 μg/mL gentamicin (GIBCO) and analyzed three to five months post-infection. PAR-CLIP data for LCL-BAC, LCL-BAC-D1 and LCL-BAC-D3 was previously published by us 12 (GEO GSE41437); PAR-CLIP data for LCL-BAC-D2 was newly generated following the same protocol, which involved treatment with double RNase T1 digestion.
LCL-BAC libraries were prepared using the original PAR-CLIP protocol 4 , which includes double RNase T1 digestion steps to fragment cross-linked RNA prior to sequencing. This has raised concerns that identified binding events may exhibit biases due to sequence preferences of the digestion enzymes. We therefore additionally analyzed previously published PAR-CLIP data from HEK 293 cells, two biological replicates involving MNase digestion of isolated RNA in place of double T1 digestion ("MNase A", GEO GSM714646; "MNase B", GSM714647). See for library and annotation metrics for all PAR-CLIP libraries.
For miRNA target prediction we considered only genomic intervals annotated as 3′ untranslated regions (UTR) in Ensembl v. 58 21 ; for genes with multiple annotated transcripts (isoforms), we kept the one with the longest 3′ UTR (measured in bases of mature transcript). For the LCL-BAC data set, the miRNA set used for prediction is based on small RNA deep-sequencing data of the same culture 12 . For the external MNase data sets, we used the miRNA set of the same cell line that was reported in a previous study 4 .
## Par-clip data processing and significance testing
PAR-CLIP data for all cell lines were processed by PARalyzer 6 to produce sequences of continuous values. PARalyzer smoothes the sequence of discrete read count values via kernel density estimation, and the resulting continuous values represent relative frequencies of T-to-C transitions. This sequence is referred to as the "signal track" for use in MUMMIE. In practice, the PARalyzer signal track for a complete UTR will typically be sparse; i.e., zero everywhere except at and around an occupied binding site. These nonzero regions, which correspond to scaffolds of aligned PAR-CLIP reads, are termed "groups." When binding sites are close together, their scaffolds may coalesce, resulting in one group for multiple occupied sites. PARalyzer therefore defines individual "interaction sites" by identifying maximal intervals in which the T-to-C transition signal is higher than the background; we term these intervals "clusters." Clusters therefore occur within groups, and generally provide for a higher resolution of RBP target sites. However, their utility in pinpointing miRNA target sites in AGO libraries is complicated due to lack of T-to-C transition signal within the miRNA-mRNA duplex site: the signal "peak" generally does not overlap the actual miRNA seed match, and the PARalyzer software therefore provides an option to "pad" and increase the size of AGO clusters. Here, other than for the baseline seed match comparison, we do not explicitly use PARalyzer clusters; instead, we run MUMMIE on the complete UTR, after normalization of the signal for individual groups. Normalization of groups ensures that the maximum signal value for each group is 1.0, and helps to control for differing binding affinities, sequencing biases, and miRNA/mRNA abundance.
To validate library-specific predictions, we compared the PAR-CLIP signal at predicted "true" sites, for a miRNA expressed in LCL-BAC, against the signal in one of the LCL-BAC-D1/2/3 libraries not expressing the miRNA of interest [fig_ref] Figure 2: Figure 2. [/fig_ref]. The significance of the signal difference was assessed by a non-parametric test, in which we first ranked target sites of all top 100 expressed miRNAs by their difference in signal and quantified the enrichment of true targets at the top, i.e. among the most-changed sites. This implicitly accounts for the overall difference in signal observed across all targets, due to incomplete saturation of the deep sequencing libraries [fig_ref] Figure 2: Figure 2. [/fig_ref]. Specifically, we started from the smoothed T-to-C transition frequencies as computed by PARalyzer, i.e. the signal track including normalization as described above. To represent a binding signal, we summed over frequencies in a local area (±3 nucleotides) around a consistent reference location, in this case the mRNA coordinate across from miRNA position 8, i.e. the beginning of the longest possible seed match. As test statistic, we used the fraction of the binding signal detected in the deletion line compared to the signal in the wildtype. Significance was assessed by the Wilcoxon rank-sum test.
## Quantification of mrna level changes
Paired-end strand-specific RNA-seq was performed for all four established LCLs. Total RNA was collected with TRIzol (Life Technologies) and libraries were prepared using ScriptSeq v2 kit and Ribozero (Epicentre). Biological replicates were performed for LCL-BAC-D1. RNA-seq data was aligned and quantified using RSEM with reference genome and transcript annotation. Differential analysis was performed using EBseq within RSEM. Real fold change (RealFC value from EBseq) for genes with median log 2 (TPM + 1) > 5 across all samples were utilized for evaluating differential expression of MUMMIE or MIRZA predictions; the same sets of genes were used to assess loss of binding events in the deletion libraries. See for specific analysis parameters and quality assessments.
To assess the enrichment of predicted targets of a specific miRNA among the top differentially regulated gene sets, genes were ranked by mRNA expression fold change (based on the data for LCL-BAC vs the corresponding deletion line) and grouped into sets of increasing size. For each top differentially regulated set, an enrichment of predicted targets was computed as a ratio of a fraction of predicted targets in the top set and a fraction of total number of predictions in the full gene set. Target sets of miRNAs with a similar number of MUMMIE predictions (between roughly half to twice as many sites) were used to quantify background expression changes of presumably unaffected targets.
## Luciferase assay
293T cells were maintained in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. miRNA expression plasmids contain ~200 nucleotides of the primary miRNA cloned from genomic DNA into pLCE at XhoI/XbaI. Expression of the miRNA was confirmed using indicator assays as previously described. Luciferase reporter assays were carried out as previously described. Briefly, 293T cells were co-transfected in 24-well plates with 1 μg miRNA expression vector (pLCE-miRNA), 12 ng pL-CMV-GL3-3′UTR, and 12 ng pL-CMV-RLUC (renilla internal control) using Fugene 6 (Promega) according to manufacturer's instructions. Lysates were harvested 48-72 hrs post-transfection and assayed for luciferase activity using the dual luciferase reporter assay kit (Promega).
## Modeling software
Binding site models were implemented within the open-source software MUMMIE (MUltivariate Markov Modeling Inference Engine), available online at http:// www.genome.duke.edu/labs/ohler/research/MUMMIE. This system permits user-specified model topologies to be designed for a wide array of potential bioinformatic applications. The software includes command-line routines for both parameter estimation ("training") and decoding ("prediction"). The following sections describe the models and algorithms used to obtain predictions for this study.
## Models
Prediction of target sites from binding and sequence data is achieved via hybrid continuousdiscrete multivariate hidden Markov models. A Markov model is a probabilistic generative model for sequential data with a fixed set of discrete states Q={q 0 , q 1 , …, q N−1 }. We here consider discrete-time models, in which observations are available for T={0,1,…,L−1} positions in a biological sequence S=s 0 s 1 …s L−1 , which the model is assumed to have emitted. Emitted sequence elements may take continuous values; for multivariate models, each sequence element is a vector of continuous values (each emitted by a separate "emission channel"). In hybrid continuous/discrete models, each emission channel is either discrete or continuous. Thus, for each vector in the emission sequence, each component of that vector will be a value that is either a symbol drawn from a discrete alphabet, or a numerical value. For example, an RNA emission channel would accept symbols from the set {A,C,G,U}, an "aligned read-count" channel would accept nonnegative integers denoting numbers of reads aligned at a given genomic position, and a "probability of being unpaired" track would accept real values between 0 and 1.
The model M emits a biological sequence S as follows. Beginning in a special silent state q 0 , at each time step t the model chooses a state to transition into, by drawing a new state x t+1 ∈Q from a transition probability distribution, P(x|x t ), which is conditional on the current state, x t . Upon entering the new state, it draws a random vector y∈V from the emission distribution P(y|x t+1 ), where V is the set of all possible emission vectors for the model. After emitting the vector's components into the respective emission channels, the model chooses the next transition according to P(x|x t ). If state q 0 is chosen, the machine terminates and S is taken to be the complete emission; otherwise, it continues its run by alternating between transitions and emissions, until state q 0 is eventually chosen. Note that q 0 never makes emissions.
Because the emission distribution is conditional only on the current state, each emission is conditionally independent of all other emissions, given the associated state. This is the case of 0 th -order emissions. A model with higher-order emissions may condition each emission on events (transitions or emissions) progressively further into the past. For the present work we consider only higher-order discrete emissions, in which the emission of a symbol may be conditioned on some number of immediately preceding symbols emitted into the same channel; this permits us to model seed matches for many different miRNAs while using a relatively small number of states (see below).
For miRNA target prediction, we designed a 47-state Markov model. The model is a hybrid continuous/discrete multivariate hidden Markov model with three emission channels. The first channel is the PARalyzer signal representing the relative abundance of T-to-C transitions in aligned CLIP scaffolds. This channel is continuous and modeled via a fourcomponent Gaussian mixture. The second channel, branch length score (BLS), provides a continuous measure of conservation; it was computed using a script from the TargetScan package. The third channel comprises the genomic DNA sequence corresponding to the mRNA, and is modeled using a 7 th -order Markov chain. Channels are pre-aligned so that for each position in a UTR we have one nucleotide and two continuous values.
The model topology is depicted schematically in [fig_ref] Figure 1: Figure 1. [/fig_ref]. State 1 models the background regions between PAR-CLIP groups. State 3 models the peak of the PAR-CLIP group, while states 2, 4, 5, and 6 model the flanking regions around the peak. State 5 is a metastatewhich expands into the 41-state submodel shown in [fig_ref] Figure 1: Figure 1. [/fig_ref] ; this metastate models the actual target site of the miRNA seed. Our default model detects seven types of miRNA binding: 6mer3-8, 6mer2-7, 7mer-m8, 7mer-m1, 7mer-A1, 8mer-A1, and 8mer-m1.
The model in [fig_ref] Figure 1: Figure 1. [/fig_ref] preferentially visits state 5 (the miRNA binding site) a short distance 3′ of a PAR-CLIP peak (which is generally matched by state 3); this preference reflects the strong enrichment of miRNA seed matches 3′ of peaks [fig_ref] Figure 1: Figure 1. [/fig_ref]. In consequence, model predictions also show a strong preference for this location relative to the peak [fig_ref] Figure 1: Figure 1. [/fig_ref]. [fig_ref] Figure 1: Figure 1. [/fig_ref] suggests a small fraction of sites 5′ of the peak, and the model predicts a fraction of these 5′ sites. This illustrates the flexibility of this class of models: when the most promising site occurs 5′ of the peak, the decoding algorithm will permit state 3 to be visited earlier in the sequence so that state 5 can match the putative target site. Likewise, when no promising seed match is present, the decoding algorithm may choose to not predict any binding site for the PAR-CLIP group. This flexibility is a function of the model parameters, in particular the variance of the emission distribution for the PAR-CLIP signal: for low variances (~0.01) the model predicts many sites (typically one per PAR-CLIP group), while for high variances (~1.0) it predicts fewer sites. The different points in [fig_ref] Figure 1: Figure 1. [/fig_ref] , g correspond to different settings of the PARalyzer signal variance, with a high variance parameter corresponding to high specificity and high SNR, and a low variance parameter corresponding to high sensitivity (the following variances were used for these plots: 1.5, 1.25, 0.75, 0.5, 0.375, 0.25, 0.20, 0.15, 0.1, 0.075, 0.05, 0.01, 0.005).
To model bulge sites with bulges at specific positions complementary to the seed, we use the same metamodel structure shown in [fig_ref] Figure 1: Figure 1. [/fig_ref] , but simplify the submodel for state 5 to include only a single linear chain of seven states to match the six seed residues plus the bulge position embedded within the seed.
## Inference algorithms
Prediction of sites can be carried out in MUMMIE using either Viterbi decoding or posterior decoding, or a combination of the two.
Viterbi decoding identifies the single most probable path through the HMM states for emitting the multivariate sequence. Any occurrence of metastate 5 in that most probable path is interpreted as a predicted miRNA binding site; the exact sequence of states within the metastate dictates the type of binding (e.g., 6mer2-7, 7mer-m1, etc.), and the matching nucleotides in the emitted sequence identify the miRNA family (seed sequences may or may not be unique to a given miRNA within a family; when multiple miRNAs share a predicted seed, all are listed as the potential binding miRNA).
In contrast to Viterbi decoding, posterior decoding utilizes the well-known forward and backward algorithms 25 to compute the posterior probability P(q,k) that state q was active at position k when the sequence was generated by the HMM. In addition to providing this standard functionality, MUMMIE also computes for each 8bp interval the posterior probability that a miRNA binds within that 8bp interval, via any of the modeled binding modes (i.e., by summing the posteriors for each binding mode for that miRNA within that 8bp interval).
Finally, MUMMIE supports a combination of posterior and Viterbi decoding via the posterior Viterbi algorithm 26 , in which emission and transition probabilities in the Viterbi algorithm are replaced with posterior probabilities. This decoding algorithm was used for the results presented here.
## Model training
Considering all states modeling the actual miRNA binding site to be foreground states and all others to be background states, training of the background states was accomplished by applying expectation maximization (EM) to entire UTRs, with the following exceptions. For the peak state (state 3), the PARalyzer signal mean was set to 1.0 and its variance was arbitrarily set to 0.01; for the flanking states (states 2, 4, and 6) the signal mean was set to 0.5 and the variance to 0.01; these mean values were chosen to have these states match the respective regions of an idealized PARalyzer signal profile at a binding site. The mean and variance of the conservation track were set to 0 and 0.01, respectively, for all background states.
For the foreground states, the nucleotide emission distribution was set so as to permit only the training miRNA seeds to be emitted; PARalyzer signal mean was set to 0.5 and variance to 0.01 as in the background flank states; conservation mean was set to 3.0. Nucleotide sequence emissions for flanking states were trained via EM on sequences within PARalyzer groups, and for the peak state on the collection of single nucleotides at each PARalyzer peak (Supplementary . Variance of the conservation track was trained on a held-out set of training data by observing the resulting sensitivity-vs.-SNR curves at different settings and selecting the value that produced the curve with the highest overall SNR. All covariances were set to 0. The Markov order of nucleotide emissions varied from zero to seven along the length of a seed, so as to enforce that only valid miRNA seeds may be emitted, as per the training set.
## Bulge site analyses
We investigated different locations for the pivot nucleotide in bulge site pairing. A bulge seed match is a perfect seed match to miRNA position 2-7 with a bulge nucleotide insertion that can pair with the pivot nucleotide. Similar to the canonical perfect seed match prediction, we computed SNR of bulge site predictions. Here, we excluded the bulge seed matches that share a 6mer with any human or EBV miRNA.
Additionally, for bulge type 2 (position 4-5) evaluation and prediction, we excluded bulge seed matches to original miRNAs with the same nucleotide at position 5 and 6, as its pairing in the in the nucleation step could extend one more position and thus provide for the same bulge seed match as in type 1 (position 5-6). Similarly, seeds for bulge type 3 (position 3-4) did not include bulge seeds that extended to bulge type 2.
## Prediction accuracy evaluation and comparison with other predictors
We computed signal-to-noise (SNR) ratios by comparing numbers of non-shuffled and shuffled sites among a set of predictions. Shuffled miRNAs were created via a random process that preserves the dinucleotide frequencies observed in real miRNAs, as described previously. These were then screened to ensure that their expected seed-match frequencies in PAR-CLIP groups did not differ by more than 15% from the same statistic for the real (unshuffled) miRNAs. The signal-to-noise ratio was averaged over 1,000 runs in which we sampled one shuffled miRNA for each original (non-shuffled) miRNA; state 5 of the model was then trained on the resulting set of shuffled and non-shuffled seeds, so that a single decoding run of the model could predict any of the seeds in the training set.
We used TargetScan release 6.1 10 to compute TargetScan predictions, "context+" scores, and branch length scores. A 23-way alignment of 3′ UTRs was used, and miRNAs were evaluated individually. We computed signal-to-noise ratio at varying context+ score thresholds for the TargetScan sensitivity-SNR plots.
MIRZA 11 was executed in its default "noupdate" mode, and optionally provided with celltype specific expression levels of miRNAs. Mirroring the example of its developers, inputs to MIRZA were 51nt sequences centered on the peak T-to-C conversion site as identified by PARalyzer, and 21nt mature miRNA sequences. MIRZA's "target frequency" score was used to rank predictions when computing sensitivity, specificity, and SNR. Identifying miRNA target sites by a joint sequence and interaction model. (a) Example of an AGO-interaction site identified by PARalyzer. Shown are the kernel density estimate of signal versus background T-to-C transitions, and the read depth of a 3′ UTR region of SPATS2 (ENST00000395063) profiled in the LCL-BAC library. Two 7mer seed matches are in close proximity, each a different distance 3′ of a signal peak; naive target prediction based on read depth alone would have incorrectly treated these two binding events as one.
(b) Across all miRNA target candidates, seed matches show the strongest enrichment immediately 3′ of the PAR-CLIP T-to-C transition peak. (c) Conservation of sites as measured by PhastCons scoresillustrate preferential conservation in the first 8nt 3′ of peaks. (d) State-transition diagram of the hidden Markov model. All states represent a joint likelihood of sequence and PAR-CLIP signal in a particular region. (e) State 5 is a metastate that specifically expands into a 41-state submodel for several types of miRNA seed matches. (f) Sensitivity-vs.-SNR tradeoff for MUMMIE, Targetscan, MIRZA, and two baselines, using the top 100 expressed miRNAs in LCL-BAC cell line. Explicit expression information was not utilized by any predictor (cf. [fig_ref] Figure 2: Figure 2. [/fig_ref] for additional results). (g) Similar to (f) for sensitivity-vs.-specificity. Validation of predicted sites and their impact on expression levels. We computed the aggregate PAR-CLIP signals at predicted target sites for specific miRNAs expressed in the LCL-BAC cell line, and compared it to the aggregate signal at the same sites in cell lines missing one of the corresponding miRNAs. (a) Loss of PAR-CLIP signal for LCL-BAC predicted target sites of BHRF1-1, BHRF1-2, and BHRF1-3 in the corresponding deletion line (LCL-BAC-D1, -D2, and -D3, respectively) (23 predicted target sites for BHRF1-1, 52 for BHRF1-2, and 10 for BHRF1-3). (b) Control: Signal difference for predicted target sites in LCL-BAC vs. deletion lines, to all but the miRNA of interest (2,159 predicted targets for the D1 control, 2,082 for the D2 control, and 2,172 for the D3 control). Binding loss of BHRF1-1 (P = 0.0006) and BHRF1-2 (P = 0.0394) were statistically significant compared to the control; loss of BHRF1-3 was consistent but not significant due to the small number of sites (P=0.1879; Wilcoxon rank-sum test). (c) Impact of site loss on steady-state mRNA expression levels, based on RNA-seq data from the LCL-BAC cell lines. The enrichment of BHRF target sites in the top gene sets ranked by differential expression is contrasted with the enrichment of other expressed miRNAs with similar numbers of predicted targets. Predictions were obtained using the top 100 expressed miRNAs, with the MUMMIE PAR-CLIP signal variance parameter set to 0.01. All predicted targets were aligned across the beginning of miRNA seed matches in the mRNAs. Vertical error bars indicate ± one standard deviation.
[fig] Figure 1: Figure 1. [/fig]
[fig] Figure 2: Figure 2. [/fig]
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Proton beam radiotherapy for patients with early-stage and advanced lung cancer: a narrative review with contemporary clinical recommendations
[bib_ref] NCCN Guidelines Insights: Non-Small Cell Lung Cancer, Version 4, Ettinger [/bib_ref] [bib_ref] Stereotactic body radiation therapy for early-stage non-small cell lung cancer: Executive Summary..., Videtic [/bib_ref] [bib_ref] PROCLAIM: Randomized Phase III Trial of Pemetrexed-Cisplatin or Etoposide-Cisplatin Plus Thoracic Radiation..., Senan [/bib_ref] [bib_ref] Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC, Antonia [/bib_ref] [bib_ref] Primary analysis of the phase II component of a phase I/II dose..., Bradley [/bib_ref] [bib_ref] Randomized phase II trial of induction chemotherapy followed by concurrent chemotherapy and..., Socinski [/bib_ref] [bib_ref] Long-Term Results of RTOG 0617: A Randomized Phase 3 Comparison of Standard..., Bradley [/bib_ref] [bib_ref] Impact of Intensity-Modulated Radiation Therapy Technique for Locally Advanced Non-Small-Cell Lung Cancer:..., Chun [/bib_ref] [bib_ref] A Meta-Analysis of Thoracic Radiotherapy for Small-Cell Lung Cancer, Pignon [/bib_ref] [bib_ref] Impact of PET staging in limited-stage small-cell lung cancer, Xanthopoulos [/bib_ref] [bib_ref] Concurrent once-daily versus twice-daily chemoradiotherapy in patients with limited-stage small-cell lung cancer..., Faivre-Finn [/bib_ref] [bib_ref] Multi-Institutional Experience of Stereotactic Ablative Radiation Therapy for Stage I Small Cell..., Verma [/bib_ref] [bib_ref] An in-silico comparison of proton beam and IMRT for postoperative radiotherapy in..., Berman [/bib_ref] [bib_ref] Comparison of 2 common radiation therapy techniques for definitive treatment of small..., Shirvani [/bib_ref] [bib_ref] Simultaneous integrated dose reduction intensity-modulated radiotherapy applied to an elective nodal area..., Liu [/bib_ref] [bib_ref] Proton beam therapy for locally advanced lung cancer: A review, Schild [/bib_ref] [fig_ref] Figure 1: Axial comparison of an IMPT plan [/fig_ref] [bib_ref] Intensity-modulated proton therapy reduces the dose to normal tissue compared with intensity-modulated..., Zhang [/bib_ref] [bib_ref] Dosimetric consequences of tumour motion due to respiration for a scanned proton..., Kraus [/bib_ref] [bib_ref] Small-spot intensitymodulated proton therapy and volumetric-modulated arc therapies for patients with locally..., Liu [/bib_ref] [bib_ref] Impact of Spot Size and Spacing on the Quality of Robustly Optimized..., Liu [/bib_ref] [bib_ref] Influence of robust optimization in intensity-modulated proton therapy with different dose delivery..., Liu [/bib_ref] [bib_ref] Impact of respiratory motion on worst-case scenario optimized intensity modulated proton therapy..., Liu [/bib_ref] [bib_ref] Exploratory Study of 4D versus 3D Robust Optimization in Intensity Modulated Proton..., Liu [/bib_ref] [bib_ref] Robust optimization in intensity-modulated proton therapy, Liu [/bib_ref] [bib_ref] Consensus Statement on Proton Therapy in Early-Stage and Locally Advanced Non-Small Cell..., Chang [/bib_ref] [bib_ref] Early findings on toxicity of proton beam therapy with concurrent chemotherapy for..., Sejpal [/bib_ref] [bib_ref] Long-Term Results of RTOG 0617: A Randomized Phase 3 Comparison of Standard..., Bradley [/bib_ref] [bib_ref] Initial evaluation of treatment-related pneumonitis in advanced-stage nonsmall-cell lung cancer patients treated..., Yom [/bib_ref] [bib_ref] Long-term clinical outcome of intensity-modulated radiotherapy for inoperable non-small cell lung cancer:..., Jiang [/bib_ref] [bib_ref] Twice-Daily Compared with Once-Daily Thoracic Radiotherapy in Limited Small-Cell Lung Cancer Treated..., Turrisi [/bib_ref] [bib_ref] Chemotherapy for small cell lung cancer: a comprehensive review, Karim [/bib_ref] [bib_ref] Dose-volume comparison of proton radiotherapy and stereotactic body radiotherapy for non-small-cell lung..., Kadoya [/bib_ref] [bib_ref] Evaluating proton stereotactic body radiotherapy to reduce chest wall dose in the..., Welsh [/bib_ref] [bib_ref] Excessive toxicity when treating central tumors in a phase II study of..., Timmerman [/bib_ref] [bib_ref] Proton stereotactic body radiation therapy for clinically challenging cases of centrally and..., Register [/bib_ref] [bib_ref] Stereotactic Body Radiation Therapy in Centrally and Superiorly Located Stage I or..., Chang [/bib_ref]
## Indications for pbt for lung cancer
## Small-cell lung cancer
Current literature on PBT for LS-SCLC is limited to two studies. In a study of 6 patients treated with PBT and cisplatin/etoposide, 1-year PFS and OS were 66% and 83%, respectively [bib_ref] Dosimetric rationale and early experience at UFPTI of thoracic proton therapy and..., Colaco [/bib_ref]. Treatment was well tolerated with no patients experiencing grade ≥3 acute or late events. Dosimetric analysis showed superior sparing of the lung and esophagus when compared to comparison IMRT plans.
In a larger study at the University of Pennsylvania, 30 patients were treated with cisplatin or carboplatin and etoposide concurrently with PBT [bib_ref] Prospective study of proton-beam radiation therapy for limited-stage small cell lung cancer, Rwigema [/bib_ref]. Patients received PBT twice daily with 45 Gy (1.5 Gy per fraction) or once daily with 59.4-66.6 Gy (1.8 Gy per fraction). Compared to IMRT, PBT plans demonstrated lower RT dose to the heart, lungs, and esophagus, with decreased lung volume IMPT, intensity-modulated proton therapy; IMRT, intensity-modulated radiation therapy; NSCLC, non-small cell lung cancer.
## Impt imrt
receiving low dose bath. In patients treated with daily RT, there was one grade 3 pneumonitis and one grade 3 anorexia. In patients treated with twice daily RT, 1 patient experienced grade 3 pericardial effusion, and another experienced grade 4 esophagitis. LC rates were 85% and 69% at one and two years, respectively, and OS rates were 72% and 58%, respectively. Five (17%) patients experienced in-field recurrence, two of which were isolated in-field failures only. Rwigema et al. demonstrated comparable survival figures to those of the CONVERT trial, and median OS and 2-year OS (28.2 months, 57.5%) were greater than those of prior studies on photon-based CRT [bib_ref] Concurrent once-daily versus twice-daily chemoradiotherapy in patients with limited-stage small-cell lung cancer..., Faivre-Finn [/bib_ref] [bib_ref] Prospective study of proton-beam radiation therapy for limited-stage small cell lung cancer, Rwigema [/bib_ref]. For example, the Intergroup 0096 study found 2-year OS rates of 41% (daily RT) and 47% (twice-daily RT) and median OS of 19 months (daily RT) and 23 months (twice-daily RT) [bib_ref] Twice-Daily Compared with Once-Daily Thoracic Radiotherapy in Limited Small-Cell Lung Cancer Treated..., Turrisi [/bib_ref]. In the Cancer and Leukemia Group B 39808 study, median OS was 22.4 months, and the 2-year OS rate was 48% [bib_ref] 70 Gy thoracic radiotherapy is feasible concurrent with chemotherapy for limited-stage small-cell..., Bogart [/bib_ref]. Importantly, all patients from the University of Pennsylvania study tolerated the regimen of CRT without RT breaks, and 93% of patients completed all four planned cycles of chemotherapy. PBT may improve tolerance of combined treatment modalities, optimizing clinical outcomes.
## Early-stage nsclc
Dosimetric studies of early-stage NSCLC demonstrated improved dose distribution with PBT compared to photonbased RT. In a study of patients with stage I NSCLC, PBT treatment plans demonstrated reduced lung V5, V10, and V20Gy when compared to SBRT treatment plans [bib_ref] Dose-volume comparison of proton radiotherapy and stereotactic body radiotherapy for non-small-cell lung..., Kadoya [/bib_ref]. In another study of patients with stage I and stage III NSCLC, comparison of dose-volume histograms similarly showed reduced mean total lung V5, V10, and V20Gy in PBT plans when compared 3DCRT plans, even with dose escalation [bib_ref] Significant reduction of normal tissue dose by proton radiotherapy compared with three-dimensional..., Chang [/bib_ref]. Furthermore, PBT reduced doses to the lung, spinal cord, heart, esophagus, and integral dose when compared with IMRT. A multicentric trial by the Radiation Oncology Collaborative Comparison (ROCOCO) compared photon, proton, and carbon-ion treatment plans for stage I NSCLC patients and reported lower mean dose and dose to 2% of CTV for proton and ion than IMRT. Doses to the spinal cord were lowest with double-scattered proton therapy plans [bib_ref] Photons, protons or carbon ions for stage I non-small cell lung cancer..., Wink [/bib_ref].
Early PBT-based clinical studies found high rates of 2-year and 3-year OS, cause-specific survival (CSS), and PFS [bib_ref] Clinical evaluation of proton radiotherapy for non-small-cell lung cancer, Shioyama [/bib_ref] [bib_ref] Hypofractionated proton beam radiotherapy for stage I lung cancer, Bush [/bib_ref] [bib_ref] High-dose proton beam therapy for Stage I non-small-cell lung cancer, Nihei [/bib_ref]. Rates of toxicity were particularly low, with two studies showing no cases of grade ≥3 lung and esophageal toxicities even with hypofractionation [bib_ref] Hypofractionated proton beam radiotherapy for stage I lung cancer, Bush [/bib_ref] [bib_ref] Hypofractionated high-dose proton beam therapy for stage I non-smallcell lung cancer: preliminary..., Hata [/bib_ref]. Two recent studies using proton and carbon ions demonstrated acceptable rates of toxicity with longer median follow-up times [bib_ref] High-dose proton therapy and carbon-ion therapy for stage I nonsmall cell lung..., Iwata [/bib_ref] [bib_ref] Long-term outcome of proton therapy and carbon-ion therapy for large (T2a-T2bN0M0) non-small-cell..., Iwata [/bib_ref]. Two Japanese studies using different proton protocols demonstrated higher rates of rib fractures [bib_ref] Outcomes and prognostic factors for recurrence after high-dose proton beam therapy for..., Kanemoto [/bib_ref] [bib_ref] High-dose proton beam therapy for stage I non-small cell lung cancer: Clinical..., Makita [/bib_ref].
In a 12-year randomized trial at Loma Linda University of 111 patients with early-stage NSCLC treated with hypofractionated PBT, increasing doses were associated with improved OS for the entire cohort [bib_ref] High-dose hypofractionated proton beam radiation therapy is safe and effective for central..., Bush [/bib_ref]. OS at four years was 18%, 32%, and 51% for patients treated with 51, 60, and 70 Gy, respectively. For a subset of patients with peripheral T1 tumors treated with 60 or 70 Gy, LC and OS at four years for were 96% and 60%, respectively. For patients with T2 tumors, LC and OS were improved with dose escalation. Notably, no patients experienced clinically significant radiation pneumonitis or significant declines in pulmonary function one year after PBT.
A study at MD Anderson Cancer Center using a hypofractionated regimen of PBT (87.5 Gy/2.5 Gy fractions) also reported limited toxicities [bib_ref] Toxicity and patterns of failure of adaptive/ablative proton therapy for earlystage, medically..., Chang [/bib_ref]. At median follow-up time of 16.3 months, LC was 89%, and distant metastasis-free survival (DMFS) was 72%. Grade 3 dermatitis was observed in 17% of patients, and no grade 4 or 5 toxicities occurred. When the study was updated in 2017 and included 35 additional patients, OS rates were 86%, 43%, and 28% at 1, 3, and 5 years, respectively [bib_ref] Long-term outcome of phase I/II prospective study of dose-escalated proton therapy for..., Chang [/bib_ref]. At a median follow-up time of 83.1 months, local recurrence-free and distant-metastasis-free survival rates were 85% and 54%, respectively. There were 1 grade 3 dermatitis and 1 grade 3 pneumonitis.
Nantavithya et al. studied the efficacy and toxicity of stereotactic body proton therapy (SBPT) delivered through passive scattering technique compared to those of photon based SBRT [bib_ref] Phase 2 Study of Stereotactic Body Radiation Therapy and Stereotactic Body Proton..., Nantavithya [/bib_ref]. While the study closed early due to poor accrual, outcomes of 19 patients who received 50 Gy in 4 fractions were analyzed. At 3 years, rates for OS were 28% and 90%, LC 88% and 90%, and regional control 48% and 90% in the SBRT and SBPT groups, respectively. In the SBPT group of patients, one case of grade 3 skin fibrosis was observed, but no grade 4 or 5 toxicities occurred.
Previous studies have reported correlations between clinical and PBT-related factors and patient outcomes. In the Loma Linda University study, stage IA patients had decreased tumor relapse rates (13%) compared to stage IB patients (51%) [bib_ref] High-dose hypofractionated proton beam radiation therapy is safe and effective for central..., Bush [/bib_ref]. Stage IA patients also experienced improved OS [bib_ref] Clinical evaluation of proton radiotherapy for non-small-cell lung cancer, Shioyama [/bib_ref] [bib_ref] High-dose hypofractionated proton beam radiation therapy is safe and effective for central..., Bush [/bib_ref]. [bib_ref] Clinical evaluation of proton radiotherapy for non-small-cell lung cancer, Shioyama [/bib_ref]. Similarly, in another study, locoregional recurrences occurred more frequently in stage IB patients (30%) than in stage IA patients (6%) [bib_ref] High-dose proton beam therapy for Stage I non-small-cell lung cancer, Nihei [/bib_ref]. In contrast, other studies found no associations between clinical substage and OS [bib_ref] Long-term outcome of phase I/II prospective study of dose-escalated proton therapy for..., Chang [/bib_ref] [bib_ref] High-dose proton therapy and carbon-ion therapy for stage I nonsmall cell lung..., Iwata [/bib_ref] [bib_ref] Proton beam therapy for patients with medically inoperable stage I nonsmall-cell lung..., Nakayama [/bib_ref] [bib_ref] High-dose proton beam therapy for stage I non-small cell lung cancer: Clinical..., Makita [/bib_ref]. While one study found radiation dose and tumor location to be significant prognostic factors for disease and local recurrence (56), another found no association between radiation dose and local control or PFS [bib_ref] High-dose proton beam therapy for stage I non-small cell lung cancer: Clinical..., Makita [/bib_ref]. Further studies are required to elucidate which clinical factors are associated with better outcomes for early-stage NSCLC patients.
## Locally advanced nsclc
Dosimetric and clinical studies suggest that PBT improved toxicity profiles compared to photon-based treatments. Compared to photon plans, proton plans for the treatment of LA-NSCLC have shown similarly robust clinical target volume (CTV) coverage and lower doses to OARs, including the heart, spinal cord, esophagus, and lung [bib_ref] Improve dosimetric outcome in stage III non-small-cell lung cancer treatment using spot-scanning..., Li [/bib_ref] [bib_ref] Protons safely allow coverage of high-risk nodes for patients with regionally advanced..., Nichols [/bib_ref]. In a prospective longitudinal observational study of patient-reported outcomes, patients treated with PBT experienced decreased esophagitis-related pain as a local symptom compared to patients treated with 3DCRT or IMRT, even though the PBT group received a significantly higher prescription dose [bib_ref] Prospective Study of Patient-Reported Symptom Burden in Patients With Non-Small-Cell Lung Cancer..., Wang [/bib_ref]. In another study, PSPT has demonstrated decreased mean doses to V5-V10 Gy but increased doses to V20-V80 Gy volumes when compared to IMRT (63); the increased V20-V80 Gy likely reflects the 3D-conformal technique of PSPT with utility of large field margins. PSPT showed decreased mean doses to the heart at all measured dose levels. Studies on patients with LA-NSCLC treated with PBT have also demonstrated excellent clinical outcomes with acceptable toxicities [fig_ref] Table 2: Proton therapy studies for locally advanced non-small cell lung cancer [/fig_ref]. A Mayo Clinic study of 79 patients demonstrated similar OS, DMFS, and freedom from locoregional recurrence in patients treated with IMPT vs. IMRT at 1 year's mark [bib_ref] Early Outcomes of Patients With Locally Advanced Non-small Cell Lung Cancer Treated..., Yu [/bib_ref]. Five IMPT patients Similar results were reported in another study of longterm clinical outcomes for 134 patients with stage II-III NSCLC treated with concurrent PBT and chemotherapy [bib_ref] Long-term outcomes after proton therapy, with concurrent chemotherapy, for stage II-III inoperable..., Nguyen [/bib_ref]. Median follow-up time was 4.7 years, and median OS were 40.4 months and 30.4 months in patients with stage II and stage III NSCLC, respectively. Disease-free survival rates were 17% (stage II) and 18% (stage III) at five years. Grade 3 pneumonitis, esophagitis, and dermatitis occurred in 1%, 4%, and 6% of patients, respectively, and one case of grade 4 esophagitis was observed.
A randomized trial comparing PSPT vs. IMRT for locally advanced NSCLC did not demonstrate any difference in the primary endpoint of grade ≥3 pneumonitis or local failure rates [bib_ref] Bayesian Adaptive Randomization Trial of Passive Scattering Proton Therapy and Intensity-Modulated Photon..., Liao [/bib_ref]. It was noted that some of the initial patients on the PSPT arm that experienced pneumonitis could have been avoided with more modern planning techniques. In a National Cancer Data Base analysis of 243,822 patients with stage I-IV NSCLC (348 patients with PBT), 5-year OS was 23.1% and 13.5% for patients treated with PBT and photon-based RT, respectively [bib_ref] National Cancer Database Analysis of Proton Versus Photon Radiation Therapy in Non-Small..., Higgins [/bib_ref]. For patients with unresectable stage II and III NSCLC, PBT was also associated with better survival compared with photon-based RT. IMPT has not been compared directly with IMRT in this setting; further study of PBT vs. IMRT for locally advanced NSCLC is needed and we encourage continued enrollment on RTOG 1308, a randomized phase III trial on stage II-IIIB NSCLC that is currently ongoing (ClinicalTrials.gov Identifier: NCT01993810, estimated completion date: Dec 31, 2025).
Proton therapy may allow for safer dose escalation or delivery of hypofractionated radiation for LA-NSCLC. In a retrospective study of 62 patients with stage III NSCLC, PBT was delivered at a higher dose (74 Gy) than photonbased radiation (63 Gy) but resulted in lower rates of esophagitis and pneumonitis [bib_ref] Early findings on toxicity of proton beam therapy with concurrent chemotherapy for..., Sejpal [/bib_ref]. Grade 3 or higher pneumonitis cases occurred in 2%, 30%, and 9% of the PBT, 3DCRT, and IMRT groups, respectively. Rates of grade 3 or higher esophagitis cases were 5%, 18%, and 44% in the PBT, 3DCRT, and IMRT groups, respectively. A multicenter phase 1 trial evaluating concurrent chemotherapy with increasing dose-per-fraction proton therapy for patients with unresectable stage II-III NSCLC found that hypofractionated proton therapy can be achieved with an acceptable rate of 1-year toxicities [bib_ref] Hypofractionated Proton Therapy with Concurrent Chemotherapy for Locally Advanced Non-Small Cell Lung..., Hoppe [/bib_ref]. Dose arms ranged from 2.5 Gy (RBE) per fraction to 4.0 Gy (RBE) per fraction to 60 or 60.01 Gy (RBE), and no maximum tolerated dose was identified. Two severe adverse events occurred among 7 patients treated at 3.53 Gy (RBE) per fraction [total 60.01 Gy (RBE)], both of which were attributed to chemotherapy. While the study closed early before accrual was met, these preliminary results are encouraging and support further study in a phase 2 trial.
## Post-operative radiotherapy (port) for nsclc
National Comprehensive Cancer Network guidelines recommend PORT for patients with N2 nodal involvement (especially for extracapsular extension) or with microscopic positive margin/residual disease. Because the primary concern regarding use of PORT is the possibility of toxicity negating the gain therapeutically, PBT becomes an attractive treatment option to minimize the risk to benefit ratio by OAR dose reduction.
Initial dosimetric and clinical data are encouraging. In a comparison of IMRT, PSPT, and IMPT plans for patients with resected stage IIA NSCLC treated with PORT, IMPT showed the greatest reduction in dose to OARs including the spinal cord, lung, and heart [bib_ref] An in-silico comparison of proton beam and IMRT for postoperative radiotherapy in..., Berman [/bib_ref]. In a study evaluating clinical outcomes of 61 patients who underwent PORT with PBT or IMRT, PBT was well tolerated with similar grade 3 pneumonitis (3.7% proton vs. 2.9% IMRT) and lower grade 3 esophagitis (3.7% proton, 11.8% IMRT) rates [bib_ref] First Clinical Report of Proton Beam Therapy for Postoperative Radiotherapy for Non-Small-Cell..., Remick [/bib_ref].
One-year OS and local recurrence-free survival rates were also similar between the 2 cohorts. The Proton Collaborative Group is currently exploring the feasibility of a prospective study evaluating cardiopulmonary toxicity after proton vs. photon therapy, which may further elucidate the benefits that may be gained from PORT with PBT (73).
## Reirradiation for recurrent nsclc
Seventy percent of NSCLC patients receive radiation to the thorax as part of definitive or adjuvant treatment [bib_ref] Feasibility of proton beam therapy for reirradiation of locoregionally recurrent non-small cell..., Mcavoy [/bib_ref] , but rates of locoregional relapse remain high (25-40%). Many patients who experience recurrences after prior radiation are not considered candidates for surgery, and response to second-line chemotherapy is generally suboptimal [bib_ref] Second-line or Subsequent Systemic Therapy for Recurrent or Progressive Non-Small Cell Lung..., Noble [/bib_ref]. Concern regarding excess toxicity to OARs often precludes reirradiation (reRT) beyond palliative doses after definitive or postoperative RT. Thus, in the setting of reRT, continued dose reductions to OARs are of particular importance with newer RT innovations. Several studies of proton-based reRT demonstrate that a majority of patients are able to complete reRT, but subsequent toxicities vary in degree and incidence [fig_ref] Table 3: Proton therapy studies for irradiation of recurrent non-small cell lung cancer [/fig_ref].
In the largest series to date of PBT reRT, 79 patients received a median reRT dose of 60 Gy with or without concurrent chemotherapy [bib_ref] Clinical Outcomes of Patients With Recurrent Lung Cancer Reirradiated With Proton Therapy..., Badiyan [/bib_ref]. Median OS (measured from time of reirradiation completion), PFS, and local relapsefree survival were 15.2, 10.5, and 12.9 months, respectively. Acute and late grade 3 toxicities were observed in 6% and 1% of patients, respectively. Three deaths occurred which the authors attributed as possibly due to RT. Two patients died of pulmonary hemorrhage after receiving reRT for recurrences near or at the right hilum. Another patient died of cardiac arrest after receiving reRT concurrently with chemotherapy, for tumor recurrence near the aortic arch.
Investigators at MD Anderson Cancer Center evaluated the toxicity and efficacy of PBT for reRT in 33 patients using a median dose of 66 Gy (RBE) [bib_ref] Feasibility of proton beam therapy for reirradiation of locoregionally recurrent non-small cell..., Mcavoy [/bib_ref]. Thirty-one patients (94%) completed reRT, and median OS was 11.1 months. One-year OS, PFS, locoregional control, and DMFS were 47%, 28%, 54%, and 39%, respectively. Grade 3 or higher esophageal and pulmonary toxicities were observed in 9% and 21% of patients, respectively. No grade 5 toxicities were found.
Another study at MD Anderson Cancer Center evaluated outcomes of reRT (IMRT versus PSPT) with or without concurrent chemotherapy in 102 patients, 97% of whom completed treatment with a median reRT dose of 60.5 Gy (76). Median OS was 14.71 months, and 1-year OS was 53%. Acute grade 3 or higher esophageal and pulmonary toxicities were 7% and 10%, respectively, and concurrent chemotherapy was associated with higher risk of acute grade 2 or higher esophageal toxicities. Lung V10, V20, and mean lung doses were associated with higher risk of grade 2 or higher pulmonary toxicities. Tumor location, IMRT vs. PSPT, and equivalent dose in 2-Gy fractions (EQD2)
were not associated with rates grade 2 or higher esophageal toxicities. No differences between LC, DMFS, or OS were found when comparing IMRT and PBT. For both RT modalities, time to reRT greater than 6 months predicted for better LC. Concurrent chemotherapy and higher EQD2 at reRT independently predicted for better OS. A multi-institutional study evaluated 57 patients treated with PBT with or without concurrent chemotherapy, and 93% of patients completed treatment [bib_ref] Multi-Institutional Prospective Study of Reirradiation with Proton Beam Radiotherapy for Locoregionally Recurrent..., Chao [/bib_ref]. At a median follow-up of 7.8 months, median OS was 14.9 months. Acute or late grade 3 or higher toxicities were observed in 42% of patients, including 6 cases of grade 5 toxicities. Increased toxicity in this experience may have been attributed to a higher rate of concurrent chemotherapy use (66%), leading to significant chemotherapy-related grade 3
or higher toxicities such as neutropenia. Increased overlap with the central airway region was associated with increasing rates of grade 3 or higher toxicities, but decreased overlap was not associated with a clear OS benefit. Increased dose to both the esophagus and heart were also associated with higher rates of grade 3 or higher toxicities, and lower mean esophageal dose, but not heart dosage, translated to improved OS. In this study, concurrent chemotherapy did not predict for better OS.
In another study at MD Anderson Cancer Center, 27 patients were treated with a median dose of 66 Gy using IMPT with or without concurrent chemotherapy [bib_ref] Reirradiation of thoracic cancers with intensity modulated proton therapy, Ho [/bib_ref]. Median OS was 18 months, and 1-year OS was 54%. The rates of freedom from local failure (LF), freedom from locoregional failure, and PFS at one year were 78%, 61% and 51%, respectively. Improved PFS, freedom from LF, and freedom from locoregional failure were associated with a dose of 66 EQD2 or higher. Only 2 patients (7%) experienced late grade 3 pulmonary toxicity, and no patients were found to have any grade 4 or 5 toxicities.
## Challenges of pbt ahead
There are various challenges of using PBT and further study defining its role in the treatment of lung cancer patients especially prospectively is urgently required. The inherent heterogeneity of tissue densities of thoracic organs may impact dose distribution. The density of lung parenchyma is about one-third of that of solid tissues, and protons travel a greater physical distance in low-density tissues. Range uncertainty resulting from uncertainty in Hounsfield units of computed tomography images may also alter proton range and dose distribution. Additional motion uncertainty in the setting of thoracic tumors must be considered as well. Setup error and organ motion from respiration cause geometric displacement of tumors and normal tissues, blurring the dose gradient from target volume to normal tissue. Finally, anatomic changes during treatment may also add to PBT's uncertainty. The interplay effect particularly influences IMPT, as misplacement of individual pencil-beam spots relative to planned positions may result in underdosing the target volume or overdosing critical structures [bib_ref] Motion interplay as a function of patient parameters and spot size in..., Grassberger [/bib_ref]. Various treatment parameters may be modified to improve dose homogeneity in moving targets, such as using larger spot sizes, changing the spot delivery sequence, and employing re-scanning [bib_ref] Motion interplay as a function of patient parameters and spot size in..., Grassberger [/bib_ref] [bib_ref] Reducing Dose Uncertainty for Spot-Scanning Proton Beam Therapy of Moving Tumors by..., Li [/bib_ref] [bib_ref] Proton pencil beam scanning for mediastinal lymphoma: the impact of interplay between..., Zeng [/bib_ref]. Most new proton centers will be using scanning beam (i.e., IMPT) technologies, instead of PSPT. Working closely with our colleagues in medical physics will be paramount in optimizing this technology for lung cancer.
PBT is also significantly more expensive than conventional RT, considering the costs of building and operating particle accelerators. However, PBT may demonstrate improved cost-effectiveness ratios with safer hypofractionation, which has already been shown in photon therapy [bib_ref] Cost Effectiveness of Modified Fractionation Radiotherapy versus Conventional Radiotherapy for Unresected Non-Small-Cell..., Ramaekers [/bib_ref]. In a meta-analysis comparing the cost-effectiveness of different modified fractionation and conventional fractionation schemes in photon therapy for unresected NSCLC patients, Ramaekers et al. demonstrated that modified fractionations yielded higher net monetary benefits, measured by multiplying the number of qualityadjusted life years with the ceiling ratio and subtracting the total costs [bib_ref] Cost Effectiveness of Modified Fractionation Radiotherapy versus Conventional Radiotherapy for Unresected Non-Small-Cell..., Ramaekers [/bib_ref]. PBT may also prevent or delay treatment failure, decreasing additional costs from added hospitalizations and outpatient visits that would otherwise occur for patients treated with photon-based RT [bib_ref] The economic burden of lung cancer and the associated costs of treatment..., Kutikova [/bib_ref]. Reduced toxicities including pneumonitis and esophagitis may also reduce costs associated with hospitalizations when comparing proton to photon therapy [bib_ref] Consensus Statement on Proton Therapy in Early-Stage and Locally Advanced Non-Small Cell..., Chang [/bib_ref]. Demonstrating cost effectiveness and delivering hypofractionated radiotherapy treatments will remain an important financial topic for consideration in adapting PBT for more clinics and hospitals in the United States and also globally [bib_ref] Cost-containment in hypofractionated radiation therapy: a literature review, Hunter [/bib_ref].
From a systemic therapy's point of view, there has been a significant effort to harness the immune system to treat lung cancer. As a result, immune checkpoint inhibitors have changed the treatment paradigm for both SCLC and NSCLC. Following the results of IMpower133, the standard of care for de novo extensive-stage SCLC is chemotherapy followed by maintenance atezolizumab; median OS was 12.3 months, a 2-month benefit compared to patients treated without immunotherapy [bib_ref] First-Line Atezolizumab plus Chemotherapy in Extensive-Stage Small-Cell Lung Cancer, Horn [/bib_ref]. In the CASPIAN trial, durvalumab plus platinum-etoposide improved OS in patients with extensive stage-SCLC compared to platinum-etoposide alone [bib_ref] Durvalumab plus platinum-etoposide versus platinum-etoposide in firstline treatment of extensive-stage small-cell lung..., Paz-Ares [/bib_ref] durvalumab significantly prolonged OS for patients with unresectable, stage III NSCLC following definitive chemoradiation [bib_ref] Overall Survival with Durvalumab after Chemoradiotherapy in Stage III NSCLC, Antonia [/bib_ref]. As immunotherapy improves survival of patients with lung cancer, there is an urgent need to optimize novel RT techniques such as PBT to improve disease control and decrease long-term cardiopulmonary toxicity. Pneumonitis is a serious treatment-related toxicity from both immunotherapy and radiation. In the setting of immunotherapy, improving the therapeutic ratio with PBT has the potential to reduce toxicities such as radiation pneumonitis. Additionally, PBT deposits energy differently than traditional photon therapy leading to a higher relative radiobiologic effect (RBE) that has traditionally been presumed with a fixed ratio of 1.1. There is enthusiasm to investigate how radiation can promote an abscopal effect, a systemic anti-tumor response, in the context of immunotherapy. Future research is needed to study the radiobiologic impact of thoracic PBT on both tumoral and normal tissues, and also the interaction between PBT and immunotherapy. Furthermore, additional studies demonstrating clinical benefit after PBT are necessary to justify the higher financial burden due to increased equipment and personnel cost. However, clinical access and accrual in PBT trials have proven to be difficult, because commercial payers have enacted prior authorization policies and other administrative barriers restricting the use and approval of PBT due to its higher cost and also a perceived lack of clinical benefits [bib_ref] The Insurance Approval Process for Proton Beam Therapy Must Change: Prior Authorization..., Yu [/bib_ref] [bib_ref] Insurance Approval for Proton Beam Therapy and its Impact on Delays in..., Gupta [/bib_ref] [bib_ref] The Insurance Approval Process for Proton Radiation Therapy: A Significant Barrier to..., Ning [/bib_ref]. A study at Rutgers Cancer Institute of New Jersey demonstrated that insurance coverage was not related to diagnosis, reRT, trial enrollment, or model policy guidelines of the American Society for Radiation Oncology [bib_ref] Insurance Approval for Proton Beam Therapy and its Impact on Delays in..., Gupta [/bib_ref]. Over three years, prior authorization (PA) delayed treatment by an average of 3 weeks, and up to 4 months for those requiring appeal. A study at MD Anderson Proton Therapy Center in Houston, Texas similarly found no associations between insurance approval rates and trial enrollment or tumor type [bib_ref] The Insurance Approval Process for Proton Radiation Therapy: A Significant Barrier to..., Ning [/bib_ref]. Ironically, submission of a comparison treatment plan (PBT vs. photon) indicating dosimetric advantages were associated with decreased likelihood of approval. Ambiguity in the decision-making process for PBT approval by commercial payers and subsequent time delays to cancer treatment serve as a significant barrier to patient care [bib_ref] The Insurance Approval Process for Proton Beam Therapy Must Change: Prior Authorization..., Yu [/bib_ref] [bib_ref] Insurance Approval for Proton Beam Therapy and its Impact on Delays in..., Gupta [/bib_ref] [bib_ref] The Insurance Approval Process for Proton Radiation Therapy: A Significant Barrier to..., Ning [/bib_ref]. The logistics and delays of insurance approval were noted to be a factor in enrollment and randomization of a phase II study of SBPT vs. SABR for early-stage NSCLC, leading to early closure [bib_ref] Phase 2 Study of Stereotactic Body Radiation Therapy and Stereotactic Body Proton..., Nantavithya [/bib_ref]. In the future, use of guidelines from professional societies [such as ASTRO PBT Model Policy (91), approved June 2017], which are updated as more evidence becomes available, should increase the efficiency and transparency of the insurance approval process, promoting timely patient care and research [bib_ref] The Insurance Approval Process for Proton Beam Therapy Must Change: Prior Authorization..., Yu [/bib_ref].
# Conclusions
PBT is a promising modality that delivers superior dose distributions and may improve local control without increasing side effects in lung cancer patients. As stage III NSCLC is a heterogeneous population with variable prognoses, we encourage future research to identify subgroups of patients that benefit from PBT based on prognostic factors, such as age, performance status, primary tumor location and size, single vs. multi-station N2 disease, individual patient anatomy, molecular factors, histopathology, re-irradiation, and cardiopulmonary comorbidities [bib_ref] Dose-escalation of locally advanced non-small cell lung cancer with proton beam therapy, Verma [/bib_ref]. As previous studies on clinical outcomes of reRT with PBT yielded variable outcomes, future studies may clarify what benefits may be gained in this subpopulation for which limiting toxicities to OARs is of primary concern. PBT may also be well suited for PORT, due to the majority of cases involving centrally located targets that are less susceptible to motion uncertainties than are peripheral lesions [bib_ref] Proton therapy for post-operative radiation therapy of non-small cell lung cancer, Shepherd [/bib_ref]. Future studies may evaluate possible reductions in cardiac damage with PBT, which is especially valuable in patients with pre-existing comorbidities. The role of PBT needs to be defined and optimized in the context of immunotherapy for lung cancer. Enthusiastic enrollment of patients in clinical trials will help establish evidence-based clinical indications and technology guidelines to clarify the benefit that PBT may offer for patients with lung cancer.
[fig] Figure 1: Axial comparison of an IMPT plan (left) vs. IMRT plan (right) of 50 Gy delivered in 20 daily fractions for a 71-year-old female with locally-advanced NSCLC. A gross tumor volume (GTV) is defined in red. Esophagus is defined in blue. Heart is defined in pink. [/fig]
[table] Table 2: Proton therapy studies for locally advanced non-small cell lung cancer [/table]
[table] Table 3: Proton therapy studies for irradiation of recurrent non-small cell lung cancer [/table]
|
# Introduction
Coronavirus disease 2019 has had a great impact on people's lives worldwide. The disease and its consequences have been the cause of death for more than 5 million individuals in the past two years . Infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for COVID-19, which is characterized by symptoms ranging from fever to the development of severe acute respiratory syndrome (SARS) . The presence of different signs and symptoms determines the different clinical conditions of the disease, which can be asymptomatic, mild, moderate, severe or critical .
In most patients, SARS-CoV-2 infection is asymptomatic, especially in children (approximately 80%), but 20% of patients require admission to the intensive care unit (ICU); the mortality rate of these patients is 25%, with most deaths attributed to severe inflammation and thromboembolic complications [bib_ref] The Dynamic Changes in Cytokine Responses in COVID-19: A Snapshot of the..., Buszko [/bib_ref].
The duration and severity of COVID-19 is related to several factors, including viral (mutations) [bib_ref] Different Mutations in SARS-CoV-2 Associate With Severe and Mild Outcome, Nagy [/bib_ref] and host (age, sex, comorbidities and immunological) factors [bib_ref] Inflammatory Markers and Cytokines in Moderate and Critical Cases of COVID-19, Chang [/bib_ref]. SARS-CoV-2 infection begins with its binding to the ACE2 protein of alveolar epithelial cells, which induces activation of the innate and adaptive immune responses through the production and interaction of chemokines, colonystimulating factors, interferons, interleukins, and tumor necrosis factor-a (TNF-a). These factors increase vascular permeability, determining COVID-19 development [bib_ref] The Looming Storm: Blood and Cytokines in COVID-19, Kaur [/bib_ref] [bib_ref] The Complexity of SARS-CoV-2 Infection and the COVID-19 Pandemic, Torres [/bib_ref].
As immunity is the essential component that determines the type of interaction between the pathogen and the host in any infectious disease; when the immune response is dysregulated, it contributes to disease pathogenesis, as in the case of a "cytokine storm" [bib_ref] Aging in COVID-19: Vulnerability, Immunity and Intervention, Chen [/bib_ref].
The term "cytokine storm" was coined to describe intense production of cytokines in infectious processes responsible for triggering immunopathological reactions [bib_ref] Cytokine Storms in Infectious Diseases, Teijaro [/bib_ref]. Among the inflammatory mediators released by immune cells, the cytokines IFN-a, IFN-g, IL-1b, IL-6, IL-12, IL-18, IL-33, TNF-a and TGF-b are highlighted, and altered levels are associated with different clinical features of COVID-19 [bib_ref] Clinical Features of Patients Infected With 2019 Novel Coronavirus in Wuhan, Huang [/bib_ref] [bib_ref] Inflammatory Markers and Cytokines in Moderate and Critical Cases of COVID-19, Chang [/bib_ref]. Indeed, cytokine storms correlate with the severity and progression of COVID-19 and can result in serious complications such as acute respiratory distress syndrome (ARDS) and multiple organ failure, which are the leading causes of death from the disease [bib_ref] COVID-19, Cytokines, Inflammation, and Spices: How are They Related?, Kunnumakkara [/bib_ref].
Another complexity of COVID-19 is the emergence of new symptoms after the acute infection or illness. Patients infected with SARS-CoV-2 may continue to experience a variety of symptoms after the established period of COVID-19, which are not explained by other causes. These symptoms include fatigue, shortness of breath, "brain fog", sleep disturbances, fevers, gastrointestinal symptoms, anxiety and depression and can persist for months and range from mild to disabling [bib_ref] Editorial: Long COVID, or Post-COVID Syndrome, and the Global Impact on Health..., Parums [/bib_ref].
The immune response is a critical factor in the evolution of COVID-19, and assessment of this response in different populations can provide a better understanding of how the host response influences the severity of the disease in some individuals, even though the majority of those infected with SARS-CoV-2 are asymptomatic or develop mild symptoms [bib_ref] The Immune Response and Immunopathology of COVID-19, Mortaz [/bib_ref]. Accordingly, we evaluated the levels of IL-17, IFN-g, TNF-a, IL-10, IL-6, IL-4 and IL-2 in patients with clinical COVID-19 and long COVID-19, with cases classified as mild, moderate and severe, and associations with risk factors for sex, age and presence of comorbidities.
# Materials and methods
## Study population and sample collection
The present study involved blood samples from 317 patients diagnosed with COVID-19 and classified according to the criteria established by the World Health Organization . The assessment included individuals of both sexes aged 18 years and over, who had not been vaccinated against SARS-CoV-2, and who were attended at the post-COVID-19 outpatient clinic at Universidade do Estado do Para, Hospital Adventista de Beleḿ or Instituto Evandro Chagas from July 2020 to May 2021. The group of post-COVID-19 patients included those who sought the long COVID-19 outpatient clinic at least 30 days after the recovery from acute COVID-19.
A total of 317 patients (n=317) were enrolled in our study aiming to access the cytokine profiles according to the symptoms severe (n=91), moderate (n=53) and mild (n=173), presented in the moment of acute disease. Aiming to analyze the cytokine profile among recovered patients of COVID-19 (n=225) that looked attending the long COVID-19 outpatient clinic at the Universidade do Estado do Para, we divided the individuals into two groups: post-COVID-19 with (n=135) and without (n=90) sequelae (long COVID-19).
A 10-mL blood sample was collected by intravenous puncture using a vacuum collection system containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant.
The samples were transported to the Virology Laboratory of the Federal University of Para, where they were processed to separate plasma and leukocytes; the former was used to determine cytokine levels.
## Serum cytokine levels
Quantification of serum cytokine levels was performed using the flow cytometry technique and Cytometric Bead Array Kit (CBA) Human Th1/Th2/Th17 (BD Biosciences, San Diego, CA, USA) with BD FACS Canto II equipment. All procedures followed the manufacturer's guidelines. The methodology used is based on beads conjugated with a capture antibody: six populations of beads with different fluorescence intensities conjugated to a specific capture antibody for each cytokine are mixed to form the CBA, and results are determined using the FL-3 channel of a flow cytometer. The bead populations were observed according to their respective fluorescence intensities: from least bright to brightest (IL-17< IFN-g < TNF-a < IL-10 < IL-6 < IL-4 < IL-2).
# Statistical analysis
The information obtained was entered into a database in Microsoft Office Excel 2013 software. Normality of the distribution of cytokine levels was analyzed using the Shapiro-Wilk test. Based on the results of the normality test, the evaluation of variations in the plasma levels of these markers between groups was performed using the nonparametric Mann-Whitney test. RStudio 4.0.2 software was used to assess the correlation (Pearson correlation) between IL-6 levels and age in the acute COVID-19 group. The frequency of epidemiological variables was estimated using direct counting, and the significance of differences between the groups was calculated using Fisher's exact test and the chi-square test. All tests were performed using the GraphPad Prism 5.0 program, and results with a p value < 0.05 were considered significant. [fig_ref] TABLE 1 |: -Median cytokine levels evaluated among patients with different clinical COVID-19 and post-COVID-19... [/fig_ref] shows the median cytokine levels of patients with severe and mild/moderate acute COVID-19 (acute SARS-CoV-2 infection) and individuals who recovered from the disease (with and without sequelae).
# Results
Assessment of cytokine levels among patients with acute COVID-19 and individuals in the post-COVID-19 period showed significantly higher levels of IL-17 (p=0.0002; , TNF-a (p<0.0001; and IL-2 (p<0.0001; in the post-COVID-19 group and higher levels of IL-6 (p<0.0001; in the acute COVID-19 group. The cytokines levels IFN-y , IL-10 and IL-4 did not show significant differences between the groups.
Comparison of cytokine levels showed that patients with severe acute COVID-19 had significantly higher levels of IL-6 (p=0.0260; as compared to mild/moderate group. Conversely, in the group of post-COVID-19 subjects, no significant differences in cytokine level among those with severe clinical forms compared to mild/moderate forms were observed .
Some variables, such as sex, age and comorbidities (diabetes mellitus, hypertension, chronic kidney disease, obesity and immunosuppression), which can influence the immune response, were evaluated in relation to the clinical manifestations of patients with COVID-19 and levels of cytokines. When comparing the frequencies of the variables between the groups of patients with COVID-19, all variables showed significant differences [fig_ref] TABLE 2 |: Distribution of frequencies of epidemiological variables between groups with different clinical conditions... [/fig_ref]. The severe group had a higher frequency of males (p=0.0027), patients aged over 60 years (p<0.0001) and comorbidities (p<0.0001).
In the group with acute COVID-19, cytokine levels were evaluated in relation to epidemiological variables [fig_ref] TABLE 3 |: Comparison of cytokine levels in relation to epidemiological variables among patients with... [/fig_ref]. Patients aged 21-40 years had higher levels of TNF-a (p= 0.0257), and those over age 60 had higher levels of IL-6, with a p value close to statistical significance (p=0.0636). Patients without comorbidities had higher levels of TNF-a (p=0.0030), IL-4 (p=0.0020) and IL-2 (p=0.0021); IL-6 levels were higher in patients with comorbidities, but without statistical significance (p=0.0891). Analysis of IL-6 levels with the age in those with acute COVID-19 showed a slightly positive correlation (r= 0.218; p= 0.0381), but there was no correlation between the variables when the evaluation was performed according to the different clinical forms of the disease [fig_ref] FIGURE 3 |: Correlation of IL-6 levels and age in acute COVID-19 [/fig_ref]. IL-6 levels were higher in hospitalized patients admitted to the ICU (median= 15.41) than in those who did not require intensive care (median= 12.59), but the difference was not significant (p= 0.3353).
The post-COVID-19 group was divided into individuals who were undergoing medical follow-up for sequelae (long COVID) and those who had recovered and had no sequelae. Assessment of cytokine levels between these groups showed that individuals with long COVID (sequelae) had higher levels of IL-17 (p=0.0035; [fig_ref] FIGURE 4 |: Cytokine profile in the long COVID-19 [/fig_ref] and IL-2 (p=0.0219; [fig_ref] FIGURE 4 |: Cytokine profile in the long COVID-19 [/fig_ref] and that individuals without sequelae had higher levels of IL-10 (p=0.0406; [fig_ref] FIGURE 4 |: Cytokine profile in the long COVID-19 [/fig_ref] and IL-4 (p<0.0001; [fig_ref] FIGURE 4 |: Cytokine profile in the long COVID-19 [/fig_ref]. A correlogram of cytokine levels in long COVID-19 patients revealed a positive correlation for most cytokines . The cytokines levels IFN-y [fig_ref] FIGURE 4 |: Cytokine profile in the long COVID-19 [/fig_ref] , TNF-a [fig_ref] FIGURE 4 |: Cytokine profile in the long COVID-19 [/fig_ref] and IL-6 [fig_ref] FIGURE 4 |: Cytokine profile in the long COVID-19 [/fig_ref] did not show significant differences between the groups.
# Discussion
The main risk factors for severe and long COVID-19 include age, male sex, smoking, presence of comorbidities (obesity, diabetes, hypertension, heart disease) and variations in the immune response of the host [bib_ref] Clinical Course and Risk Factors for Mortality of Adult Inpatients With COVID-19..., Zhou [/bib_ref] [bib_ref] COVID-19 and Comorbidities: Deleterious Impact on Infected Patients, Ejaz [/bib_ref] [bib_ref] Inflammatory Markers and Cytokines in Moderate and Critical Cases of COVID-19, Chang [/bib_ref]. Evidence suggests that age is the most significant risk factor for the severe form of COVID-19 and related complications [bib_ref] Aging in COVID-19: Vulnerability, Immunity and Intervention, Chen [/bib_ref]. In the present study, subjects in the post-COVID-19 group had higher levels of IL-17, TNF-a and IL-2 as compared to the acute COVID-19 group. In contrast, patients in the acute COVID-19 group had higher levels of IL-6. Younger patients had higher levels of TNF-a, and patients without comorbidities had higher levels of TNF-a, IL-4 and IL-2. Patients over age 60, with comorbidities, had higher levels of IL-6. In the post-COVID-19 group, subjects with long COVID-19 had higher levels of IL-17 and IL-2 and subjects without sequelae had higher levels of IL-10, IL-6 and IL-4. The death rate from COVID-19 is directly related to age, whereby older age is associated with greater risk. The rate varies from 0.4 to 3.3% in patients aged 40 years or younger, from 1.3 to 4.85% in those aged 40-50 years, 3.6-6.4% in those aged 60-70, 8.0 to 12.6% in those aged 70-80, and 14.8%-25.9% in those older than 80 [bib_ref] Aging in COVID-19: Vulnerability, Immunity and Intervention, Chen [/bib_ref]. Overall, rates of hospitalizations, ICU admissions, and death from COVID-19 increase with age (CDC COVID-19 Response Team, 2020), which occurs because of immune system remodeling or immunosenescence, rendering individuals more susceptible to infections, with more severe disease symptoms and lower response to vaccination [bib_ref] Age and Immunity: What Is "Immunosenescence, Pawelec [/bib_ref] [bib_ref] Dynamics of Anti-SARS-CoV-2 IgG Antibodies Post-COVID-19 in a Brazilian Amazon Population, Bichara [/bib_ref]. IL-6 levels showed a slight correlation with advanced age in acute COVID-19. However, there was no correlation of cytokine level with age according to the different clinical forms, which suggests that age may contribute to the increase in IL-6 levels but that these two factors are not sufficient to define the severity of the disease. Although IL-6 levels were higher in older patients with acute COVID-19, cytokine levels showed no statistically significant difference between age groups in this group, possibly due to the small sample size, which is a limitation of the study. In our study, patients with the severe form of acute COVID-19 had higher levels of IL-6 and had some type of comorbidity (diabetes mellitus, hypertension, obesity, and immunosuppression). In the evaluation of patients with acute COVID-19, regardless of disease severity, individuals with comorbidities had higher levels of IL-6 (but without statistical significance). Comorbidities can contribute to the development of COVID-19 and are associated with worse prognosis due to the establishment of complex inflammatory symptoms [bib_ref] COVID-19 and Comorbidities: Deleterious Impact on Infected Patients, Ejaz [/bib_ref] [bib_ref] Epidemiological, Comorbidity Factors With Severity and Prognosis of COVID-19: A Systematic Review..., Fang [/bib_ref].
The IL-6 contributes to inflammation in all insulin target tissues, including fat, the liver, and muscle, indicating the role of IL-6 in driving obesity and in the pathogenesis of systemic insulin resistance [bib_ref] Inflammatory Concepts of Obesity, Rocha [/bib_ref] [bib_ref] Interleukin-6 and Obesity: The Crosstalk Between Intestine, El-Kadre [/bib_ref]. In addition to IL-6, variations in the levels of different cytokines are involved in the development of different comorbidities [bib_ref] An Imbalance in Serum Concentrations of Inflammatory and Anti-Inflammatory Cytokines in Hypertension, Mirhafez [/bib_ref] [bib_ref] Targeting Innate Immune Mediators in Type 1 and Type 2 Diabetes, Donath [/bib_ref] [bib_ref] Cytokines and the Immune Response in Obesity-Related Disorders, Moghbeli [/bib_ref]. Although the cytokines TNF-a, IL-4 and IL-2 were not associated with severe COVID-19 in the present study, lower levels of these cytokines were observed in patients with comorbidities who had acute COVID-19.
The main risk factors associated with COVID-19, related to advanced age and the presence of comorbidities, are conditions characterized by constant low-grade activation of the inflammatory response [bib_ref] Immune Dysfunction in Patients With Diabetes Mellitus (DM), Geerlings [/bib_ref] [bib_ref] Interleukin-6 in Aging and Chronic Disease: A Magnificent Pathway, Maggio [/bib_ref] [bib_ref] Chronic Inflammation (Inflammaging) and its Potential Contribution to Age-Associated Diseases, Franceschi [/bib_ref] [bib_ref] The Immunological Basis of Hypertension, Rodrıǵuez-Iturbe [/bib_ref] , which promote impairment of the general immune response to infection. This suggests that although the clinical profile of severe COVID-19 is induced by an increase in cytokine production, it may be that this increase occurs in a moderate way and is sufficient to intensify inflammatory processes already existing in patients with the aforementioned risk factors and deregulate homeostasis. Furthermore, corticosteroid use in critically ill patients may also be associated with lower levels of cytokines compared to those with less aggressive forms of the disease. IL-6 also contributes to increase vascular permeability and cause interstitial edema and tissue damage [bib_ref] The IL-6-Soluble IL-6Ralpha Autocrine Loop of Endothelial Activation as an Intermediate Between..., Marin [/bib_ref] [bib_ref] Protective Effects of IL-6 Blockade in Sepsis are Linked to Reduced C5a..., Riedemann [/bib_ref] [bib_ref] Interleukin-6, Fibrin D-Dimer, and Coagulation Factors VII and XIIa in Prediction of..., Lowe [/bib_ref]. An increase in these events is related to the severity of COVID-19 and is responsible for many deaths [bib_ref] Incidence of Thrombotic Complications in Critically Ill ICU Patients With COVID-19, Klok [/bib_ref] [bib_ref] High Risk of Thrombosis in Patients With Severe SARS-CoV-2 Infection: A Multicenter..., Helms [/bib_ref] [bib_ref] The Impact of COVID-19 Disease on Platelets and Coagulation, Wool [/bib_ref].
Although the severity of COVID-19 is related to risk factors such as advanced age and comorbidities, Del [bib_ref] An Inflammatory Cytokine Signature Predicts COVID-19 Severity and Survival, Valle [/bib_ref] noted that elevated serum levels of IL-6, IL-8, and TNF-a in COVID-19 patients at the time of hospitalization were strong and independent predictors of patient survival. IL-6 was one of the most robust prognostic markers of survival (surpassing CRP, D-dimer and ferritin), and elevated IL-6 levels were associated with severity and predictive of poor outcome. In our study, among the cytokines evaluated, only high levels of IL-6 were associated with the severity of COVID-19, confirming the relevance of this cytokine in the outcome of the disease. In this sense, maintenance of high levels of IL-6 in patients with the severe form of COVID-19 might be mainly related to the consequence of the inflammatory process resulting from the main risk factors for the disease, the presence of different comorbidities and advanced age and will contribute to intensifying the inflammatory process and the development of severe clotting events.
Some studies have evaluated symptoms related to long [bib_ref] Symptoms, Pulmonary Function, and Functional Capacity Four Months After COVID-19, Abdallah [/bib_ref] [bib_ref] Clinical Features of Patients Infected With 2019 Novel Coronavirus in Wuhan, Huang [/bib_ref] [bib_ref] 6-Month Consequences of COVID-19 in Patients Discharged From Hospital: A Cohort Study, Huang [/bib_ref] [bib_ref] Sequelae in Adults at 6 Months After COVID-19 Infection, Logue [/bib_ref] [bib_ref] Post-COVID Syndrome in non-Hospitalised Patients With COVID-19: A Longitudinal Prospective Cohort Study, Augustin [/bib_ref]. However, most of these studies evaluated the long COVID-19 symptoms among patients who were hospitalized, i.e., only from patients who had severe COVID-19. [bib_ref] Post-COVID Syndrome in non-Hospitalised Patients With COVID-19: A Longitudinal Prospective Cohort Study, Augustin [/bib_ref] evaluated post-COVID syndrome in outpatients, but in order to correlate clinical symptoms with IgG antibodies, they did not evaluate molecular markers of inflammation. Cytokine IL-6 has been proposed as a potential mediator of neuropsychiatric symptoms of long COVID-19, possibly related to its persistence [bib_ref] Interleukin-6 as Potential Mediator of Long-Term Neuropsychiatric Symptoms of COVID-19, Kappelmann [/bib_ref]. This relationship was not observed in our study, since individuals in the post-COVID-19 group had lower levels of IL-6, compared to patients with acute COVID-19. Furthermore, in the post-COVID group there was no significant difference in IL-6 levels between individuals with and without sequelae in the COVID-19 group. The relationship of IL-6 levels with the development of symptoms in long COVID-10, including neuropsychiatric symptoms, needs to be better investigated.identified a likely risk of developing prolonged symptoms of COVID-19 in hospitalized patients, noting that convalescent patients had a lower frequency of IL-10 + CD4 + T cells, IL-17 + CD4 + T cells, and IL-6 + B cells, compared to individuals with acute COVID-19. Although in our study we identified lower levels of IL-10 in the long COVID-19 group compared to subjects without sequelae in the post-COVID-19 group, IL-17 levels were higher in those subjects with long COVID-19. Furthermore, no difference was observed in IL-6 levels between the groups. The difference between the results presented here and those observed bymay be due to the clinical moment at which the patients were analyzed. In our study, evaluated individuals in the post-COVID-19 period, including individuals with and without sequelae, as well as individuals who had acute COVID-19 with different clinical manifestations (mild, moderate and severe), showing that the development of long COVID-19 may not be related only with the severity of acute COVID-19. In addition, it was possible to identify immuno-inflammatory molecular patterns, through the characterization of the cytokine profile, which may be related to long COVID-19.
In relation to individuals in the post-COVID-19 group, higher levels of IL-10 and IL-4 were observed in the group that did not present sequelae after the disease. This suggests better control of the inflammatory process due to increased levels of anti-inflammatory cytokines in these individuals. Furthermore, individuals who continued to experience sequelae after infection (long COVID) had significantly higher levels of IL-17 and IL-2 and lower levels of Il-4 and IL-10, suggesting a possible "molecular signature" for long COVID characterized by a Th17 inflammatory profile with a reduced anti-inflammatory response mediated by IL-4 and IL-10, that must be confirmed by other studies enrolling a largest sample size. The correlation of cytokine levels in the long COVID-19 group was positive among most of the cytokines evaluated. Hence, although the disease is associated with variations in levels of certain cytokines, it appears to induce an increase in levels of cytokines related to different types of inflammatory response. Post-COVID syndrome (long COVID) has been associated with residual inflammation, organ damage, preexisting health conditions or nonspecific effects due to hospitalization or prolonged ventilation [bib_ref] Post-Acute COVID-19 Syndrome. Incidence and Risk Factors: A Mediterranean Cohort Study, Moreno-Peŕez [/bib_ref] , conditions easily related to the severe form of the disease. Nonetheless, it is important to emphasize that SARS-CoV-2 shows tropism for the nervous system, and neurological manifestations can be observed in patients with different clinical forms of the disease (mild, moderate and severe), ranging from anosmia, ageusia, headache, stroke, Guillain-Barrésyndrome, seizure and encephalopathy. Thus, it is possible that microvascular The consequences of the long COVID-19 are impacting health care resources, as up to 30% of the associated health burden may be due to prolonged disability induced by COVID-19 rather than mortality [bib_ref] Editorial: Long COVID, or Post-COVID Syndrome, and the Global Impact on Health..., Parums [/bib_ref]. Therefore, understanding the biological basis of long COVID-19 will allow us to identify factors that predispose to the development of long-term complications and guide effective therapies. In this sense, several studies have been developed, such as the REACT-GE study (in partnership with Genomics England) that aims to look for biological signatures that may be linked to the development of long COVID-19 and whether genes affect the severity of the COVID-19 and its long-term progression (Post-COVID-19 syndrome: in it for the long haul, 2021) Some studies have evaluated the molecular signature of acute COVID-19 cytokines by different methodologies [bib_ref] The Relationship Between Cytokine and Neutrophil Gene Network Distinguishes SARS-CoV-2-Infected Patients by..., Freire [/bib_ref] [bib_ref] Longitudinal Profiling of Respiratory and Systemic Immune Responses Reveals Myeloid Cell-Driven Lung..., Szabo [/bib_ref] [bib_ref] Severe COVID-19 Shares a Common Neutrophil Activation Signature With Other Acute Inflammatory..., Schimke [/bib_ref]. In our study, IL-6 levels were associated with the severe form of acute COVID-19.also identified IL-6 persistence in severe disease, along with the TNF-a. In contrast, other studies have shown that disease severity was related to elevated levels of myeloid chemoattractants and neutrophil activation [bib_ref] The Relationship Between Cytokine and Neutrophil Gene Network Distinguishes SARS-CoV-2-Infected Patients by..., Freire [/bib_ref] [bib_ref] Longitudinal Profiling of Respiratory and Systemic Immune Responses Reveals Myeloid Cell-Driven Lung..., Szabo [/bib_ref] [bib_ref] Severe COVID-19 Shares a Common Neutrophil Activation Signature With Other Acute Inflammatory..., Schimke [/bib_ref]. Possible differences in results (molecular profiles) observed between different studies may be due to differences in methods used for investigation, different times of sample collection during infection, or even due to differences in the immunogenetic background of the investigated population. Therefore, identification of a molecular profile in relation to long COVID-19 still needs to be further investigated. Even though several studies have evaluated the main symptoms of long COVID-19 [bib_ref] Symptoms, Pulmonary Function, and Functional Capacity Four Months After COVID-19, Abdallah [/bib_ref] [bib_ref] Post-COVID Syndrome in non-Hospitalised Patients With COVID-19: A Longitudinal Prospective Cohort Study, Augustin [/bib_ref] [bib_ref] Sequelae in Adults at 6 Months After COVID-19 Infection, Logue [/bib_ref] [bib_ref] Persistence of Symptoms After Discharge of Patients Hospitalized Due to COVID-19, Wu [/bib_ref] [bib_ref] Clinical Features of Patients Infected With 2019 Novel Coronavirus in Wuhan, Huang [/bib_ref] [bib_ref] 6-Month Consequences of COVID-19 in Patients Discharged From Hospital: A Cohort Study, Huang [/bib_ref] , none of them investigated the association of clinical manifestations with the immunoinflammatory profile of cytokines. Our study showed, for the first time, evidence for the existence of a long COVID-19associated cytokine profile in a cohort of the Brazilian Amazon region. Further evaluated in studies conducted with larger cohorts from distinct geographic areas is needed.
Our results are important because it was possible to identify differences in cytokine synthesis and identify characteristic profiles in acute COVID-19 (higher levels of IL-6) and long COVID-19 (high levels of IL-2 and IL-17).
In conclusion, the present study shows that advanced age, the presence of comorbidities and elevated serum IL-6 levels are associated with the severity of COVID-19 and represent good markers to differentiate severe COVID-19 from mild clinical forms. Furthermore, high serum levels of IL-17 and IL-2, as well as low levels of IL-4 and IL-10, appear to constitute a long COVID-19 cytokine profile (molecular signature), and identification of these markers as a potential target may establish more adequate treatment and prevention strategies for specific groups.
# Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
# Ethics statement
The studies involving human participants were reviewed and approved by National Research Ethics Committee (CAEE: 33470020.1001.0018). The patients/participants provided their written informed consent to participate in this study.
# Author contributions
# Funding
The study was supported by the National Council for Scientific and Technological Development (CNPQ #401235/2020-3); Fundacão Amazônia de Amparo a Estudos e Pesquisa do Para( FAPESPA #005/2020 and #006/2020) and Secretaria de Estado de Ciência, Tecnologia e Educação Profissional e Tecnológica (#09/2021).
[fig] FIGURE 1 |FIGURE 2 |: Cytokine levels according to different clinical manifestations of COVID-19. Comparison of cytokine levels (A) IL-17, (B) IFN-y, (C) TNF-a, (D) IL-10, (E) IL-6, (F) IL-4 and (G) IL-2, between patients with acute COVID-19 and individuals in the post-COVID-19 period. Mann-Whitney test. Cytokine profiles in acute and post-COVID-19 syndrome. Comparison of cytokine levels of patients with severe and mild/moderate COVID-19 and post-COVID-19. Mild/Mod: mild/moderate. Mann-Whitney Test. (A) IL-17, (B) IFN-y, (C) TNF-a, (D) IL-10, (E) IL-6, (F) IL-4 and (G) IL-2. [/fig]
[fig] FIGURE 3 |: Correlation of IL-6 levels and age in acute COVID-19. Correlation of all patients and patients with mild/moderate and severe forms of the disease. Cellular and Infection Microbiology | www.frontiersin.org June 2022 | Volume 12 | Article 922422 [/fig]
[fig] FIGURE 4 |: Cytokine profile in the long COVID-19. Comparison of cytokine levels (A) IL-17, (B) IFN-y, (C) TNF-a, (D) IL-10, (E) IL-6, (F) IL-4 and (G) IL-2, between individuals with and without post-COVID-19 symptoms (sequelae). Mann-Whitney test. Cellular and Infection Microbiology | www.frontiersin.org June 2022 | Volume 12 | Article 922422 inflammation in cells of the nervous system during infection may trigger mild symptoms of the disease (Lechner-Scott et al., 2021), which may persist even after the infection has resolved. [/fig]
[table] TABLE 1 |: -Median cytokine levels evaluated among patients with different clinical COVID-19 and post-COVID-19 conditions. [/table]
[table] TABLE 2 |: Distribution of frequencies of epidemiological variables between groups with different clinical conditions of COVID-19. [/table]
[table] TABLE 3 |: Comparison of cytokine levels in relation to epidemiological variables among patients with acute COVID-19. [/table]
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Intraosseous Leiomyoma of the Tibia. A Case Report
# Introduction
Leiomyoma is a benign smooth-muscle tumor that most commonly arises in the uterus, the gastrointestinaltract, and the skin. It constitutes 70% to 80% of all benign mesenchymal tumors, with uterine leiomyomata being the most common smooth-muscle tumor in women. Leiomyomata are infrequently seen in the extremities and rarely seen in bone [bib_ref] Leiomyoma of the hand: a case report and review of the literature, Boyd [/bib_ref]. To the best of our knowledge, the eleven reported cases occurred in peripheral skeleton. Five cases were intraosseous lesion in long bones, including neck of the femur,ulna [bib_ref] Intraosseous leiomyoma of the ulna: a case report, Zikria [/bib_ref] , fibula [bib_ref] Intraosseous leiomyoma: a report of two cases, Lafosse [/bib_ref] , tibia [bib_ref] Intraosseous angioleiomyoma the tibia: A case report, Lau [/bib_ref] and distal femur [bib_ref] Intraosseous leiomyoma of the distal femur: a case report and review of..., Sung [/bib_ref]. Three cases were periosteal leiomyomata [bib_ref] Hameed MD A Painful Tibial Mass in a 37-year-old Man, Seena [/bib_ref] and two cases arose from the hip bone [bib_ref] Intraosseous angioleiomyoma the tibia: A case report, Lau [/bib_ref]. In addition to three cases of intraosseous leiomyomatosis secondary to disseminated leiomyomatosis have been reported [bib_ref] Serial spread of benign metastasizing leiomyoma to the thoracic spine, Jayakody [/bib_ref]. We describe a case of a patient with painful intraosseous leiomyoma involving the proximal aspect of the tibia and review the literature about intraosseous leiomyoma.
## Case presentation
A 73-year-old female presented with a four year history of a painful swelling in the left knee that was slowly growing, with a gradual increase in pain at the same time.. She had unnoticeable medical history. Pain was aggravated by weight bearing, sitting straight, but she enjoyed full range of motion, and her general health was not affected. Osteoarthritis of left knee joint was suspected in another clinic, and she was treated conservatively with analgesic drugs. The pain persisted despite administration of nonsteroidal anti-inflammatory drugs (NSAIDs) for long time; she visited our outpatient clinic for consultation. Clinical examination revealed local tenderness over medial aspect of proximal tibia, small well defined swelling over the medial proximal tibia was palpable. It was firm in consistency and was not red or hot to touch. Roentgenography revealed a small well-defined osteolytic lesion with thinning out of the cortex over medial part of proximal tibia, surrounded by a thin sclerotic margin . Computed tomography (CT) scan revealed radiolucent lesion eroding the cortex, with central calcification . Magnetic resonance imaging (MRI) revealed a hypoechoic mass in both T1and T2 weighed mages, not suppressed on T2 fat suppression view T2FS, no fluid-fluid level, it was enhanced with Gadolinium, the lesion had extra osseous extension .Technetium99 (Tc99) bone scan revealed an increased uptake. Laboratory studies were within normal range. CT-guided biopsy was performed, and the pathology showed a picture of leiomyoma, consisting of moderate cellular proliferation of smooth-muscle cells with little cellular pleomorphism, with no mitoses or necrosis. Immunohistochemical stains showed positive stain for smoothmuscle actin (SMA). The diagnosis of a benign intraosseous leiomyoma was made. Since the definite diagnosis was established, surgical excision with wide margin was decided to avoid local recurrence. The specimen consisted of gray-white soft tissue mass without apparent central necrosis or fluid content by macroscopic ex amination. [fig_ref] Figure 3: Intraoperative [/fig_ref]. Microscopically, the tumor composed of fascicles of spindleshaped cells with elongated nuclei and fusiformor blunt ends. No atypical mitosis was seen [fig_ref] Figure 3: Intraoperative [/fig_ref]. Immunohistochemical stains to SMA were diffusely positive. The surgical margin was free of tumor. Otherwise, the histological exams were compatible with the results of biopsy and confirmed the diagnosis of intraosseous leiomyoma. The patient symptoms had improved after surgery, pain gradually improved postoperatively until it disappeared by two months after surgery, weight bearing was allowed from next postoperative day, convalescence passed uneventfully. At the latest follow-up after 18 months following surgery, the range of motion of left knee achieved full extension and flexion to 130 degrees without discomfort without any instability or residual pain of the knee. No local recurrence, malignant change, or distal metastasis had occurred.
# Discussion
Leiomyomata are very rare in the bone. Most of reported intraosseous leiomyomata arise from axial skeleton. First case of intraosseous leiomyoma was in the maxillary tooth socket and it was described in 1976 by Rhatigan and Kim [bib_ref] Leiomyoma arising adjacent to a maxillary tooth socket: an intraosseous leiomyoma presenting..., Rhatigan [/bib_ref]. Loyola et al. revised 11cases involving the mandible and maxilla [bib_ref] Intraosseous leiomyoma of the mandible, Loyola [/bib_ref]. The lesion is recorded in other areas of axial skeleton such as skull, spine, as well as a single case of the rib has been reported [bib_ref] Intraosseous leiomyoma in a rib. A case report, Ganyusufoglu [/bib_ref] , while only limited cases of intraosseous leiomyomata in peripheral skeleton have been published. In published English literature, eleven cases were found with peripheral skeleton Journal of Orthopaedic Case Reports Volume 6 Issue 2 April -June 2016 Page 81-85 | | | | (Limbs and pelvis except sacrum). Average age for the reported cases including our case was 44.5y±10y, 4male representing 33.3% and 8 female representing 66.6%, with range between 37-73years; our case is the oldest, at 73years, among all reported cases of intraosseous leiomyoma. Bone predilection was noticed among the reported cases, as in six out of twelve cases the lesion was in the tibia 50%, two cases of femur 16.6%, two cases in the pelvic bone 16.6%, one case of fibula (8.3%) and ulna (8.3%) each. Another trend that deserves attention is location of tumor within the bone; we noticed that primary intraosseous leiomyomata of peripheral skeleton seem to arise from epi-metaphyseal region, while four cases were in the distal epimetaphysis, three were in the proximal metaphysis, three cases with periosteal located lesion at diaphyseal level, one case in the pubic bone and another case was in in the iliac crest [fig_ref] Table 1: Table 1 [/fig_ref]. Diagnosis of this tumor as well as of the previous reported cases was not straightforward, it included many differential diagnoses, nopathognomonic clinical sign was related to intraosseous leiomyoma though nonspecific pain is the main presentation of intraosseous leiomyoma. Swelling and difficult weight bearing are also less frequent complaints. Radiographic differential diagnosis includes a variety of benign primary bone tumors, i.e. osteoid osteoma, non-ossifying fibroma, hemangioma, adamantinoma in addition to malignant tumors such as leiomyosarcoma, and metastasis. In osteoid osteoma, there is little sclerosis surrounding a central calcified nidus which is also surrounded by new bone formation. CAT scan clearly reveals the nidus in most cases though it might be easily missed in MRI which usually reveals bone marrow edema. In non-ossifying fibroma, the lesion is typically sharply demarcated, asymmetrical, cortically based lucency with a thin sclerotic rim. It often appears as a multiloculated lesion. It is located in the metaphysis adjacent to the physis. As the patient ages it seems as if it migrates away from the physis, there is no associated periosteal reaction, cortical breach or associated soft tissue mass. On MRI appearances are variable, and depend on when along the development and healing phase the lesion is imaged. Initially the lesion has high or intermediate T2 signal with a peripheral low signal rim corresponding to the sclerotic border as it matures and begins to ossify, the signal becomes low on all sequences, and contrast enhancement is also variable. In surface hemangiomas, it could be associated with cortical thickening, cortical erosion, and a lytic focus resembling a nidus on CT. A soft tissue mass extending from the cortex might be seen but is an infrequent finding [bib_ref] Leiomyoma arising adjacent to a maxillary tooth socket: an intraosseous leiomyoma presenting..., Rhatigan [/bib_ref]. In adamantinoma which was included in differential diagnosis because of the tibial location of the lesion, it appears as a multilocular or slightly expansible osteolytic lesion. This may be visualized as areas of lysis interspersed with areas of sclerosis. Lesions tend to have an eccentric epicenter and a lack of periosteal reaction. There may be a locally aggressive disease at presentation. On MRI, it might be presented as one of two morphologic patterns either a solitary lobulated focus or multiple small nodules in one or more foci [bib_ref] Cortical lesions of the tibia: characteristic appearances at conventional radiography, Levine [/bib_ref]. In leiomyosarcoma, radiological feature depends on grade of malignancy, in high grade leiomyosarcomas there is illdefined, irregular osteolytic lesion with a moth-eaten or permeative pattern of osseous destruction radiographically, and diffuse involvement and destruction of the medullary bone histologically. On the other hand, low grade tumors exhibit diffuse involvement of the marrow spaces, and have radiographic features, including a geographic pattern of bone destruction and a sclerotic rim [bib_ref] Primary leiomyosarcoma of bone: a clinicopathologic, immunohistochemical, and ultrastructural study of 33..., Antonescu [/bib_ref]. Calcification is exceedingly rare in leiomyosarcoma. In MRI, it may frequently demonstrate cystic foci within. The lesion appears so intense to muscle T1, while inT2 intermediate to hypo intense to neighboring fat, and predominantly hyper intense in T2FS. Metastasis is not a far possibility, it would occur from primary carcinomas, including lung, breast, kidney, and pancreas. The thin capsule seen around the lesion, as well as its oval shape and sharply marginated edge made metastasis less likely. We considered adamantinoma far less likely in the differential diagnosis, as histologically this neoplasm is noted for its heterogeneity and presence of epithelial islands. A malignant spindle cell tumor was not considered because the tumor lacked pleomorphism, high rate of mitoses, and necrosis. The sclerotic margins seen on the conventional radiograph and in the histologic sections also supported the benign nature of this tumor. However, absence of signs of malignancy must be carefully documented in order to rule out leiomyosarcoma. Histologically; leiomyoma is classified into three histological categories: solid leiomyoma, vascular leiomyoma (or angioleiomyoma), and epithelioid leiomyoma. While angioleiomyoma is the most prevalent type in soft tissues, Abdelaal A et al solid leiomyoma represents the majority of cases of intraosseous leiomyomata, it exhibits the same histological features as leiomyoma at other sites. It is composed of a proliferation of spindle-shaped cells arranged in thick intersecting bundles. Mitoses are very rarely seen (low rate of mitosis 0-4per ten high power fields) with moderate cellularity and no necrosis.
Angioleiomyomas are further classified into three histological categories: solid (the most common type in soft tissues), venous, and cavernous. However, all three patterns are often present within the same tumor. Similarly, reported cases of intraosseous angioleiomyoma showed no distinctive histological features compared to soft-tissue angioleiomyomas. In published cases, three cases out of eleven were histologically angioleiomyoma, two were in tibia and one in the iliac bone. The use of immunohistochemical staining was helpful in this case, the tumor cells stained strongly for muscle markers, including smooth muscle actin and desmin, thus confirming this tumor's smooth muscle origin. The neoplastic cells did not stain for neural, fibrous, or epithelial markers (S-100, CD34, and AE1/3, respectively). The standard treatment for intraosseous leiomyoma is extensive tumor excision with wide margin followed by packing of the cavity with autologous cancellous bone graft [bib_ref] Intraosseous leiomyoma of the ulna: a case report, Zikria [/bib_ref]. Internal fixation is required when the residualbony defect is large. Complete excision of intraosseous leiomyoma is typically curative with excellent prognosis. Till now, no cases of recurrences have been reported after complete excision [bib_ref] Intraosseous leiomyoma of the ulna: a case report, Zikria [/bib_ref] [bib_ref] Intraosseous leiomyoma: a report of two cases, Lafosse [/bib_ref] , however, regular follow-up is necessary to observe the risk of recurrence.
In only one of the previously reported cases, reconstruction using bone graft alone was not enough, and reconstruction by TKA was performed due to difficulty in preserving the articular cartilage and thinning out of the bone [bib_ref] Hameed MD A Painful Tibial Mass in a 37-year-old Man, Seena [/bib_ref].
Otherwise, all known cases were treated either by en-bloc excision, surgical resection or curettage. In addition to bone graft, and in all cases, results were satisfactory to excellent with no recurrence, malignant change, or distant metastases, early recovery and restoration of function.
In our case, we performed wide tumor excision, the bone defect was not large enough to necessitate internal fixation. Patient satisfaction after surgery was good, with improvement of pain, and preserved function of the knee. Pain was completely relieved by two months post-operatively. There was no recurrence, malignant change or distant metastases at the latest follow up visits.
# Conclusion
Diagnosis of Intraosseous leiomyoma of the extremities is difficult due to extreme rarity of the tumor and absence of pathognomonic radiological signs in X-ray, CAT, or even MRI. The exact diagnosis is only achieved by histopathological examination and with immunohistochemistry stains, which can differentiate it from malignancy, especially from the much less rare leiomyosarcoma. Orthopedic oncologists have to include this rare benign tumor in the differential diagnosis of any intraosseous lesion with gradually worsening and long-standing pain, despite of benign imaging characters. Different histological patterns of leiomyoma do exist, however there is no difference in prognosis or treatment options. Treatment standard includes wide excision with autologous bone graft whenever possible. Internal fixation may be necessary if the bone defect is large or there is thinning out of the cortex which may lead to pathological fracture. Intraosseous leiomyoma is a rare tumor; it lacks a pathognomonic radiological sign which makes its diagnosis quite difficult. It is better to include this tumor in the differential diagnosis of painful benign intraosseous lesions.
## Clinical message
[fig] Figure 3: Intraoperative.A: Intraoperative photo showing the mass extruding from the anteromedial surface of the tibia. B: Immediate photo after tumour excision. C: Microscopic picture of the lesion with spindle cells, low cellularity and smooth muscle stroma. D: Immunohistochemical stain (α-SMA) which is specific for smooth muscles. [/fig]
[fig] Figure 4: Postoperative XP (last follow up). A: Anteroposterior view of the knee joint, showing the filled bone defect in the upper medial part of tibial metaphysis B: Lateral view of the knee joint, showing the filled bone defect in the upper medial part of tibial metaphysis [/fig]
[table] Table 1: Table 1: Summary of all reported cases of intraosseousleiomyomata [/table]
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Alimentary and Pharmaceutical Approach to Natural Antimicrobials against Clostridioides difficile Gastrointestinal Infection
Citation: Tortajada-Girbés, M.; Rivas, A.; Hernández, M.; González, A.; Ferrús, M.A.; Pina-Pérez, M.C.
# Introduction
Since 2017, following the first publication of the most antibiotic-resistant bacteria by the World Health Organization (WHO) [1], there has been an urgent call internationally to boost research and development on novel strategies based on natural or synthetic antibiotics to effectively fight against these microorganisms. In fact, it is expected that by 2050, more people will die due to multiresistant bacteria than cancer pathologies [1]. Among these highly antimicrobial-resistant bacteria, Clostridioides difficile is becoming a concerning threat worldwide. These Gram-positive anaerobic and spore-forming bacteria has become the most frequent causal agent of hospital-acquired intestinal infection in Europe and all over the world, causing close to 30,000 death per year in the US (estimated mortality close to.7%). According to a recent surveillance (2019) developed by the European Centre for Disease Prevention and Control (ECDC) in collaboration with the US Centers for Disease Control and Prevention (CDC), the incidence of C. difficile infections (CDI) has increased close to 70% in relation to values included in the previous European surveillance study . The consequences of CDI are fatal in some cases, with high rates of morbidity and mortality (16-23%), starting with diarrhea, which can result in major complications including loss of intestinal barrier function, pseudomembranous colitis, toxic megacolon, colon perforations, and sepsis. CDI is caused by the bacterial production of two toxins, A and B, and also a third binary toxin produced by some strains of C. difficile (including the hypervirulent NAP1/027 epidemic strain). High rates of CDI recurrence have also been progressively detected in primary infected patients managed under an antibiotic treatment, with close to 20-25% of infected and recovered patients suffering a second episode.
The bacterium C. difficile is spread via the fecal-oral route. The progression of colonization and infection occurs via two routes, namely the presence (endogenous infection) or acquisition (exogenous infection) of CD and the altered composition of gastrointestinal microbiota. Several factors have been described as being responsible for the increased incidence of CDI and its fatal consequences in recent years. Among them, one of the most significant reasons for CD microbiota domination and severe gut infection is linked to the exposure of patients to broad-spectrum antibiotics against which CD is resistant, thereby favoring extensive gastrointestinal colonization and toxin production. Some of the antibiotics able to disrupt the healthy microbiota balance in the gastrointestinal tract (GIT), thereby allowing proliferation of C. difficile, are ampicillin, amoxicillin, cephalosporins, clindamycin, and fluoroquinolones.
Other relevant factors in relation to CDI progression and the severity of its consequences include (i) age, (ii) disruption of the host defense (low serum antibody response to C. difficile toxins), and (iii) previous health status of patients. Important research efforts are nowadays focused on further understanding the observed increase in CDI infection within communities rather than just in specific healthcare or hospital facilities . Asymptomatic patients act as agents of pathogen reservoirs and vehicles of infection transmission to immune-compromised individuals.
## Current antibiotic therapies applied against clostridioides difficile
Among the most effective antibiotics used to date against CDI are vancomycin, fidaxomicin, and metronidazole, which have been applied as the first line of therapy in the last 30 years. Antibiotics targeted at inactivating C. difficile act mainly against bacterial DNA (by means of DNA damage), causing inhibition of protein synthesis and enzymatic activity (pyruvate and ferredoxin oxidoreductase) or disruption of the membrane potential and peptidoglycan synthesis. A significant reduction in the effectiveness of antibiotic therapies has been observed in recent years. This includes increased resistance and "resistome" transference in C. difficile as well as the emergence of new hypervirulent strains. According to Peng et al., in recent years, causative events resulting in increased C. difficle antibiotic resistance include (i) transfer of mobile genetic elements, (ii) selective pressure in vivo resulting in gene mutations, (iii) altered expression of redox-active proteins, (iv) iron metabolism, (v) DNA repair, and (vi) biofilm formation. Failure rates with authorized treatments are in the range of 14-22% (14% with vancomycin and 22% with metronidazole), while the recurrence rate is also high (25-30%). Similar results have been observed for the most recently applied treatment with fidaxomicin, with 12-15% infection recurrence observed in recent decades .
In the last 20 years, very few new antibiotics have been successfully developed against CDI. Among the most recently developed antibiotics are cadazolid, surotomycin, ramoplanin, nitazoxanide, rifampin, and rifaximin. Unique properties to fight against C. difficile have been attributed to cadazolid (minimum inhibitory concentration (MIC) = 0.125 µg/mL; minimum bactericidal concentration (MBC) (3 log 10 reduction) = 2 × MIC). The mode of action of this antibiotic is focused on protein synthesis inhi- bition (toxin inhibition) and suppression of spore formation, which increases C. difficile susceptibility to treatment. Among the main advantages of this compound are (i) strong in vitro and in vivo effectiveness; (ii) the capability to inhibit C. difficile infection in a gut model, thereby maintaining normal microbiota at correct levels; and (iii) reduced rates of infection recurrence. Other novel drugs are nowadays in Phase 1, 2, and 3 trials, with ibezapolstat (ACX-362E), CRS3123, and NVB302 the most recently developed drugs for the oral treatment of CDI.
However, under pressure, the C. difficile genome sets up a variety of resistance mechanisms responsible for the observed capability of CD to persist and be recurrent even when clinic antimicrobial strategies are applied. In fact, conjugation, transduction, and/or transformation of mobile genetic entities, and specifically transposons, within different C. difficile strains and/or between C. difficile and other bacterial species are among the remarkable resistance mechanisms associated with this microorganism. Additionally, acquired antibiotic resistance by means of alterations in antibiotic targets and/or metabolic pathways has been described as significant contributing factors to proliferation and increased incidence of CDI, particularly in the last decade. In fact, aggressive symptomatology hypervirulent strains have been emerging in recent times (2010-2020), including C. difficile BI/NAP1/ribotype 027 and BK/NAP7/ribotype 078, which are resistant to fluoroquinolones and cephalosporins. This has contributed to the increase in antibiotic resistance, along with other relevant exacerbating virulence factors such as increased sporulation and surface layer protein adherence capability of these strains.
The biofilm-forming capacity of C. difficile has significantly contributed to the increase in antibiotic resistance. In fact, according to Semenyuk et al., C. difficile biofilms confer a 100-fold increase in metronidazole resistance. It has been proven that the capacity of sessile bacteria to form biofilms in the mucus layer of the gut plays a fundamental role in gut health and disease. Although very little information has been published to date regarding the in vivo biofilm-forming capacity of C. difficile, it is well known that these multicellular structures could potentially protect bacteria from cellular immune responses and from antibiotics. Moreover, the recurrence of CDI can be associated with biofilm persistence. At present, among the most concerning unknown aspects of clostridial pathogenesis (gut colonization and infection progression) is the biofilm-forming capacity of C. difficile in vivo and how this multicellular intraspecific "dialogue" can interact with the host immune system.
In spite of the urgent need to develop novel antimicrobial therapies against this pathogen and the recent technological advancements in vaccination, the process of research, development, validation, authorization, and launch of any novel drug represents an average cost of USD 2-3 billion and takes up to 13-15 years. A very common approach to find new antimicrobial options is the study of currently authorized drugs, even those applied in other clinical areas (e.g., oncology, dermatology, and digestive medicine), as well as the search for synergies between effective antibiotics that are currently used. In this regard, Pal and SeelemThe anti-CD mitomycin effect is exerted with a MIC value of 0.25 µg/mL. Naclerio et al.recently developed one of the most effective antimicrobials against CD, the trifluoromethylthio-containing N-(1,3,4-oxadiazol-2-yl)benzamides, which displayed very potent activity with MIC values as low as 0.003 (µg/mL). According to the study by Naclerio et al., this compound (which is nontoxic to mammalian cells) can be obtained by the replacement of the thiophene toxicophore molecule in TFOB (named as compound 12 by Naclerio et al.) to generate the HSGN-218 product. The principal antimicrobial potential of this compound is mainly attributed to the (trifluoromethylthio)phenyl group, which shows even more effectiveness than vancomycin against C. difficile (MIC values ranging between 0.25 and 1 µg/mL). Another chemically potent compound, 2-(4-(3-(trifluoromethoxy)phenoxy)picolinamido)benzo[d]oxazole-5-carboxylate, with high selectivity against C. difficile was discovered by Speri et al.. The selectivity of this compound to exclusively target C. difficile was indicated by an MIC value of 0.125 µg/mL compared to MIC values against beneficial microbiota (Bifidobacterium fragilis, Lactobacillus reuteri, and Bifidobacterium longum) of 2-126 µg/mL.
In the search for alternative natural antimicrobial strategies, other molecules have demonstrated bactericidal or bacteriostatic effect against C. difficile. To date, these emerging studies to test the antimicrobial potential of different natural antimicrobial compounds against C. difficile have mainly been based on the in vitro disk diffusion test methodology followed by comparison with the Clinical and Laboratory Standards Institute (CLSI) breakpoint methodology that is applied for conventionally used antibiotics, with some studies validating their findings by means of in vivo animal models.
The present review aims to provide a global view on the most effective alternative antimicrobials found in vegetable, bacterial, and marine sources against C. difficile and the possibilities of these materials to exert inhibitory and bactericidal potential in order to contribute to increasing the current knowledge on future clinic and nutraceutical supplements to be administered as therapy in CDI mitigation. In the present study, a review was performed based on published literature on PubMed, Google Scholar, EMBASE, BIOSIS, and Web of Science databases from 2000 to 2021. The terms included to obtain results were as follows: "natural antimicrobials", "marine bioactives", "Clostridium difficile", "therapy", "marine antimicrobials", "marine drugs", "algae", and "gastrointestinal disease". to generate the HSGN-218 product. The principal antimicrobial potential of this compound is mainly attributed to the (trifluoromethylthio)phenyl group, which shows even more effectiveness than vancomycin against C. difficile (MIC values ranging between 0.25 and 1 μg/mL). Another chemically potent compound, 2-(4-(3-(trifluoromethoxy)phenoxy)picolinamido)benzo[d]oxazole-5-carboxylate, with high selectivity against C. difficile was discovered by Speri et al.. The selectivity of this compound to exclusively target C. difficile was indicated by an MIC value of 0.125 µ g/mL compared to MIC values against beneficial microbiota (Bifidobacterium fragilis, Lactobacillus reuteri, and Bifidobacterium longum) of 2-126 µ g/mL.
In the search for alternative natural antimicrobial strategies, other molecules have demonstrated bactericidal or bacteriostatic effect against C. difficile. To date, these emerging studies to test the antimicrobial potential of different natural antimicrobial compounds against C. difficile have mainly been based on the in vitro disk diffusion test methodology followed by comparison with the Clinical and Laboratory Standards Institute (CLSI) breakpoint methodology that is applied for conventionally used antibiotics, with some studies validating their findings by means of in vivo animal models.
The present review aims to provide a global view on the most effective alternative antimicrobials found in vegetable, bacterial, and marine sources against C. difficile and the possibilities of these materials to exert inhibitory and bactericidal potential in order to contribute to increasing the current knowledge on future clinic and nutraceutical supplements to be administered as therapy in CDI mitigation. In the present study, a review was performed based on published literature on PubMed, Google Scholar, EM-BASE, BIOSIS, and Web of Science databases from 2000 to 2021. The terms included to obtain results were as follows: "natural antimicrobials", "marine bioactives", "Clostridium difficile", "therapy", "marine antimicrobials", "marine drugs", "algae", and "gastrointestinal disease".
## Natural antimicrobials against clostridioides difficile infection (cdi): nutraceutical and pharmaceutical approach
Nowadays, in addition to previously detailed antibiotic therapies authorized and generally used in CDI treatment, novel materials and bioactives are being investigated. Among the most innovative novel substances with possible application in
## Natural antimicrobials against clostridioides difficile infection (cdi): nutraceutical and pharmaceutical approach
Nowadays, in addition to previously detailed antibiotic therapies authorized and generally used in CDI treatment, novel materials and bioactives are being investigated. Among the most innovative novel substances with possible application in nutraceutical CDI management are (i) natural compounds from vegetable origin, (ii) restoration of beneficial microbiota, and (iii) marine (bacterial and algae) bioactive compounds. MIC: minimum inhibitory concentration; EOs: essential oils; md: growth inhibition assayed by microdilution method.
[formula] Onion juice 100% (v/v) 10.3 ± 0.6 Garlic juice 100% (v/v) 27.0 ± 1.0 Ginger juice 100% (v/v) - Garlic powder (20% w/v) 26.6 ± 0.6 Cinnamon powder (20% w/v) 20.9 ± 0.9 Curcumin 4-32 µg/mL md [25] Manuka honey 50% (v/v) 11.4 ± 0.5 [26] Nigella sativa (black seed oil) 2% (v/v) >15 [27] Commiphora myrrha (water extract) 2% (v/v) >15 EOs (Satureia montana, [/formula]
## Vegetable compounds in clostridioides difficile infection mitigation: research advances
Natural antimicrobials extracted from vegetable materials (fruits, seeds, grains, leaves, roots, and vegetables) or by-products are representing an innovative and sustainable pathway to fight against human clinical and foodborne pathogens. Effectively, natural vegetable raw materials (and extracted/processed products) have demonstrated antimicrobial potential against C. difficile. Recently, Roshan et al.assayed the in vitro antimicrobial potential of natural onion and garlic juices (100% v/v), onion and garlic powders (20% w/v), ginger, artichoke, honey, cinnamon powder (20% w/v), turmeric powder (20% w/v), and aloe vera compounds against different pathogenic strains of C. difficile (via disk diffusion method and microdilution test). Results revealed that, among the assayed products, garlic juice (100% v/v) was the most effective in inhibiting C. difficile growth (MIC ≈ 9.4 mg/mL) and even showed similar inhibiting potential to that obtained by vancomycin treatment (30 µg/disc; control) (≈30 mm inhibition zone, tested by means of Kirby-Bauer diffusion disc methodology). Moreover, aloe vera (14-19 mm inhibition zone) and artichoke products (12.7-13.9 mm inhibition zone) showed high antimicrobial potential against C. difficile. With regard to processed products (with dimethyl sulfoxide (DMSO) 20% primary solvent used in the extraction process), trans-cynnamaldehide (0.02% v/v), peppermint oil (8% v/v), coconut oil (32% v/v), allicin (MIC = 4.7 mg/mL; MBC = 37.5 mg/mL), and menthol (MIC = 9.4 mg/mL; MBC = 18.8 mg/mL) showed the most effective potential with the lowest required dosage (99.9% reduction of bacterial counts in microdilution test). The study by Roshan et al.also revealed the synergistic effect between the natural compounds that were studied and conventional antibiotic therapies that are currently applied (vancomycin and metronidazole) (trans-cinnamaldehyde with metronidazole and trans-cinnamaldehyde with vancomycin), thereby opening new avenues for future treatment. Moreover, hypervirulent (BI/NAP1/027) C. difficile strains and clinical toxigenic isolates showed susceptibility to curcuminoids, the major phytoconstituents of turmeric, at concentrations ranging from 4 to 32 µg/mL. Curcumin was more effective than fidaxomicin in inhibiting C. difficile toxin production, with no negative effect on beneficial gut microbiota. Possible synergistic effects between curcumin and the most effective antibiotic therapies against C. difficile were also evaluated in vitro. Fidaxomicin (ranging from 0.0005 to 0.5 µg/mL), vancomycin, and metronidazole (at a range of 0.015-8 µg/mL) were tested in combination with curcumin (at a concentration range of 2-64 µg/mL). Although, no synergistic effect was detected between curcumin and the studied antibiotics, antagonist effects also did not manifest.
The studies by Aljarallahand Num and Usehalso revealed the potential of natural herbal extracts to ameliorate possible CDI. In fact, bioactive molecules in herbal extract from Nigella sativa L. (including thymoquinone TQ) and Myrrh (Commiphora myrrha) showed a broad spectrum of antibacterial and antifungal activity against C. difficile (strains JIR and VPI). Both black seed oil (2% v/v) and Myrrh water extract (2% v/v) were effective in inhibiting growth of C. difficile in vitro (via the in vitro agar diffusion method). These results were consistent with those previously obtained by different researchers in relation to the effectiveness of these natural extracts against other gastrointestinal habitual pathogens, such as E. coli, S. aureus, P. aeruginosa, Salmonella Typhimurium, S. flexneri, Bacillus circulans, Enterococcus faecalis, and Helicobacter pylori.
A recent study by Yu et al.demonstrated the effectiveness of manuka honey against 20 C. difficile clinical isolates, with MIC values for aqueous extracts in the range of 4 to >30% (w/v). Manuka honey (produced by Apis mellifera foraging Leptospermum scoparium flowers) demonstrated both bacteriostatic and bactericidal effects against this pathogen. It not only worked against planktonic cells but also inhibited C. difficile biofilmforming capacity. Manuka honey also demonstrated optimum activity at 40-50% (v/v) concentration in inhibiting biofilm formation in four C. difficile ribotypes studied, namely R017, R023, R027, and R046. Manuka honey has been described as a nonallergenic product that does not have a negative impact on the gastrointestinal tract (GIT) microbiome. Its administration is also associated with stimulation of the epithelial cells and fibroblasts in the human host (increased resistance, thus preventing CDI). Additionally, manuka honey has been shown to be a more potent antimicrobial agent against Gram-positive than Gram-negative bacteria, which may be beneficial as adjunct therapy against CDI (preserving normal gut flora, which is predominantly Gram-negative).
Natural essential oils (EOs) with well-recognized antimicrobial potential have also demonstrated an effective capacity for Clostridium spp. inhibition (C. butyricum, C. intestinale, C. hystoliticum, C. perfringens, and C. ramosum), but it has not yet been tested against C. difficile. Among the studied EOs, Satureia montana, Abies alba Mill., and Thymus vulgaris were especially effective, with the lowest minimum inhibitory concentrations against Clostridium spp. (0.38-76 µL/mL). Mechanisms of action of natural antimicrobials in reducing bacterial cell viability include (i) effect on pH homeostasis and equilibrium of inorganic ions, (ii) inhibition of NADH oxidation, and (iii) structural and functional damage of the cell membrane. The cell wall of Gram-positive bacteria is constituted by a thick layer of peptidoglycan, contrary to Gram-negative bacteria that have a cell wall composed of a thin layer of peptidoglycan surrounded by an outer membrane (that is rich in lipopolysaccharides, in addition to proteins and phospholipids). The outer membrane of Gram-negative bacteria is often hidden by a slime layer, which in turn hides the antigens of the cell. This different structure (outer membrane of Gram-negative bacteria) prevents certain drugs and antibiotics from entering the cell, which means these bacteria have increased resistance to drugs. According to a recent study by Roshan et al., one of the main advantages of these natural antimicrobials is that the antibiotic resistance mecha-nisms developed by C. difficile are not cross-protective for natural products. Furthermore, among the advantages derived from the application of naturally extracted antimicrobials is the minimal effect on gut microbiota by these treatments (Bifidobacterium spp., Lactobacillus spp., and Bacterioides spp. are less affected compared to conventional antibiotic strategies). This aspect is crucial in CDI progression and recurrence. Several studies have described dysbiosis in the GIT microbiome as a determinant factor in C. difficile colonization and subsequent infection. Infected patients with C. difficile showed lower richness and diversity of beneficial gut bacteria (Lactobacillus spp. and Bifidobacterium genera) and also relative reduced abundance of Bacteroidetes, Ruminococcaceae, and Lachnospiraceae members. A specific example was demonstrated by Crobach et al.between control (noninfected CD individuals) and CDI patients. According to Crobach and co-workers, the presence of Eubacterium hallii and Fusicatenibacter contributed to generating resistance against C. difficile colonization and infection. In contrast, Veillonella is a genus that is always present in infected patients and related with susceptibility to CDI.
## Probiotic administration, microbiota restoration (fecal transplantation), and microbiota diet modulation: a biological strategy to improve cdi resistance
Among the main disadvantages associated with antibiotic therapies in CDI management is the negative effect on normal microbiome of the host, which reduces a wide spectrum of protective microbiota (short-chain fatty acids (SCFA) producers and carbohydrate degraders such as Eubacterium Hallii, Fusicatenibacter, several Enterococci, Ruminococcus gnavus, and Lachnoclostridium) at the gastrointestinal level. It also reduces complete absorption of antibiotic from the intestinal tract, thereby restricting its concentration in the colon. The International Human Microbiome Consortium and the National Institute of Health's Human Microbiome Project (HMP) are undertaking research to explain how microbiome could play a critical role in human health and disease. Bacteroidetes (defined as groups able to break down host glycans and nondigestible carbohydrates, specifically resistant starches and plant cell wall polysaccharides) and Firmicutes (which form 50-70% of the colonic bacterial community), especially members of the Clostridium genus, are known for their ability to degrade polysaccharides and ferment amino acids (members of the Lachnospiraceae and Ruminococcaceae families) and have been described as always being present and predominant in healthy individuals. As CDI progresses, Proteobacteria and Bacteroidetes decrease.
Biological strategies such as microbiota transplantation and probiotic administration have also emerged as being effective in reducing and mitigating C. difficile infection. Transplantation of fecal healthy microbiota (TFM) is currently being studied but is not yet a regulated strategy. According to in vivo studies carried out by Cammarota et al.in CDI patients, TFM was effective in 90% of treated patients after just 1 year, with no adverse effects manifested.
Regarding restoration of microbiota equilibrium in the gut, promising results have been obtained for biotherapeutic preparations of probiotics, which have been standardized and launched as nutraceuticals to combat recurrent C. difficile infections. Examples of these preparations are RBX2660 and SER-109, which are in phase 3 (PUNCH CD (NCT03244644), 127 patients enrolled) and phase 2 (ECOSPORE, 87 patients enrolled) clinical trials, respectively. Rates of success close to 87% in CDI treatment were obtained using these biotherapeutic preparations, even in three times recurrent Clostridioides infection. No toxigenic effects were observed in any of the standardized microbiota mixtures, including purified Firmicutes spores (in the case of SER-109). At present, several companies are working on the development of similar biotherapeutic products (among them Pfizer, Nanotherapeutics, and Viropharma) to treat and reduce possible recurrent infection with C. difficile, such as the newly proposed product RBX7455 for oral C. difficile prevention, which is a first of its kind nonfrozen, room temperature stable oral microbiota-based formulation under the MRT™ drug platform. Most of them are at least in phase 2 clinical trials (2017-2020 period) and include both probiotic strategies to displace and mitigate CDI and vaccines specifically developed to prevent CDI. Directly administered probiotics such as Saccharomyces boulardii l-745, Lactobacillus rhamnosus GG, Lactobacillus plantarum 299v, Clostridium butyricum, and Lactobacillus acidophillus have demonstrated good prospects in vitro in preventing C. difficile growth by means of an established competition between bacterial species in the media. However, to date, evidence from clinical trials regarding the potential benefits of probiotics against C. difficile is based exclusively on a few bacterial strains, meaning there is not enough data to generally accept and explain the positive in vivo potential. In fact, to date, little is known about how the antagonism is established between probiotic bacteria and C. difficile proliferation. The study by revealed Lactobacillus (L. agilis), Enterococcus, and Clostridium (mainly, C. butyricum) genera as having antagonistic potential against C. difficile by synthesis of extracellular thermostable antimicrobials.
The beneficial effect of S. boulardii and L. rhamnosus GG has been further confirmed by the prevention of antibiotic-associated diarrhea, leading the ESPGHAN (European Society for Pediatric Gastroenterology Hepatology and Nutrition) to recommend the use of probiotics for the prevention of antibiotic-associated diarrhea in children. Furthermore, the study by Chen et al.revealed that genetically modified probiotic S. boulardii was able to constitutively secrete a single tetra-specific antibody that potently and broadly neutralized toxins secreted by C. difficile (TcdA and TcdB), demonstrating protection against primary and recurrent CDI in both prophylactic and therapeutic mouse models of disease.
Modulation of beneficial gastrointestinal bacteria by diet has also been described as a critical aspect contributing to preventing CDI. Jochems et al.evaluated 18 dietary proteins (from protein sources whey, pea, egg, soyabean, insect, potato, fungi, corn, and yeast) to test the impact on epithelial cell colonization and toxin (TdcA and TdcB) production by C. difficile. According to the authors, diet supplementation with certain proteins can enhance the mitigating potential of host immune system to react and restore faster when CDI occurs. Egg-white protein increased IL-6 and IL-8 release (beneficial immunomodulatory effect of protein supplementation but preventing TcdA-induced disruptive consequences), while wheat, lesser mealworm, and yeast proteins increased nitric oxide levels after TcdA exposure. In the same research line, the study by Mefferd et al.supported these previous conclusions. In addition to the specific effect of proteins in immune system reinforcement, Mefferd et al. demonstrated that carbohydrate-based diets exerted a protective effect against C. difficile gut colonization; in contrast, high fat/high protein diets, such as the Atkins diet, greatly exacerbated antibiotic-induced CDI. Hryckowian et al.also found that mixtures of microbiota-accessible carbohydrates (MACs), specifically inulin, decreased C. difficile in vivo (humanized mice) by growth stimulation of carbohydrate-utilizing bacteria and SCFA production. The influence of carbohydrate-based diet on CDI prevention was also recently studied by Schnizlein et al.. Xanthan gum (5% administered in the diet) was evaluated in vivo (C57BL/6 mice model) in terms of microbiota impact (16S rRNA gene amplicon sequencing). According to the results obtained in mice, the administration of xanthan gum increased fiber-degrading taxa and SCFA concentrations, altering mice susceptibility to C. difficile colonization (maintaining balanced microbiota).
Modulation of gut microbial shape to reduce the ability of C. difficile to colonize and establish is among the most promising initiatives to prevent infection. For this task, diet can play a significant role as it can reduce C. difficile pathogenicity by not only regulating the ecological-microbial interactions in the gut but also altering the expression of pathogenesis factors.
## Marine natural compounds as antimicrobials: future niche strategy against c. difficile
In recent years (2010-2020), marine organisms have been increasingly considered as sustainable sources of food and pharmaceutical potential bioactives. Bacteria, fish, shellfish, seaweed, microalgae, mollusks, crustaceans, and cephalopods, among others, are some of the biological matrices that have been identified as being able to produce or synthesize high added value metabolites with potential health benefits for humans. Proteins, peptides, vitamins, carbohydrates, polyphenols, and terpenes are examples of marine molecules with demonstrated functional effects when accurately extracted, purified, and administered as food ingredients or pharmaceutical carriers. Prebiotic, antimicrobial, antioxidant, immunomodulatory, anticancerigen, lipidolemic, and angiotensin I-converting enzyme (ACE) activities are among the most relevant health benefits that have been exerted to date in vitro and in vivo by some of these molecules.
Algae marine organisms offer higher productivity rates than terrestrial plants (close to 12,000 dry tons of microalgal biomass is produced worldwide; protein efficiency/area unit macroalgae = 2.5-7.5 tn/ha/year; microalgae: 4-1 tn/ha/year) and can be sustainably produced as a source of valuable bioactives. The increasing pharmaceutical application of marine algae bioactives is mainly based on their demonstrated antioxidant, antimicrobial, and anticancerigen properties. Moreover, food and nutraceutical supplements based on raw or purified algae compounds are being developed.
Algae compounds have shown antibacterial potential against a wide range of Grampositive and Gram-negative microorganisms. The antimicrobial potential of algae materials is based on the (i) type and algae matrix source (e.g., different algae taxonomic groups, culture conditions, seasonal harvest, and accumulation of bioactives), (ii) structural chemical diversity of compounds, (iii) molecular weight of compounds, (iv) type of extraction and purification methods employed, and (v) modification and way of administration. Among the most relevant antimicrobial bioactives from macro-and microalgae are phlorotannins, laminarin, sargafuran, peyssonoic acid, bromophycolides, neurymenolides, acetylmajapolene, phycobiliproteins, scytonemines, carotenoids, polysaccharides, phytohormones, cyanotoxins, phytol, fucosterol, neophytadiene, palmitic, palmitoleic, and oleic acids.
Seaweeds are classified into green algae, red algae, and brown algae based on their pigmentation. The most promising antimicrobial potential has been found in brown algae, namely Phaeophyceae (84% of species with demonstrated antimicrobial capability), followed by Rhodophyceae (67%) and Chlorophyceae (44%). Regarding microalgae, Spirulina platensis and Chlorella vulgaris are nowadays the most studied algae substrates in terms of their antibacterial/antiviral capacity.
Among the studied bacteria, some of the Gram-positive bacteria that are sensitive to algae compounds are strains of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, and Micrococcus luteus, while the Gram-negative bacteria include Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium, and Vibrio cholerae. Clinical human pathogens, such as P. aeruginosa, E. coli, S. aureus, E. faecalis, group B Streptococcus (GBS), and Proteus mirabilis, are among pathogens that most frequently affect hospitalized patients, and all of them have demonstrated sensitivity to exposure to natural compounds from marine algae sources. Five microalgae cultures (Chlorella minutissima, Tetraselmis chui, Nannochloropsis sp., Arthrospira platensis, and Isochrysis sp.) were effective in inhibiting Gram-positive and Gram-negative nosocomial pathogens, with MIC value equal to 300 µg/mL for Chlorella vulgaris and Spirulina platensis against the most resistant clinical pathogens under study. The chemical characterization of algae extracts demonstrated that volatile algae oils contained in Chlorella spp. and Spirulina platensis, including linalool, geraniol, citronellol, monocyclic limonene, 1-8-cineol, p-cymene, bicyclic α-and β-pinene, cadinene, aromatic eugenol, and isoeugenol, exerted a potent antimicrobial effect against the studied bacteria. Moreover, terpenes (such as π-cymene (+), limonene, β-myrcene, β-pinene, and linalool) have shown active antimicrobial potential toward drug-resistant pathogens.
To our knowledge, in spite of the extensive existing literature related to the assessment of natural algae antimicrobial bioactives against a wide spectrum of human/animal pathogens, nothing has been previously reported in relation to the effectiveness of algae compounds in inhibiting C. difficile proliferation. We can, however, consider that algae compounds with demonstrated prebiotic potential (mainly polysaccharides and complex sulfated bioactives) in promoting significant improvement of healthy microbiota could consequently also improve resistance of the GIT microbial population against CD coloniza-tion and reinforcement of the host immune system, thereby preventing several infection recurrence episodes. Similar approaches have been developed to study and prevent one of the most concerning gastrointestinal pathogens, the unique biological carcinogenic agent Helicobacter pylori.
Algae compounds with recognized antimicrobial and protective digestive effects as well as matrices rich in algae polysaccharides matrices have been described as being useful as prebiotics. Laminaria, Saccharina, Spirulina platensis, Chlorella species, Dunaliella salina, and Scenedesmus species have been shown to exert a potent prebiotic capability by oral administration and also by integration in the diet of in vivo animal models. Several studies focusing on algae polysaccharides have recently been published dealing with the positive impact of diet rich in algae polysaccharides on human gut microbiota balance and its possible capacity to reinforce the host response against C. difficile invasion. According to the study by Han et al., the abundance of Ruminococcaceae, Coprococcus, Roseburia, and Faecalibacterium in an animal model was increased by diet supplementation of polysaccharides and oligosaccharides. Meanwhile, diet supplementation of algae polysaccharides and oligosaccharides has been shown to have a positive impact in preventing proliferation of opportunistic pathogenic bacteria Escherichia, Shigella, and Peptoniphilus. Among macroalgae polysaccharides, special attention has been paid in the last decade to fucoidan (sulfated polysaccharide rich in fucose) from brown macroalgae (Phaeophyceae). The antimicrobial potential of fucoidan has been recognized in several high-impact studies against gastrointestinal pathogens. Purified fucoidan showed effective MIC concentrations in the range of 25-100 µg/mL against Salmonella enterica serovar Typhimurium and Helicobacter pylori depending on the algae source (Fucus vesiculosus, Undaria pinnatifida, and Macrocystis pyrifera). Fucoidan from Fucus vesiculosus was most effective against the studied gastrointestinal pathogens. Since 2017, fucoidan from Fucus vesiculosus and Undaria pinnatifida have been granted "Generally Recognized as Safe" (GRAS) designation by the US FDA and received EU Novel Foods approval. Another study on supplementation of fucoidan to human diet revealed how fecal innate immunity indicators were improved (e.g., lysozyme concentrations, expression of key intestinal tight junction proteins, and secretion of antimicrobial peptides in the gut mucosa).
Moreover, polysaccharide chitosan (natural cationic polysaccharide composed of randomly repeating units of β-(1,4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) nanofibers with extended application in food formulation and packaging were recently (2020) described as having antimicrobial properties against clinical toxigenic isolates of C. difficile with promising in vitro results (MIC values of 0.25 µg/mL).
Phocoenamicin, a novel natural compounds from marine mammal microbiota (from Micromonospora auratinigra, Actinobacteria), has recently been extracted, purified, and characterized with a potent selective antimicrobial activity against C. difficile. In fact, marine ecological habitats have huge microbial diversity, with high capability to synthesize antimicrobial substances. Among these natural sources of antimicrobials, Actinobacteria (from marine sediment habitat) have been recognized as major producers of antimicrobial compounds. Actinobacteria (Actinomadura, Actinoplanes, Amycolatopsis, Marinispora, Micromonospora, Nocardiopsis, Saccharopolyspora, Salinispora, Streptomyces, and Verrucosispora), as prolific producers of pharmaceutical metabolites (70% of bioactives produced by Actinobacteria are currently in clinical use), are producing potent antimicrobials that could be applied in nutraceuticals for future prevention of CDI. Thiocoraline (peptide) from Micromonospora sp., bonactin (esters) from Streptomyces sp., and chinikomycins A from Streptomyces sp. have been shown to exert potent antimicrobial activity against Grampositive bacteria (MIC ≈ 4 µg/mL). In the period, 2015-2018, 45 patents have been launched claiming therapeutically active biomolecules from marine sources, mainly aimed at treating or preventing cancer, infectious diseases, and cardiovascular disorders. Among these novel products with unique structures and novel bioactivity are, isoquinoline alkaloid; trabectedin, the polyether macrolide; halichondrin B, and peptide dolastatin 10.
Recently presented results on marine substrates are opening new possibilities in terms of CDI treatment by means of different strategies, namely (i) pharmaceutical products designed to be applied to complement antibiotic therapies against C. difficile and (ii) possible diet supplementation with these marine prebiotics and highly nutritional molecules (e.g., peptides) that may exert an additional antimicrobial effect against C. difficile gut invasion.
# Conclusions
Novel developments in the field of antimicrobial therapies against C. difficile are now under way. The urgent need to find effective antimicrobial strategies to fight against this pathogen without affecting beneficial microbiota at the GIT level is one of the main challenges to achieve highly specific treatment. Tailor-made antimicrobial strategies should be developed against CDI for both (i) prevention and (ii) treatment (depending on the severity of symptoms manifested and previous clinical history of the patient). Natural compounds from vegetable and marine origin are being investigated due to their anticlostridial bacteriostatic and bactericidal potential as well as their capacity to maintain healthy microbiota equilibrium. Special attention should be paid to algae compounds as sustainable and worthy sources of unexplored antimicrobials. Fucoidan from Phaeophyceae is among these valuable compounds with demonstrated prebiotic potential. It promotes the proliferation of beneficial bacteria while exerting antimicrobial effect against gastrointestinal pathogens such as Helicobacter pylori and Salmonella enterica. Further research is required on the use of algae antimicrobials as nutraceuticals in CDI management. Structurally effective, sustainable, easily extracted, and cost-effective purified natural biomolecules will be a reality in the short to medium term based on these antimicrobial compounds from vegetable and algae origins, which can be used an alternative to antibiotic-based therapy (diet or nutraceutical administration of natural compounds alone) or as a supplement to drugs (with possible synergistic effects) against C. difficile. In vivo studies to further understand (i) to what extent these compounds are available and effective in the digestive tract to exert antimicrobial functionality and (ii) how long nutraceuticals should be administered to ensure a protective effect on C. difficile colonization are required for safe and effective risk mitigation against CDI. |
Seronegative Arthritis and Whipple Disease: Risk of Misdiagnosis in the Era of Biologic Agents
We report 2 cases of Whipple disease (WD), previously diagnosed as seronegative polyarthritis and treated for several years with immunosuppressive agents, accordingly. Both cases had been treated over years with cDMARDs and bDMARDs. e first patient was a 48-year-old male, who developed a life-threatening disease characterized by fever, significant weight loss, and bloody diarrhoea, supported with RBC transfusions. e second patient was a 55-year-old man, presenting with arthritis, fever, serositis, lymphadenopathy, thoracic rash, and systemic inflammation; at the beginning he was diagnosed as adult onset Still's disease. He was treated with steroids and antitumour necrosis factor agents, but showed no improvement. Both patients were eventually treated with antimicrobial therapy for WD with dramatic improvement and no clinical relapse in 6 months. is paper reviews the literature on WD mimicking chronic inflammatory arthritis. WD may lead to chronic seronegative arthritis that might often be misrecognized. Importantly, patients treated with bDMARDs and glucocorticoids might develop a life-threatening disease. erefore, WD should be suspected and excluded in patients showing resistance or frequent recurrence of chronic arthritis, if seronegative, under treatment with bDMARDs, especially in the presence of new, unexpected sign and/or symptoms.
# Introduction
Whipple disease (WD) is a rare infection disorder, first described by George Whipple in 1907, caused by the rodshaped actinomycete, Tropheryma whipplei, and characterized by diarrhoea, weight loss, and arthralgia. Human beings are the only known host of T. whipplei and the spread of this infectious disease might be related to human-to-human transmission .
T. whipplei is commonly infecting humans, while WD is rare. Cases are rare and disproportionately associated with occupational exposure to soil or animalsand may mimic more common conditions. According to a report regarding 142 patients affected by T. whipplei, the main symptom is arthralgia (88/113, 78%), which might explain the frequent misdiagnosis as chronic inflammatory arthritis, such as rheumatoid arthritis (56/113, 50%). Fifty percent of patients receive immunosuppressive treatments which are responsible for a more rapid clinical progression (43%). Endocarditis is the second most frequent manifestation of T. whipplei, followed by neurologic symptoms. Other localized infections such as adenopathy, uveitis, pulmonary involvement, or frank arthritis are sporadic, but still may cause misdiagnoses. Diagnosis of WD can be established by periodic acid-Schiff (PAS) staining of inclusion bodies within lamina propria macrophages in biopsies of the small intestine and by polymerase chain reaction (PCR) . In the absence of duodenal histologic involvement, localized infections were defined by specific positive T. whipplei PCR results obtained using samples of other tissues and body fluids. Early diagnosis should enable appropriate treatment and improve the prognosis, and prolonged antibiotic treatment often leads to complete remission . e disease could develop in decades because of a very long doubling time (up to 18 days) and only in predisposed patients. Patients showing seronegative oligoarthritis or polyarthritis could be mistakenly treated with immunosuppressive agents, including biologic drugs, even for a long time. Here, we report two cases which we consider emblematic to include WD as mandatory in the differential diagnosis of seronegative arthritis.
## Case presentation
All procedures performed in our study involving human subjects were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the patients included in the study.
## Patient 1.
e first patient was a 48-year-old Caucasian male with progressive and unintended weight loss, daily diarrhoea (initially without bleeding), and fever.
In the past medical history, he had a previous diagnosis of seronegative spondyloarthritis because of 7-year intermittent joint swelling, early morning stiffness associated with inflammatory back pain and increased C-reactive protein (CRP), and a familiar history of psoriasis. No history of travel or surgery was recorded. erapeutic trials with different antirheumatic drugs included methotrexate, hydroxychloroquine, etanercept (ETN), and finally, tocilizumab (TCZ). e most recent medical history of the first patient was characterized by disease relapse with fever and systemic inflammation (CRP 20 mg/l) without any response to a medium dose of glucocorticoids in May 2017. Before May 2017, he was finally treated with monthly TCZ 8 mg/kg intravenously, which was effective on his clinical manifestations, allowing the resolution of arthritis, and fever and led to normalization of CRP within 3 months of therapy and, notably, lasting 2 years. In April 2017, abdominal pain, daily fever (around 38°C), and daily diarrhoea without bleeding started. e patient reported a relevant weight loss of 8 kg during the last 3 months.
Importantly, physical examination showed no evidence of an arthritis flare, while cutaneous lesions on the lower limbs that may resemble a cutaneous vasculitis or atypical psoriasis. Laboratory exams revealed CRP 72 mg/l, hemoglobin 10.7 g/l, HLA-B38 positive, and HLA-B27 and B51 negative. Rheumatoid factor (RF), anticitrullinated protein antibodies (ACPAs), ANA, and ANCA autoantibodies were negative. e day after the admission in our hospital ward, the patient presented bloody diarrhoea with a significant decrease in the hemoglobin level in 24 hours (from 10.7 g/dl to 8.4 g/dl). us, he urgently underwent colonoscopy and gastrointestinal arteriography, but without revealing the site of bleeding.
We tested also Vidal-Wright reaction, CMV, HBV, HCV, H. pylori, TB test, rotavirus, adenovirus, norovirus, Yersinia, Shigella, Salmonella, Campylobacter, Clostridium, and blood cultures, but all tests resulted negative.
Suspecting an intestinal vasculitis or a chronic inflammatory bowel disease, we administered methylprednisolone 1 g daily for three days with the regression of diarrhoea and fever. In order to find the site of bleeding, he underwent a gastrointestinal bleeding scintigraphy and CT-PET, which revealed a diffuse uptake in the small bowel.
Because of persistent weight loss, we introduced parenteral nutrition and explored other causes of malabsorption syndrome, such as WD; thus, the patient underwent gastroscopy. e histological findings resulted in PAS-positive granules in macrophages (intestinal lipodystrophy). T. whipplei was then detected in blood, feces, and saliva samples by PCR in an international reference laboratory.
Intravenous ceftriaxone (2 g daily for one week) was started followed by trimethoprim-sulfamethoxazole 160/ 800 mg 3 times a day with dramatic improvement. ere were no relapses after 6 months and was a significant weight recovery.
## Patient 2.
e second patient was a 55-year-old Caucasian male, with a history of symmetric polyarthritis and systemic inflammation (CRP 130 mg/l) without fever, previously diagnosed as seronegative rheumatoid arthritis in 2012. No history of travel or surgery was recorded. Over the years, he was treated with cDMARDs (methotrexate) and bDMARDs (certolizumab, ETN, TCZ, abatacept, and adalimumab). e disease relapsed with arthritis (synovitis demonstrated by US), fever (39°C), and thoracic discomfort, suspicious for pleurisy in September 2017. Previous treatments provided only a partial clinical benefit; however, the systemic inflammation with CRP ranging from 20 to 40 mg/l persisted.
ere was not any improvement with the introduction of colchicine. After switching to anakinra, we even registered a clinical worsening with fever, arthritis, laterocervical lymphadenopathy, and thoracic rash along with a higher systemic inflammation (CRP 213 mg/l).
Many infections were excluded (i.e., CMV, EBV, chlamydia, mycoplasma, TB, Salmonella, Shigella, and Borrelia burgdorferi) as well as autoinflammatory diseases (negative genetic tests for Mediterranean fever and no improvement with colchicine and anakinra treatment, as reported) and, initially, WD by negative duodenum histopathology (no signs of PAS-positive granules in the duodenum samples).
In the absence of other diagnosis, the adult onset Still's disease appeared the most probable one. e patient was then treated with steroid 0.5 mg/kg/day, with clinical improvement. Infliximab was started and steroid progressively tapered, but with recurrence of fever and systemic inflammation (CRP 68 mg/l).
Despite the PAS negativity in the duodenal biopsy, the patient was then treated with ceftriaxone, showing a dramatic and rapid clinical and laboratory improvement. us, the duodenal samples, as well as other samples from stool and saliva, collected before the introduction of the antibiotic therapy, were analyzed for T. whipplei by PCR in the Reference Center of Marseille, resulting all positive. Finally, the patient was treated with doxycycline 100 mg twice daily and hydroxychloroquine 6 mg/kg/day, with no relapse after 6 months.
# Discussion
T. whipplei is a ubiquitous Gram-positive microorganism that is rarely associated with symptomatic disease, namely, WD [1]. T. whipplei is a commensal organism and not an obligate pathogen. Up to 7% of individuals are healthy carriers with positive stool PCR tests. e systemic infection involves especially the digestive tract. e epidemiology of WD is limited by small sample size and case series design. e incidence of WD has been estimated at about 0.5 to 1/1000000 people. In a recent large population-based study, the overall prevalence of WD in the USA was 9.8 cases per 1 million people. It equally affects men and women and is more common in Caucasians and middleaged people.
e way of transmission may involve passage of the bacterial agent from the environment into the Case Reports in Rheumatology body through the gastrointestinal tract, followed by fecal-oral transmission. However, person-to-person transmission through the oral-oral route cannot be ruled out. Farmers and individuals exposed to soil and animals may be at higher risk. Besides the classical presentations with chronic diarrhoea and malabsorption, clinical presentations include endocarditis, central nervous system (CNS) involvement, uveitis, arthritis, and diskitis without gastrointestinal manifestations and/or with normal gastrointestinal histology. Indeed, patients with WD were more likely to have associated arthritis, CNS manifestations, endocarditis, diabetes, malignancy, dementia, vitamin D deficiency, iron deficiency, chemotherapy, weight loss, abdominal pain, and lymphadenopathy. WD often leads to a chronic seronegative arthritis that could be often misdiagnosed. Interestingly, 1.58% of patients with unexplained arthritis and no other evidence of WD were positive for T. whipplei. Articular involvement often characterizes the onset of WD. e mean time from the development of articular symptoms to the diagnosis is almost 7 years, but there are some reports focusing the attention on the very delay of the diagnosis of WD, in the absence of gastrointestinal symptoms. Arthritis has been reported in 41%-61% of cases. In a retrospective single-center cohort study of 7 patients, all patients presented with polyarthritis with a predominantly symmetric pattern, with erosion in 3 out of 7 patients. e most affected joints were wrists, metacarpophalangeal joints, and knees, followed by proximal interphalangeal joints, hips, elbow, and shoulder. All patients had increased CRP, while RF and ACPA were absent, and all were initially misclassified as affected by seronegative RA. Six patients received DMARD treatment consisting of methotrexate and/or leflunomide, and three were also treated with at least one bDMARD. Most patients showed an inadequate response. In all patients, T. whipplei was detected in synovial fluid by PCR. Notably, gastrointestinal symptoms and other extra-articular manifestations were absent, mild, or nonspecific. All patients had good treatment responses with improvement of arthritis and extra-articular manifestations upon started antibiotic therapy. Even if gastrointestinal involvement is usually demonstrated by histological or PCR tests, a few patients, however, have chronic focal joint infection with normal gastrointestinal biopsy specimens, even by PCR. us, unexplained intermittent oligoarthritis or polyarthritis, especially of the large joints, in middle-aged men should suggest excluding WD, even in the absence of gastrointestinal symptoms. Chronic polyarthritis in a symmetric distribution is less common, and the small joints are usually spared. Rarely, patients with prolonged untreated WD may develop erosions, and the progression to the late stage of ankylosis may occur only after many years. Notably, patients treated with TNF-alpha inhibitors and glucocorticoids could reveal a life-threatening disease, triggering visceral disorders. On the contrary, the present case reports showing a clear, even if transient, clinical response for a long time under immunosuppressive agents, demonstrate that the course of arthritis does not really allow the clinician to definitely rule out such a chronic infection. Notably, clinicians should be aware that especially TCZ as a potent inhibitor of IL-6 is able to abolish systemic features, such as fever or CRP elevation, along with arthritis, while the infection spreads slowly, as clearly reported in our first patient.
A recent literature search on 19 studies reported the use of immunosuppressive drugs, particularly therapy with TNF inhibitors, before the diagnosis in 41 patients with WD. As arthritis may precede the diagnosis of WD by many years, a relevant percentage (up to 50% in some reports) of patients is treated with immunomodulatory drugs or with biologics. Indeed, complicated WD course or T. whipplei endocarditis following medical immunosuppression, particularly after TNF inhibitors, have been reported. e histological detection of macrophage-containing PAS-positive granules in the duodenum lamina propria is considered the standard diagnostic method . Notably, as in our second case, negative PAS particles do not completely rule out WD. Another diagnostic procedure, critical in immunosuppressed patients, is the T. whipplei PCR, especially in strong clinical suspicion. is test can be made on various samples, from saliva to stools and also on synovial fluid, but the highest specificity relies on the duodenal biopsy, even if negative small bowel PAS and PCR do not definitely exclude the diagnosis of WD, and blood PCR alone is insensitive for active infection. Importantly, in the case of seronegative arthritis (especially in the absence of systemic symptoms), synovial fluid should always be collected, if possible, and then analyzed also by T. whipplei PCR, based on clinical suspicion, since in Whipple arthritis, the diagnosis is made by PCR analysis of the synovial fluid. e gastrointestinal analyses (PAS staining, histology, and PCR) are done in order to exclude additional systemic or intestinal manifestations. Doxycycline together with hydroxychloroquine is the first-line choice for WD without neurological involvement (defined as absence of clinical neurological abnormalities and a negative PCR assay on the cerebrospinal fluid). In the past, the most widely recommended regimen was oral trimethoprim 160 mg and sulfamethoxazole 800 mg twice daily for 1 to 2 years. In any case, treatment generally includes primary therapy for 2 weeks with intravenous antibiotics capable of reaching high levels in the cerebrospinal fluid, such as ceftriaxone. Association with glucocorticoids has been suggested to avoid the immune reconstitution syndrome, which affects 2%-10% of patients. Antibiotic therapy usually promptly provides improvement in the clinical and laboratory abnormalities, but it is of great importance to continue the treatment for at least 2 years or even preferably throughout life.
# Conclusions
WD should be suspected and definitely ruled out in patients affected by seronegative arthritis, who show a disease course characterized by resistance or recurrence of the disease, along with the development of new, unexpected symptoms or signs (in particular fever, serositis, and/or gastrointestinal symptoms). Duodenal biopsy is the standard diagnostic procedure; however, in those patients with negative result on duodenal biopsy, but high level of suspicion and risk of complication due to immunosuppression, T. whipplei PCR should be performed on all the available body tissues or fluids, in particular the synovial fluid. Finally, a correct disease classification of patients remains an essential step for an appropriate use of targeting treatments in systemic rheumatic diseases, especially in seronegative arthritis since selective inhibitors of proinflammatory cytokines may masquerade symptoms and signs of chronic systemic infections.
## Additional points
(1) Whipple disease should be considered in the differential diagnosis of seronegative arthritis. (2) All the available body tissues or fluids should be investigated by T. whipplei PCR.
(3) Biologic agents can mask symptoms and signs of such a chronic infection, in particular IL-6 inhibitors. (4) Correct classification is an essential step for using biologics properly.Systemic symptoms along with arthritis should alert the clinician to suspect Whipple disease. |
COVID-19 and Sick Leave: An Analysis of the Ibermutua Cohort of Over 1,651,305 Spanish Workers in the First Trimester of 2020
Objectives: The worldwide SARS-COV2 pandemic has impacted the health of workers and companies. The aim is to quantify it according to sick leave.Methods: Using ICD-9 codes, we analyzed Ibermutua records of all sick leaves during the first trimester of 2020, compared to during the same months of 2017, 2018, and 2019. We stratified the analysis by causes, patient sex, activity sectors, and regional data. All sick leaves were adjusted by the number of Ibermutua-affiliated persons in each period.Results: In March 2020, there was an unprecedented (116%) increase in total sick leaves, mainly due to infectious and respiratory diseases. Men and women were equally affected. All activity sectors were impacted, with the highest increase (457%) observed among health-related workers, especially due to contagious disease. The incidences of sick leaves were heterogeneous among different regions. Cost-analysis of sick leaves during the first trimester of 2020 compared with in previous years showed 40.3% increment (mean 2,813 vs. 2,005 e per 100 affiliated workers).Conclusions:The SARS-COV2 pandemic is having a huge impact on workers' health, as shown by data regarding sick leaves in March 2020. This is associated with greater economic burden for companies, both due to the cost associated with sick leaves and the losses in productivity due to confinement.
# Introduction
Since December 2019, starting in the city of Wuhan, China, the world has been suffering an outbreak of the new coronavirus SARS-COV2, which induces acute respiratory infection, bilateral pneumonia, respiratory failure, and in some cases death. The rapid dissemination of this virus has forced many national authorities to restrict individuals' movements and to close industries that are considered non-essential, including tourism companies and air travel. In Spain, such measures have been mandatory since .
The first two cases identified in Spain were imported from Germany (January 31, 2020) and the UK (February 10, 2020). These cases were followed by a sharp rise in the number of infected individuals. At the end of March 2020, there were 85,195 known cases, and the mortality was 34% higher than expected. This rapid spread overwhelmed many health facilities, necessitating the construction of auxiliary sanitary centers. In the acute setting, COVID-19 diagnosis relies on PCR-based viral detection from nasopharyngeal swabs. However, access to these kits has been limited and some infected individuals remain asymptomatic or with only mild symptoms; thus, it is generally agreed that the disease is markedly underdiagnosed.
Sick leave (SL) is a complex indicator of the working population's well-being and also a predictor for health consequences and mortality. Moreover, if paid sick leave is guaranteed from the 1st day of illness, it encourage workers to stay at home preventing the spread of virus infection.
Ibermutua is a mutual insurance company in occupational medicine, which collaborates with the National Public Health System in Spain to provide healthcare for the working population. Ibermutua covers over 1.6 million workers, and has nearly 100 of its own health centers and 1,000 external health centers, spread throughout Spain. Regarding non-work-related or common diseases (CD), Ibermutua receives daily information from the National Public Health System about its covered workers who are on sick leave, and the cause of it. This enables assessment of the impacts of CD on health in a large sample of workers, and the associated economic burden on society.
The impact of COVID-19 pneumonia on SL and its costs have not been reported in our knowledge, despite of the important component of the economic burden of the disease and the loss of productivity due to morbidity and premature death. In the present study, we aimed to analyze the CD-related SL (CD-SL) of workers during the first trimester of 2020. We compared these data with the findings from previous years, with special focus on health-related workers.
# Methods
## Study design and participants
We performed a retrospective comparative analysis that included all CD-SL episodes started between January and March 2020 among workers covered by Ibermutua. We compared these data with the CD-SL episodes during the same months in the years 2017-2019. Data on these episodes were obtained from the National Public Health System Register for SL due to CD.
On January 31, 2020, Ibermutua covered a total of 1,651,305 workers (55.1% men; 44.9% women). The mean age was 42 years (SD, 11.36; range, 16-80 years) among all workers, 42.2 years among men, and 41.9 years among women. On this date, the Spanish Social Security covered 19,171,039 workers (53.4% men; 46.3% women), which included the workers affiliated with Ibermutua (9) The Ibermutua-affiliated sample is considered to provide adequate representation of the Spanish working population, as previously published.
Ibermutua is a mutual insurance company that provides healthcare for work-related SL episodes and occupational illnesses. They are also responsible for the management of CD-SL for some companies. Mutual insurance companies in Spain collaborate with National Social Security to administer statutory sick pay. Among its preventive activities, it launched the ICARIA (Ibermutuamur CArdiovascular RIsk Assessment) project in 2004.
In the present study, workers were classified into four major activity sectors (agriculture, construction, industry, and service) according to the Spanish Classification of Economic Activities. The sample included workers from all activity sectors and occupational categories, with a broad age range, and from both genders. We performed separate analysis of CD-SL in health-related workers.
## Sick leave definitions and cost
SL episodes due to infectious disease, cardiovascular disease, respiratory disease, and undefined symptoms were identified based on the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) codes 001-139, 390-459, 460-519, and 780-799, respectively. Additionally, cases of COVID-19 infection were identified using the specific ICD-9-CM codes released on March 6, 2020: non-specified viral infection (codes 079) and exposure to contagious disease (codeV01). SL cost was calculated by multiplying the regulatory base (contribution base of the month prior to the SL divided by 30 days) by the SL duration. To compare SL cost in 2020 with costs in 2017, 2018, and 2019, we adjusted total costs in every trimester by the number of workers affiliated in each period.
# Statistical analysis
Data analyses were performed using Excel 2016 (Microsoft Suite, United States). Data are shown as absolute values and percentages. For comparison with the SL in the first 3 months of 2020, to balance the year-to-year variability, we used the mean SL corresponding to the same months of the years 2017-2019. Data on common SL were adjusted by the number of Ibermutua-affiliated workers every month, which is shown in Supplementary File. We also performed analyses with stratification by gender, activity sector, and autonomous communities.
# Ethical issues
The research described herein adhered to the tenets of the Declaration of Helsinki. All medical records were anonymized; only statistical information was provided by Ibermutua for research purposes.shows total and specific diagnosis of CD-SL during the first trimester of each year from 2017-2020. In 2020, the number of SL in March was increased compared with during the same period in 2017, 2018, and 2019. This increase was especially notable among the codes of infectious disease, undefined symptoms (including malaise, feverishness, myalgia, arthralgia, etc.), and respiratory disease. During the same time period, SL for cardiovascular codes remained stable across all years. We observed a particularly dramatic increase of SL for respiratory diseases in March 2020, accounting for 4.9 cases per 1,000 workers, compared to 2.5 cases per 1,000 workers during this period in 2017, 2018, and 2019.shows changes across the study period. In 2017-2019, SL decreased with progression through the first trimester, and this expected decrease was not observed in 2020. SL clearly increased in March, by 96% for respiratory diseases and by 264% for infectious diseases. The most striking finding of our study was the SL for workers exposed to contagious diseases, which was scarcely reported in 2017-2019, while 4,539 such cases were reported in March 2020, 2.9 cases per 1,000 workers.
# Results
At the end of March 2020, there were 136,977 Ibermutuaaffiliated workers in the sanitary and social services (see Supplementary File).shows the total and specific causes of SL among these workers. They exhibited increased total SL, especially associated with respiratory disease, infectious disease, undefined symptoms, and non-specified viral infections. The code associated with SARS-COV2 infection (V01. SL among health related workers in the first trimester of the years 2017-2020).shows the distribution of SL during the studied period according to gender and economic activity sector (EAS). No differences were observed between men and women. Additionally, the increase of SL in March 2020 was seen in all EAS, and was higher in construction (284%), agriculture (218%), and industry (163%), compared to in services (85%). SL differed among autonomous regions of the country, with the greatest increases observed in the Basque region, Castilla La Mancha, Madrid, and Navarra. In contrast, the island territories (Balearic and Canary) did not show any SL increase .
Finally, the increased SL translated into an 40.3% increase of associated costs during the first trimester of 2020 compared with the same period of 2017-2019 (mean 2,813 vs. 2,005 e per 100 affiliated workers).shows the increased cost of SL per 100 workers between January and March 2020 with respect to the same months of the years 2017-2019.
# Discussion
The findings of our present study confirm that in March 2020, there was an unprecedented increase in the number of SL compare with in previous years with the greatest impact on exposed workers. The increased SL were mainly due to SL related to respiratory disease, infectious disease, and undefined symptoms, which is concordant with the SARS-COV2 outbreak. Considering the epidemiological context, our results suggest that most of these SL might be related to mild or moderate symptomatic cases of COVID-19. In this context, it is interesting to note that during March 2020, general practitioners in Spain cared for 26 subjects with COVID-19-like symptoms in their clinics for every two patients diagnosed with severe forms of COVID-19 and admitted to hospitals. There was no increase of SL in February 2020, even though the first Spanish case of COVID-19 death was identified post-mortem in a patient who died in February, and phylogenetic data indicates that SARS-COV2 was already present in Spain at that time.
Increasing clinical and epidemiological evidence suggests that SARS-COV2 infection can induce cardiovascular complications that are associated with a worse prognosis. However, we did not find an increase in SL related to cardiovascular disease during 2020. It is possible that cardiovascular diseases associated with COVID-19 might be linked with the severe forms of the disease during evolution, and are rarely the cause of SL.
Concerning economic activity sectors, most SL (in absolute terms) affected the service sector. However, when data were adjusted by affiliated workers to each sector, the SL increase was lowest in the service sector (85%), and the highest increase was observed in the construction sector (284%). Importantly, this finding indicates that no activity sector was safe. It is interesting to note that many of the SL, especially in March 2020, were among health-related workers. Of the 136,977 healthrelated workers in our study cohort, 6.78% had a SL, compared to 1.22% in previous years. Additionally, 1,574 SL among health-related workers in March 2020 were coded as due to exposure to biological agents. This code has been very rarely used in past years. These results are in agreement with data on confirmed cases of COVID-19 in Spain, where nurses and doctors comprise almost 20% of affected people up to March 2020 (3). In that line, a recent publication in USA found a significant increase in absenteeism in workers in personal care and service groups, including those giving healthcare support during April 2020.
In terms of occupational health, the present situation is a very important issue and a challenge for companies, as they should adapt the production of goods to keep their workers at the lowest risk possible, and following the recommendations of the WHO. Additionally, regional and national authorities should provide workers with the personal protection equipment required to avoid contact and the widespread transmission of infection at work. This is especially relevant to health-related workers at hospitals and primary care centers, as well as at socio-sanitary institutions (elderly care facilities, etc.). The capacity of health-related workers to face the SARS-COV2 pandemic in Spain has already been recognized.
Based on the increased total number of SL in the first trimester of 2020 compared with in previous years, the total cost of SL has also increased by 40.3% reaching 137,722,056 e. This data underestimates the actual cost, since it does not include additional costs faced by workers and companies. Since the Spanish Government decreed confinement starting from March 14, 2020 (2), many non-essential industries have been closed, up to 800,000 workers have become newly unemployed, and many other have been affected by temporal regulation of their contracts (furlough). This adds to the fact that over the last 5 years, SL has substantial increased in terms of prevalence and duration, with corresponding increases of associated healthcare costs and productivity losses. We found that SL were heterogeneous between regional areas, being lower in the Balearic and Canary islands (despite having more tourism activities) and Catalonia, and higher in the Basque region, Madrid, and Castilla-La Mancha. In some instances, our data showed a mismatch with reports of illness in the general population. For instance, Catalonia had the second highest number of patients affected by COVID-19 pneumonia (3), but showed a very small increase in SL. This discrepancy might be related to differences in activity sectors or in the mean age of the population. It is well-known that severe COVID-19 pneumonia predominantly affects elderly people, who are not often affiliated with Ibermutua. Additionally, SL may be affected by the mobility of workers between close regions. This might the case for Castilla-La Mancha and Castilla León, which are very close to Madrid, the largest focal point of COVID-19, with thousands of workers moving between regions every day on high-speed trains. Similarly, Cantabria is close to the Basque region.
One strength of this analysis of non-work-related (common) diseases, is that Ibermutua receives daily official reports from the National Public Health System of Spain about its covered workers who are on a SL and the cause of it. This encoded information (ICD-9-CM) becomes part of the Ibermutua official records. These data were anonymized and used for the present analyses.
As a limitation of this study, it should be noted that during the months of January and February and mid-March, primary care physicians did not have an official regulation for the codification of SL related to the COVID-19 pandemic. This came into use in early March. Thus, especially prior to March, many cases of this infection would have been encoded as undefined symptoms, viral infections, or respiratory infections.
In conclusion, in just the first trimester of 2020, the outbreak of SARS-COV2 has had a dramatic impact on the health of the Spanish working population and on their companies. This influence has been especially severe for health-related workers. Moreover, the impact on health is associated with a significant economic burden on society.
# Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
# Ethics statement
Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for |
DNA methyltransferases and their roles in tumorigenesis
DNA methylation plays an important role in gene expression, chromatin stability, and genetic imprinting. In mammals, DNA methylation patterns are written and regulated by DNA methyltransferases (DNMTs), including DNMT1, DNMT3A and DNMT3B. Recent emerging evidence shows that defects in DNMTs are involved in tumor transformation and progression, thus indicating that epigenetic disruptions caused by DNMT abnormalities are associated with tumorigenesis. Herein, we review the latest findings related to DNMT alterations in cancer cells and discuss the contributions of these effects to oncogenic phenotypes.
Background DNA methylation is one of the most important epigenetic modifications [bib_ref] Epigenetic Determinants of Cancer, Baylin [/bib_ref] , playing key roles in the regulation of gene expression, genomic imprinting, X chromosome inactivation, and tumorigenesis [bib_ref] DNA methylation: roles in mammalian development, Smith [/bib_ref]. In mammals, DNMT1, DNMT3A and DNMT3B, the generally recognized three types of DNA methyltransferases (DNMTs), execute the genomic methylation process [bib_ref] Genetic alterations of DNA methylation machinery in human diseases, Hamidi [/bib_ref]. These proteins are highly conserved and have similar amino acid sequences. The N-terminus contains a regulatory domain, which allows DNMTs to anchor in the nucleus and recognize nucleic acids or nucleoproteins, and the Cterminus possesses a catalytic domain, which is responsible for the enzymatic activity [bib_ref] Structure and function of eukaryotic DNA methyltransferases, Chen [/bib_ref]. DNMT1, DNMT3A and DNMT3B have different functions in the methylation process. DNMT1 is required for the maintenance of all methylation in the genome. During replication, DNMT1 restores the specific methylation pattern on the daughter strand in accordance with that of the parental DNA. DNMT3A and DNMT3B are referred to as de novo methyltransferases, which are responsible for establishing DNA methylation patterns during embryogenesis and setting up genomic imprints during germ cell development [bib_ref] DNA methylation in mammals, Li [/bib_ref]. Although they are highly expressed in early mammalian embryos, DNMT3A and DNMT3B decrease in expression over the course of cell differentiation. These two proteins have distinct functions throughout embryonic development, showing both spatial and temporal differences. DNMT3A primarily methylates a set of genes and sequences at the late stage of embryonic development and especially after birth, whereas DNMT3B modifies a broader region of genomic sequences in early embryos [bib_ref] DNA methylation: roles in mammalian development, Smith [/bib_ref] [bib_ref] DNA methylation in mammals, Li [/bib_ref]. Very recently, one study identified a new de novo DNA methyltransferase DNMT3C in murine germ cells. DNMT3C exhibits high identity with DNMT3B, and is specialized at methylating the young retrotransposons [bib_ref] The DNA methyltransferase DNMT3C protects male germ cells from transposon activity, Barau [/bib_ref]. Beside the above-mentioned enzymes, which are essential for the methylation of mammalian DNA, the DNMT family also includes two additional members, DNMT2 and DNMT3L. Although DNMT2 is not currently considered to be a DNA methylase, this enzyme methylates small transfer RNAs (tRNAs) [bib_ref] Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2, Goll [/bib_ref]. DNMT3L, an important regulator without catalytic activity, operates in the form of DNMT3L-DNMT3A heterotetramers and facilitates the methylation of cytosine residues [bib_ref] DNA methylation: roles in mammalian development, Smith [/bib_ref] [bib_ref] Structure and function of eukaryotic DNA methyltransferases, Chen [/bib_ref] [bib_ref] DNA methylation in mammals, Li [/bib_ref]. In animal models, Dnmt3a knockout mice have been found to exhibit postnatal growth retardation and dysplasia and to die by 4 weeks of age [bib_ref] DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and..., Okano [/bib_ref]. Mice deficient in either Dnmt1 or Dnmt3b exhibit embryonic lethality [bib_ref] DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and..., Okano [/bib_ref] [bib_ref] Targeted mutation of the DNA methyltransferase gene results in embryonic lethality, Li [/bib_ref]. Male mice without Dnmt3c are sterile [bib_ref] The DNA methyltransferase DNMT3C protects male germ cells from transposon activity, Barau [/bib_ref]. Thus, these phenotypes demonstrate that the establishment and maintenance of global genomic methylation processes is the basis for cell proliferation and differentiation.
In recent years, interest in the relationship between DNA methylation and human diseases has increased. Alterations in DNA methylation patterns have been implicated in tumorigenesis in several studies [bib_ref] Epigenetics in cancer, Esteller [/bib_ref] [bib_ref] Epigenetic reprogramming in cancer, Suva [/bib_ref] [bib_ref] Epigenetic modulators, modifiers and mediators in cancer aetiology and progression, Feinberg [/bib_ref].
Owing to the revolutionary progress of next-generation sequencing technology, a variety of genomic landscapes of human tumor tissues have been described, and a number of defective genes associated with illnesses have been discovered [bib_ref] Genetic alterations of DNA methylation machinery in human diseases, Hamidi [/bib_ref] [bib_ref] Epigenetic modulators, modifiers and mediators in cancer aetiology and progression, Feinberg [/bib_ref]. Sequencing studies on hematologic disorders achieve big success in identifying previously unrecognized mutated genes [bib_ref] Emerging concepts of epigenetic dysregulation in hematological malignancies, Ntziachristos [/bib_ref]. Among these mutated genes, many, such as DNMT3A, TET2, and IDH1, are involved in epigenetic processes [bib_ref] DNMT3A mutations in acute myeloid leukemia, Ley [/bib_ref] [bib_ref] Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute..., Yan [/bib_ref] [bib_ref] Recurring mutations found by sequencing an acute myeloid leukemia genome, Mardis [/bib_ref] [bib_ref] Mutation in TET2 in myeloid cancers, Delhommeau [/bib_ref] and are directly or indirectly related to DNA methylation. These discoveries bring new prospects for cancer diagnosis and treatment, enabling researchers to fully realize the enormous potential of genomic methylation abnormalities in tumorigenesis.
The following content will describe the relationship between defective DNMTs and tumorigenesis, and finally will focus on the DNMT3A alteration that has been especially well studied.
## Emerging evidence of dnmts in malignant transformation
Tumor cells typically exhibit aberrant DNA methylation patterns during malignant transformation [bib_ref] Gene silencing in cancer in association with promoter hypermethylation, Herman [/bib_ref]. Although this phenomenon is generally attributed to different mechanisms, alteration in the DNMT family of genes and the resulting dysregulation of genomic methylation is a primary causative factor [bib_ref] Cancer epigenetics: from mechanism to therapy, Dawson [/bib_ref] [bib_ref] Cancer epigenetics reaches mainstream oncology, Rodriguez-Paredes [/bib_ref]. Numerous samples with lesions in the DNMT genes have been studied to identify methylation changes and to evaluate cancer development. These lesions can be classified into three categories: overexpression, mutation and deletion [fig_ref] Table 1: Emerging evidence of DNMTs in malignant transformation [/fig_ref].
## Overexpression
Overexpression of DNMTs (DNMT1, DNMT3A, and DNMT3B) in a variety of tumors results in hypermethylation and oncogenic activation [bib_ref] Epigenetics in cancer, Esteller [/bib_ref]. DNMT1 overexpression correlates well with aberrant DNA methylation in solid tumors, thus resulting in lymph node metastasis and poor prognosis in patients [bib_ref] Effects of DNA methyltransferase 1 inhibition on esophageal squamous cell carcinoma, Zhao [/bib_ref] [bib_ref] DNA methylation of multiple tumor-related genes in association with overexpression of DNA..., Peng [/bib_ref] [bib_ref] Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with..., Saito [/bib_ref]. Similarly, highly expressed DNMT3A or DNMT3B has been found in a large number of patient specimens, and increased DNMT3A expression is involved in hepatocellular carcinogenesis [bib_ref] Depletion of DNMT3A suppressed cell proliferation and restored PTEN in hepatocellular carcinoma..., Zhao [/bib_ref]. Moreover, high expression levels of DNMT3B and CTCF are critical in the epigenetic inactivation of BRCA1 in sporadic breast tumors [bib_ref] Epigenetic inactivation of BRCA1 is associated with aberrant expression of CTCF and..., Butcher [/bib_ref]. Additional studies have suggested that DNMT3B is required for the outgrowth of colonic micro-adenomas [bib_ref] Suppression of intestinal neoplasia by deletion of Dnmt3b, Lin [/bib_ref] [bib_ref] Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional..., Linhart [/bib_ref]. Several studies have provided explanations for the relationship between overexpressed DNMTs and tumorigenesis. Zhao et al. have shown that DNMT1 knockdown has an inhibitory effect on the cell cycle in esophageal squamous cell carcinoma, indicating that increased methylation levels promote cell mitosis [bib_ref] Effects of DNA methyltransferase 1 inhibition on esophageal squamous cell carcinoma, Zhao [/bib_ref]. Two groups have demonstrated that DNMT3B overexpression is closely related to CIMP-high in colon cancers [bib_ref] Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic..., Ibrahim [/bib_ref] [bib_ref] DNMT3B expression might contribute to CpG island methylator phenotype in colorectal cancer, Nosho [/bib_ref]. Additional studies performed on cultured primary prostate cells have shown that the overexpression of DNMT3B1 and DNMT3B2, the two subtypes of DNMT3B, leads to an increase in methylation [bib_ref] DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases..., Kobayashi [/bib_ref].
## Mutation
Somatic mutations in DNMTs are the prominent features of many tumors and substantially contribute to malignant transformation [bib_ref] A decade of exploring the cancer epigenomebiological and translational implications, Baylin [/bib_ref]. As shown in [fig_ref] Table 1: Emerging evidence of DNMTs in malignant transformation [/fig_ref] , DNMT1 mutations in colon tumors and DNMT3A mutations in hematological malignancies have been observed in the cancer genome. Kanai et al. have shown that DNMT1 inactivation due to mutational changes in colon cancers results in genome-wide alterations of the DNA methylation status [bib_ref] Mutation of the DNA methyltransferase (DNMT) 1 gene in human colorectal cancers, Kanai [/bib_ref]. Critical findings on DNMT3A variation have suggested that DNMT3A is frequently mutated in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and adult early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) and is associated with disease aggressiveness and treatment resistance [bib_ref] DNMT3A mutations in acute myeloid leukemia, Ley [/bib_ref] [bib_ref] Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute..., Yan [/bib_ref] [bib_ref] Array-based genomic resequencing of human leukemia, Yamashita [/bib_ref] [bib_ref] Recurrent DNMT3A mutations in patients with myelodysplastic syndromes, Walter [/bib_ref] [bib_ref] Whole-exome sequencing in adult ETP-ALL reveals a high rate of DNMT3A mutations, Neumann [/bib_ref]. Mice expressing the Dnmt3a Arg882 mutant protein developed chronic myelomonocytic leukemia with thrombocytosis [bib_ref] DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation..., Xu [/bib_ref]. Moreover, DNMT3A mutations, particularly those in the catalytic domain, substantially decrease enzymatic activity [bib_ref] Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute..., Yan [/bib_ref] [bib_ref] Array-based genomic resequencing of human leukemia, Yamashita [/bib_ref]. In DNMT3A-mutated AML samples and relevant mouse models, such loss of function results in the hypomethylation of HOX family genes [bib_ref] Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute..., Yan [/bib_ref] [bib_ref] DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation..., Xu [/bib_ref]. Together, these studies suggest that mutated DNMTs disrupt genomic methylation and play significant roles in tumor formation.
## Deletion
An in vivo mouse model with embryonically inactive DNMT3A and DNMT3B has shown that the deletion of de novo methyltransferases leads to lethal phenotypes [bib_ref] DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and..., Okano [/bib_ref]. Recently, the effects of de novo methyltransferase on hematopoiesis have been evaluated through conditional knockout technology. The deletion of Dnmt3a in adult mice induces the proliferation of hematopoietic progenitors [bib_ref] Dnmt3a is essential for hematopoietic stem cell differentiation, Challen [/bib_ref]. On the basis of this abnormality, researchers then demonstrated that mutated NRAS-or FLT3-ITDdriven malignancy is accelerated by a lack of Dnmt3a [bib_ref] DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias, Yang [/bib_ref] [bib_ref] Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation, Mayle [/bib_ref] [bib_ref] Loss of Dnmt3a and endogenous Kras(G12D/+) cooperate to regulate hematopoietic stem and..., Chang [/bib_ref] [bib_ref] DNMT3A Haploinsufficiency Transforms FLT3ITD Myeloproliferative Disease into a Rapid, Spontaneous, and Fully..., Meyer [/bib_ref]. Furthermore, the ectopic introduction of c-Kit variants into a Dnmt3a-deficient background produces acute leukemia [bib_ref] Enforced differentiation of Dnmt3a-null bone marrow leads to failure with c-Kit mutations..., Celik [/bib_ref]. Moreover, DNMT3A inactivation leads to the progression of peripheral T cell lymphoma (PTCL) and lung tumors, thus indicating that DNMT3A may act as a tumor-suppressor gene [bib_ref] Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma, Haney [/bib_ref] [bib_ref] Deletion of the de novo DNA methyltransferase Dnmt3a promotes lung tumor progression, Gao [/bib_ref]. Studies have also shown that DNMT3B acts as a tumor suppressor in Myc-induced lymphomas and MLL-AF9-driven AML [bib_ref] Loss of Dnmt3b accelerates MLL-AF9 leukemia progression, Zheng [/bib_ref]. A lack of maintenance methyltransferase activity is also related to carcinogenesis. Studies have shown that DNMT1 deletion leads to DNA demethylation and that DNMT1 is critical for T-cell lymphoma prevention and maintenance, contributing to aberrant methylation by de novo and maintenance methylation [bib_ref] Essential role for Dnmt1 in the prevention and maintenance of MYCinduced T-cell..., Peters [/bib_ref]. Therefore, deletion of genes encoding DNMTs also participates in tumor development.
## Epigenetic disruptions involving dnmts in tumorigenesis
Epigenetic disorders, which are commonly found in cancer, are attributed in part to DNMT dysfunction [bib_ref] Genetic alterations of DNA methylation machinery in human diseases, Hamidi [/bib_ref]. Because of its catalytic role and inhibition of target gene transcription, DNMTs play a significant role in the maintenance of chromosomal homeostasis [bib_ref] DNA methylation in mammals, Li [/bib_ref]. Defective DNMTs induce imbalances in DNA and/or histone modification, thus resulting in chromatin remodeling, genomic instability and gene inactivation. Unlike the genomes in normal tissue, the genomes of tumor cells generally display global hypomethylation throughout, with localized hypermethylation in particular regions [bib_ref] Cancer epigenetics: from mechanism to therapy, Dawson [/bib_ref]. Moreover, crosstalk between DNMTs and other chromatin regulators, such as histone methyltransferases and transcriptional co-suppressors, is highly important in epigenetic disruption [bib_ref] Cancer epigenomics: DNA methylomes and histone-modification maps, Esteller [/bib_ref] [bib_ref] Linking DNA methylation and histone modification: patterns and paradigms, Cedar [/bib_ref] [bib_ref] DNA methylation pathways and their crosstalk with histone methylation, Du [/bib_ref]. These characteristics may contribute to diagnosis and targeted therapy in clinical applications .
## Global hypomethylation
DNA hypomethylation of tumor cells is the first process characterized as an epigenetic abnormality [bib_ref] Gene silencing in cancer in association with promoter hypermethylation, Herman [/bib_ref]. The genome-wide hypomethylation of tumor cells results in a reduction of 5-mC, mainly in genecoding regions and satellite repeats . These changes cause mitotic recombination, copy number deletion and chromosomal rearrangement, and even genomic imprinting annihilation. Gaudet et al. have demonstrated that deletion or reduction of DNMT1 leads to substantial genome-wide hypomethylation and chromosomal instability [bib_ref] Induction of tumors in mice by genomic hypomethylation, Gaudet [/bib_ref]. Through methylated DNA immunoprecipitation (MeDIP)-chip analysis, hypomethylated CpG islands (CGIs) of the HOXB cluster have been found in AML samples with DNMT3A mutations [bib_ref] Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute..., Yan [/bib_ref]. Although the underlying mechanism governing the effects of genome-wide hypomethylation on the process of tumorigenesis is not fully understood, these limited data have provided alternative insight into a relationship between aberrant DNMTs and global hypomethylation, along with subsequent tumor occurrence [bib_ref] Epigenetic Determinants of Cancer, Baylin [/bib_ref].
## Localized hypermethylation
In normal somatic cells, DNA methylation occurs primarily in dinucleotides containing less CpG, whereas the CpG-enriched region is unmethylated [bib_ref] DNA methylation in mammals, Li [/bib_ref] [bib_ref] DNA methylation pathways and their crosstalk with histone methylation, Du [/bib_ref]. Throughout malignant transformation, the global methylation level of DNA changes, thus leading to non-CpG island hypomethylation and CGI hypermethylation. As a result, the number of genes that are hypermethylated on their promoters increases. In particular, hypermethylation induces the silencing of several key tumor-suppressor genes (TSGs), which play important roles in tumor progression . Generally, abnormal CGI hypermethylation is an epigenetic characteristic of tumors, of which hypermethylated TSGs are the most common feature [bib_ref] Epigenetic Determinants of Cancer, Baylin [/bib_ref] [bib_ref] Epigenetic modulators, modifiers and mediators in cancer aetiology and progression, Feinberg [/bib_ref]. A great deal of research has been performed to explore the mechanism of aberrant TSG methylation in tumor tissue. As expected, DNMTs have been included in the aforementioned studies. In leukemia, deletions or mutations in DNMTs often disrupt the distribution of 5-mC in the genome [bib_ref] DNA methylation in normal and malignant hematopoiesis, Celik [/bib_ref]. Butcher et al. have shown that in some sporadic breast tumors, hypermethylation of the BRCA1 promoter is partially due to DNMT3B overexpression [bib_ref] Epigenetic inactivation of BRCA1 is associated with aberrant expression of CTCF and..., Butcher [/bib_ref]. Using a conditional Dnmt3a knockout mouse model, researchers have observed a general decrease in hypomethylation in the transcription factor-binding sites of cross-regions (Canyons) [bib_ref] Large conserved domains of low DNA methylation maintained by Dnmt3a, Jeong [/bib_ref]. Additionally, canyonassociated genes, including HOX genes, are markedly enriched in DNMT3A mutant AMLs [bib_ref] Large conserved domains of low DNA methylation maintained by Dnmt3a, Jeong [/bib_ref].
Tumor-associated DNA methylation generally occurs in the promoter regions of TSGs [bib_ref] Epigenetic modulators, modifiers and mediators in cancer aetiology and progression, Feinberg [/bib_ref]. However, owing to rapid advancements in methylation sequencing, data increasingly indicate that a large number of non-TSGs are methylated at the early stage of tumor initiation, and methylation changes within the gene body have a substantial effect on the process of transcription. The TCGA network has reported the integrated methylation profiles of AML samples with mutations in DNMT3A, as determined with Human Methylation 450 Bead Chip arrays. This complete epigenomic landscape reveals a large amount of hypermethylated cytosine bases in the gene body and intergenic regions. Similarly, in a Dnmt3a mutant-transduced mouse model, hypermethylation is greater in the intergenic regions, and a cluster of suppressed genes related to lymphocyte development, such as Notch1 and Gata3, are hypermethylated in the gene body regions [bib_ref] DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation..., Xu [/bib_ref]. Furthermore, Yang et al. have suggested that DNMT3B-dependent gene body methylation enhances transcription and may be a potential therapeutic target in cancer [bib_ref] Gene body methylation can alter gene expression and is a therapeutic target..., Yang [/bib_ref].
## Interaction with histone modifications
The entire epigenetic profile of the genome shows that active chromatin regions are generally characterized by acetylated histones and unmethylated DNA, whereas methylated histones associated with repressed chromatin and methylated DNA are enriched in suppressed regions [bib_ref] DNA methylation pathways and their crosstalk with histone methylation, Du [/bib_ref]. Thus, the two chromatin markers interact in a highly orchestrated manner and are closely linked: DNA methylation helps guide histone modification, and histone modification directs DNA methylation . For example, DNMT1 is required for the maintenance of H3K9 methylation in human cancer cells [bib_ref] Human DNA methyltransferase 1 is required for maintenance of the histone H3..., Espada [/bib_ref] , and DNMT3A PWWP interacts with H3K36me3 and consequently enhances DNMT3A activity [bib_ref] The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides..., Dhayalan [/bib_ref]. These effects can be regarded as the outcome of cooperation between histone methyltransferase (HMT) and DNMTs. Indeed, DNMTs form complexes with HMTs and consequently regulate transcription. Both the H3K36 methyltransferase Epigenetic alterations involving DNMTs in tumorigenesis. Numerous clinical and experimental data suggest that tumor cells generally exhibit genome-wide hypomethylation and localized hypermethylation, in contrast with normal cells. Interactions between DNMTs and histone methyltransferases, such as EZH2 and SETD2, play critical roles in epigenetic disruption during malignancy. Thus, the identification of epigenetic alterations involving DNMTs in tumorigenesis may contribute to improved cancer diagnosis and effective treatments SETD2 and the PWWP domain of DNMT3B are required for the de novo methylation of transcribed genes [bib_ref] Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic..., Baubec [/bib_ref]. Likewise, the ADD domain of DNMT3A recognizes unmodified H3, which is repressed by H3K4 methylation [bib_ref] Structural insight into autoinhibition and histone H3-induced activation of DNMT3A, Guo [/bib_ref]. In undifferentiated human embryonic carcinoma cells, promoter-related DNMTs overlap with different histone modifications [bib_ref] Linking DNA methyltransferases to epigenetic marks and nucleosome structure genome-wide in human..., Jin [/bib_ref]. Two groups have demonstrated that DNMT1 improves genomic methylation through enhanced histone modification by EZH2. EZH2 polycomb group protein mediates H3K27 methylation and recruits and directly controls DNA methylation [bib_ref] The Polycomb group protein EZH2 directly controls DNA methylation, Vire [/bib_ref] [bib_ref] Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo..., Schlesinger [/bib_ref]. Thus, the above-mentioned investigations confirm that abnormal DNA methylation in tumor cells is closely related to histone modification. The relationship between DNA methylation and histone modification should provide more comprehensive insights into epigenetic regulation in tumorigenesis.
## Dnmt3a alterations lead to epigenetic reprogramming in leukemia
In recent years, mutated genes encoding a group of epigenetic modification regulators have attracted attention because of their high frequency of variation in hematological diseases [bib_ref] The role of mutations in epigenetic regulators in myeloid malignancies, Shih [/bib_ref]. Epigenetic disruption due to genetic alterations is the root cause of malignant transformation, particularly in hematologic malignancies [bib_ref] The origin and evolution of mutations in acute myeloid leukemia, Welch [/bib_ref]. Most notably, through a variety of high-throughput techniques, somatic mutations involving the DNMT3A gene have been identified in AML at a mutation rate of~20%, and the prognosis for mutant patients is relatively poor [bib_ref] DNMT3A in haematological malignancies, Yang [/bib_ref]. Currently, DNMT3A abnormalities are the most common subject in the field of epigenetic medical research, because of its significance in tumor pathogenesis and the potential for target medication. Herein, the organization characteristic of DNMT3A and critical implications of DNMT3A alterations in hematological cancers are highlighted.
## Dnmt3a structure and function
As a member of the DNMT family, DNMT3A possesses the characteristic peptide structure: its catalytic domain directly binds to S-adenosyl-L-methionine (SAM) and DNA strands, and the N-terminus regulation domain is primarily involved in nuclear localization and protein interactions, in which the PWWP domain interacts with methyl lysine histones, and the PHD domain recognizes unmethylated histones. These functions serve as a signal of the histone transfer effect, thus ensuring diverse epigenetic modification [bib_ref] Structure and function of eukaryotic DNA methyltransferases, Chen [/bib_ref]. Specifically, DNMT3A forms a butterfly-shaped tetramer (DNMT3L-DNMT3A-DNM T3A-DNMT3L) in the C-terminus with DNMT3L, thus changing the conformation of DNMT3A and facilitating its catalytic activity. The N-terminus of DNMT3A also operates as a transcriptional repressor.
The regulatory domain recruits nucleoproteins into the complex and performs histone modifications, chromatin remodeling and gene transcription. A range of partners is known to interact with DNMT3A, including histone methyltransferases, histone deacetylases, and various transcription factors, even enzymes in the DNMT family [bib_ref] DNA methylation pathways and their crosstalk with histone methylation, Du [/bib_ref]. DNMTs are bound to each other, and de novo methyltransferase said in coordination during methylation maintenance [bib_ref] Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo..., Jia [/bib_ref]. Studies have shown that H3K9 methyltransferases, such as SUV39H1 and SETDB1, can directly bind to the PHD domain of DNMT3A and improve each other's catalytic activity, thus indicating that different epigenetic modifications can enhance chromatin inhibition by cooperating together [bib_ref] Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET, Matsui [/bib_ref].
## Dnmt3a mutation in leukemia
Clinically, in patients with DNMT3A mutations, the number of leukocytes present at diagnosis is relatively higher, and survival is comparatively shorter [bib_ref] DNMT3A mutations in acute myeloid leukemia, Ley [/bib_ref] [bib_ref] Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute..., Yan [/bib_ref]. To date, numerous functional experiments have provided a better understanding of the effects of DNMT3A mutation on leukemia pathogenesis [fig_ref] Figure 2: DNMT3A alterations lead to epigenetic reprogramming in leukemia [/fig_ref]. For instance, DNMT3A mutation is an early event in the initiation of hematopoietic disorders and is one of several causative factors for the establishment of founder clones and the transformation of hematopoietic stem cells (HSCs) to pre-leukemic stem cells (Pre-LSCs) [bib_ref] Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia, Shlush [/bib_ref]. In addition, DNMT3A mutants harbor dominant-negative effects, such as those exhibited by DNMT3A R882H (R878H in mouse) mutated protein against wild-type DNMT3A [bib_ref] A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES..., Kim [/bib_ref] [bib_ref] The R882H DNMT3A mutation associated with AML dominantly inhibits wild-type DNMT3A by..., Russler-Germain [/bib_ref]. DNMT3A mutations also disrupt hematopoiesis. Researchers have used a bone marrow transplantation mouse model to determine that the function of Dnmt3a mutants in blood cell production is aberrant [bib_ref] DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation..., Xu [/bib_ref]. Additional studies have shown that mutated DNMT3A disrupts normal hematopoiesis and promotes the transformation to malignant cells, in combination with other epigenetic regulators [bib_ref] DNMT3A R882 mutants interact with polycomb proteins to block haematopoietic stem and..., Koya [/bib_ref]. In Dnmt3a-mutated models, a double-hit is essential for clonal expansion. In vivo experiments suggest that the mutation or deletion of Dnmt3a induces the development of leukemia by cooperating with oncogenic factors, such as RAS mutation, c-Kit variation, or FLT3-ITD abnormalities [bib_ref] DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias, Yang [/bib_ref] [bib_ref] Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation, Mayle [/bib_ref] [bib_ref] Loss of Dnmt3a and endogenous Kras(G12D/+) cooperate to regulate hematopoietic stem and..., Chang [/bib_ref] [bib_ref] DNMT3A Haploinsufficiency Transforms FLT3ITD Myeloproliferative Disease into a Rapid, Spontaneous, and Fully..., Meyer [/bib_ref] [bib_ref] Enforced differentiation of Dnmt3a-null bone marrow leads to failure with c-Kit mutations..., Celik [/bib_ref] [bib_ref] Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and..., Lu [/bib_ref]. DNMT3A mutations may also play an important role in tumor metastasis. For example, DNMT3A mutant leukemia cells may undergo leukemic extramedullary infiltration in NOD/SCID mice, a result partially linked to high expression levels of TWIST1, an epithelial-mesenchymal transition (EMT) inducer [bib_ref] DNMT3A mutation leads to leukemic extramedullary infiltration mediated by TWIST1, Xu [/bib_ref]. In summary, DNMT3A mutation exerts a great influence in hematological malignancy. A variety of small molecule compounds targeting relevant epigenetic disruptions have been developed and applied in the treatment of leukemia, which also provide a comprehensive innovation for the study of pathogenesis and targeted therapy of solid tumors.
# Conclusions
Owing to advances in sequencing technologies, numerous gene alterations associated with epigenetics have been identified in cancer genomes. Furthermore, wholeepigenome approaches, including array-based methylation profiling and bisulfite sequencing, afford a comprehensive view of the tumor methylome, and potential mechanisms of epigenetic disruption caused by DNMT changes have been explored. However, the effects of DNMT aberrations in the promotion of tumorigenesis are not entirely clear, and novel strategies for relevant targeted therapies must be developed. Future work should focus on the elucidation of tumorigenic mechanisms induced by defective DNMTs and the production of effective therapeutic approaches. Leukemia is a heterogeneous disease caused by cumulative multi-step disruption. In the initial stage of leukemogenesis, accumulated DNA lesions, emergent stimuli, and metabolic stress are observed in hematopoietic stem cells (HSCs). These conditions lead to gene alterations and link epigenetic reprogramming to leukemia development. Currently, DNMT3A gene lesions are considered to be critical epigenetic alterations in the occurrence of leukemia. In patient specimens and mouse models, the mutation or deletion of DNMT3A causes the apparent reversal of normal HSCs into pre-leukemia stem cells (Pre-LSCs). Frequently, Pre-LSCs are quiescent and stable in the early phases of leukemia. The accumulation of other transformative changes, such as a series of mutations (RAS mut , NPM1 mut , c-Kit mut ) or oncogenic alterations (FLT3 ITD ) causes Pre-LSCs to undergo malignant transformation into leukemia stem cells (LSCs), which finally enter the clonal expansion stage. Furthermore, during the aggressive progression of leukemia in a xenograft mouse model of OCI-AML3 with mutated DNMT3A, DNMT3A mutation promotes leukemic extramedullary infiltration by up-regulating the expression of the EMT inducer TWIST1. HSCs: hematopoietic stem cells; Pre-LSCs: pre-leukemia stem cells; LSCs: leukemia stem cells; mut: mutation; del: deletion; ITD: internal tandem duplication; OE: overexpression; EMT: epithelial-mesenchymal transition
[fig] Figure 2: DNMT3A alterations lead to epigenetic reprogramming in leukemia. [/fig]
[table] Table 1: Emerging evidence of DNMTs in malignant transformation [/table]
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Variation in use of non-surgical treatments among osteoarthritis patients in orthopaedic practice in the Netherlands
# Introduction
Patients with symptomatic hip or knee osteoarthritis (OA) suffer from pain and loss of function for which treatment is required. Different treatment options are available, surgical and non-surgical treatments. For patients, good quality of care is achieved if their symptoms are managed in the short term, but also taking into account which treatment option results in the best longterm patient outcomes. As the lifespan of a prosthesis is limited, and patient outcomes after revision arthroplasty are not as good as after primary surgery, [bib_ref] Management of osteoarthritis of the knee, Bennell [/bib_ref] it is generally acknowledged that total hip arthroplasty (THA) and total knee arthroplasty (TKA) should not be performed too early. For this reason, evidence-based guidelines recommend to start with non-surgical treatments (eg, education, physical therapy, non-steroidal antiinflammatory drugs (NSAIDs)), [bib_ref] OARSI recommendations for the management of hip and knee osteoarthritis: part III:..., Zhang [/bib_ref] [bib_ref] American College of Rheumatology 2012 recommendations for the use of nonpharmacologic and..., Hochberg [/bib_ref] [bib_ref] Care and management of osteoarthritis in adults: summary of NICE guidance, Conaghan [/bib_ref] [bib_ref] EULAR recommendations for the non-pharmacological core management of hip and knee osteoarthritis, Fernandes [/bib_ref] and to use surgical intervention only if a patient does not respond sufficiently to non-surgical treatment options in the context of end stage OA. [bib_ref] Evidence-based tailored conservative treatment of knee and hip osteoarthritis: between knowing and..., Snijders [/bib_ref] [bib_ref] Conservative non-pharmacological treatment options are not frequently used in the management of..., Shrier [/bib_ref] [bib_ref] Knee osteoarthritis clinical practice guidelines-how are we doing?, Dehaan [/bib_ref] [bib_ref] Quality of osteoarthritis care for community-dwelling older adults, Hunter [/bib_ref] [bib_ref] The quality of health care delivered to adults in the United States, Mcglynn [/bib_ref] [bib_ref] The use of conventional and complementary treatments for knee osteoarthritis in the..., Jordan [/bib_ref] [bib_ref] First line treatment of knee osteoarthritis in outpatients in France: adherence to..., Denoeud [/bib_ref] These recommendations are all based on the large body of evidence supporting the effectiveness of these non-surgical treatments to help patients with hip and knee OA manage their symptoms and preserve joint function. [bib_ref] A systematic review of recommendations and guidelines for the management of osteoarthritis:..., Nelson [/bib_ref] Despite these guidelines, several studies have suggested that the use of non-surgical
## Strengths and limitations of this study
▪ This study assesses the use of non-surgical treatments in orthopaedic practice, which has not been done before. ▪ This study includes the perspective of orthopaedic surgeons and patients. ▪ Owing to the retrospective nature of our study and the reliance on self-reported data, it is susceptible to recall bias.
treatments in patients with hip or knee OA can be improved. For instance, Snijders et al 7 demonstrated that 81% of patients with hip or knee OA did not receive all non-surgical treatments in the primary care setting. However, patients with OA may receive nonsurgical treatments later on in the care trajectory from their orthopaedic surgeon once referred to specialist care. In the Netherlands, patients with OA are usually treated by the general practitioner. According to guidelines, patients should be referred to the orthopaedic surgeon if they do not respond sufficiently to nonsurgical treatment options. In orthopaedic practice, the decision will be made to start/continue non-surgical treatments or to perform a surgery depending on previous received treatments and disease severity. Therefore, orthopaedic surgeons have an important role in ensuring optimal care of patients with OA by confirming whether recommended non-surgical treatments have been exhaustedly used before considering a surgical intervention. Furthermore, surgical interventions, like a THA and TKA do not have good patient reported outcome in about 10-20% of patients. [bib_ref] Knee arthroplasty: are patients' expectations fulfilled? A prospective study of pain and..., Nilsdotter [/bib_ref] [bib_ref] Functional outcome and patient satisfaction in total knee patients over the age..., Anderson [/bib_ref] [bib_ref] The role of pain and function in determining patient satisfaction after total..., Baker [/bib_ref] [bib_ref] Predicting patient dissatisfaction following joint replacement surgery, Gandhi [/bib_ref] This stresses even more the importance of good non-surgical treatment modalities before implant surgery is encountered.
McHugh et al 23 examined which treatments patients with OA used throughout the care trajectory, before and while on the waiting list for a TKA or THA, and showed that only 10% of the patients had received information on pain management with the consequence that some patients used their own regime to take analgesics. This in turn may have led to insufficient effects of analgesics. However, they did not investigate the full range of nonsurgical treatments and the estimates were reported by patients, so that the information may have been given to them but not remembered. Given the known effectiveness of non-surgical treatments individually and for some combinations (eg, physical therapy with dietary therapy [bib_ref] Exercise and dietary weight loss in overweight and obese older adults with..., Messier [/bib_ref] , use of the full range of available recommended non-surgical treatments may improve patient outcomes, 2-6 thereby improving quality of care, and postpone the need for surgery which would reduce chances for a revision with worse patient outcomes than primary surgery. What is currently lacking and needed to provide a complete view, is an assessment of the full range of non-surgical treatments as well as including both patients' and orthopaedic surgeons' perspectives, as these may differ. Therefore, the aim of this study was to assess the extent to which all recommended non-surgical treatments were used by patients with hip or knee OA as reported by patients and orthopaedic surgeons, both as a single option and in combination.
## Materials and methods study design
We performed two cross-sectional internet-based surveys in November and December 2013 and January 2014 to assess the use of non-surgical treatments in orthopaedic practice as reported by patients and orthopaedic surgeons.
## Population patients
A total of 195 patients were invited by email to participate in the survey, to estimate a previously reported 19% use of non-surgical treatments among 47 000 patients with hip and knee OA annually in the Netherlands,with a 5% margin of error. Patients were recruited via advertisements in local newspapers across the Netherlands, and through the websites or newsletters of patient associations. Patients who volunteered to participate in the survey in reaction to the advertisements were dialed by the research team to provide information about the study, to answer questions and to ask whether they approved for participation. In addition, patients received written information before the start of the survey and the availability to stop during the study. In addition, patients received written information before the start of the survey and the availability to stop during the study. Inclusion criteria for patients were: age ≥18 years, a doctor's diagnosis of hip or knee OA, and either have undergone TKA or THA no longer than 12 months ago or being on the waiting list for surgery with a confirmed date within 3 months. Patients who were unable to understand written Dutch or who had undergone or were scheduled for revision surgery were excluded from the study. Patients who initially indicated that they wanted to participate but did not respond, were sent two reminders, one after one and a half weeks, and if still no response again after 3 weeks. Participants who completed the questionnaire received a €10 gift card as an incentive.
## Orthopaedic surgeons
All 482 Dutch orthopaedic surgeons listed in the registry of the Netherlands Orthopaedic Association (NOV) and/or the Dutch medical address book with an email address received an invitation to participate. All orthopaedic surgeons who treated patients with hip or knee OA were eligible. Orthopaedic surgeons who did not respond received two reminders, one after one and a half weeks and if still no response again after 3 weeks. Orthopaedic surgeons did not receive an incentive for their participation.
## Survey development survey for patients
The survey for patients included questions about general patient characteristics, general health and symptoms of OA, and non-surgical treatment for OA. Patient characteristics included: age, gender, region of residence (north, middle, and south), educational level (basic education (none or only primary education), intermediate education ( prevocational secondary education, senior secondary vocational training, senior secondary general education, pre-university education), or high education (higher professional education or university (bachelor, master, or PhD degree)), work situation ( paid work or not), height (cm) and weight (kg) to calculate the body mass index (BMI), and type of insurance (basic and/or additional coverage for care such as physical therapy, glucosamine sulfate and hyaluronic acid). Furthermore, the survey included general and disease-specific health questions, such as duration of OA and duration of complaints of the affected joint, comorbidities, average pain during 6 months before surgery, measured on a 0 (no pain)-10 (unbearable pain) scale, and patient-perceived reasons for surgery.
Questions about healthcare use included all nonsurgical options before joint replacement surgery as described in the Dutch stepped-care strategy (SCS) and were formulated as follows: "Did you receive the following treatments for the complaints of your affected joint before joint replacement surgery?." The SCS is based on (inter)national guidelines. The first step consists of education, life style advice and acetaminophen. If the treatment options in the first step are not sufficient, treatment options in the second step can be considered (exercise therapy, dietary therapy and NSAIDs).
Multidisciplinary care, intra-articular injections, and transcutaneous electrical nerve stimulation (TENS) are treatment options in the third step and could be considered if treatment options in step one or two are ineffective.
In the survey these non-surgical treatments were formulated as follows: education about the disease OA, education about the possible treatment options in OA, lifestyle advices (ie, stay active, lose weight), physical therapy/ exercise therapy, acetaminophen, anti-inflammatory painkillers (eg, NSAIDS such as Celebrex, Diclofenac, Cataflam, Voltaren), tramadol (eg, Tramal, Tramagetic, Tradonal, Zaldiar), multidisciplinary care (care of different healthcare providers at the same time, eg, in a revalidation centre), injections in the knee, TENS (therapy that uses electrical current on the skin). Patients could choose one or more of the following answers: yes, received from the orthopaedic surgeon; yes, received from another healthcare provider; yes, received on my own initiative; no.
## Survey for orthopaedic surgeons
The survey for orthopaedic surgeons included questions about their background characteristics, and the prescription of non-surgical treatments. Characteristics of orthopaedic surgeons included: age, gender, work region, work setting, years of working experience as an orthopaedic surgeon, number of new patients with hip/knee OA seen per month. Questions about prescribed treatments included all non-surgical options described in the SCS and were formulated as follows: "If patients did not receive the following non-surgical treatments, do you prescribe these treatments?" In case of physical therapy or dietary therapy we asked whether they referred patients, rather than initiating this treatment themselves. Orthopaedic surgeons could choose one of the following answers: never, sometimes, often or (almost) always.
# Analysis
Descriptive statistics were used to describe the characteristics of respondents, and the use of non-surgical treatments from the patients' or orthopaedic surgeons' perspectives. From the patient perspective we distinguished the use of non-surgical treatments prescribed by any healthcare provider, by the orthopaedic surgeon, or undertaken by their own initiative. From the orthopaedic surgeon perspective we dichotomised the answers into 'prescribed' (often/almost always) and 'not prescribed' (never/sometimes).
To assess the use of non-surgical treatments, we made a distinction between non-surgical treatments recommended by various organisations (eg, OARSI, EULAR, AAOS, NOV) [bib_ref] A systematic review of recommendations and guidelines for the management of osteoarthritis:..., Nelson [/bib_ref] [bib_ref] Health care use of patients with osteoarthritis of the hip or knee..., Smink [/bib_ref] and other non-surgical treatments. The recommended non-surgical treatments were education about OA, education about different treatment options, lifestyle advice, (referral to) dietary therapy, physical therapy containing exercises, acetaminophen, NSAIDs and glucocorticoid injections. Other nonsurgical treatments included glucosamine sulfate, tramadol, multidisciplinary care, TENS, and hyaluronic acid injections (for knee OA). These treatments are not supported by high quality evidence or clinical guidelines, but are nevertheless sometimes recommended and used by patients with OA. BMI of patients was classified into normal weight if BMI <25 kg/m 2 , overweight if BMI ≥25 <30 kg/m 2 , and obese if BMI ≥30 kg/m 2 to assess whether dietary therapy was indicated. If BMI was unknown, we assumed that dietary therapy was not indicated for that patient.
For each non-surgical treatment, we calculated the percentage of patients who had received this treatment, and the percentage of orthopaedic surgeons who always/ often prescribed this treatment for their patients. In addition, we calculated the percentage of participants who received/prescribed the recommended non-surgical treatments listed in each step of the Dutch SCS including the previous steps (conditional percentage). The proportion of patients and orthopaedic surgeons using each non-surgical treatment was compared using the χ 2 test.
In addition, we explored whether patients and orthopaedic surgeons using all recommended treatments differed from those who did not, in age, gender, region of residence, BMI and level of education (for patients) and on differences in age, gender, work region, work setting, years of working experience and number of new patients with hip/knee OA seen per month (for orthopaedic surgeons). We also explored differences in use of each treatment between patients with THA and TKA. The independent t test or Mann Whitney U tests for continuous variables and χ 2 tests or Fisher's exact tests for proportions was used to compare differences between subgroups. Significance testing was done two-sided at α=0.05. SPSS V.20.0 was used for analyses.
## Ethics
This study protocol was presented to the Medical Ethical Committee of the Leiden University Medical Center (CME P13.087/NV/nv). An exemption was obtained, as ethical approval for this type of study is not required under the Dutch law.
# Results
## Response
A total of 182 patients (response rate of 93%) completed the survey. Eight patients were subsequently excluded from the analyses, because they did not fulfil the inclusion criteria. This left 174 patients (89%) included in the final analyses.
One hundred and eighty one (response rate of 38%) orthopaedic surgeons completed the questionnaire. Nine orthopaedic surgeons were excluded because they indicated they did not see patients with OA in consultations. Thus a total of 172 (36%) orthopaedic surgeons were included in the final analyses.
## Characteristics of the population patients
Characteristics of patients who completed the questionnaire are described in table 1. The majority of the participants were female, 26% were obese (BMI ≥30 kg/m 2 ) and thus indicated for dietary therapy. Most of the respondents had already undergone THA or TKA at the time of recruitment, and a significant proportion of patients reported a duration of symptoms for more than 5 years. Almost all patients had additional insurance coverage, meaning that physical and dietary therapy was likely to be covered by their insurance rather than being subject to out of pocket expenses.
## Orthopaedic surgeons
The characteristics of the orthopaedic surgeons who completed the questionnaires are presented in table 2. On average, they had been working for 13 years (SD 8) as an orthopaedic surgeon, and saw an average of 25 new patients with hip OA (SD 24) and 31 (SD 22) new patients with knee OA per month. Orthopaedic surgeons from various parts of the country and different hospital types were included in the sample. [fig_ref] Table 3: Received recommended and non-recommended non-surgical treatments by patients as reported by patients [/fig_ref] shows the percentage of patients that received recommended and non-recommended non-surgical treatments as reported by patients.
## Use of recommended non-surgical treatments
The most frequently received non-surgical treatments were education about OA (80%), physical therapy (73%), acetaminophen (72%), education about different treatment options (66%) and NSAIDs (64%). Of these, education about OA and education about different treatment options were mostly received from the orthopaedic surgeon [fig_ref] Table 3: Received recommended and non-recommended non-surgical treatments by patients as reported by patients [/fig_ref] , whereas the other treatments were received from another healthcare professional, or patients own initiative. Dietary therapy was used least frequently, even when non-overweight patients were excluded. Only 11% of overweight patients and 30% of the obese patients reported they had received dietary therapy. A minority of these patients was referred to a dietician by their orthopaedic surgeon [fig_ref] Table 3: Received recommended and non-recommended non-surgical treatments by patients as reported by patients [/fig_ref]. In addition, looking at the conditional percentage in table 4, only 33% of the patients received all recommended treatments in step 1 of the SCS, and eleven (6%) patients reported to have received all recommended treatments in step 1 and 2 of the SCS. Because many patients did not remember which type of injection they received, we excluded glucocorticoid injections and did not calculated the conditional percentage of step 1, 2 and 3 together.
Reasons for surgery according to patients (multiple answers possible) were: pain could not be controlled with painkillers (55% of the patients), insufficient effect of other treatments (eg, physical therapy, dietary advice) (51%), duration of symptoms (41%), difficulties with daily activities (75%), or other reasons (17%) (eg, severe cartilage loss, difficulties with sports, immobility).shows the percentage of orthopaedic surgeons that often or always prescribes recommended nonsurgical treatments as reported by orthopaedic surgeons. Orthopaedic surgeons often reported to prescribe lifestyle advice (98%), education about different treatment options (95%), education about OA (87%) and acetaminophen (64%). However, table 6 shows that only 96 (56%) of the orthopaedic surgeons reported prescribing all recommended treatments in step 1 of the SCS, 17 (10%) reported prescribing all recommended treatments in step 1 and 2, and 10 (6%) reported prescribing all recommended treatments in step 1, 2 and 3, if the patient had not received these treatments in their previous care trajectory. As among patients, dietary therapy was reported as the least prescribed treatment (reported by 28% of the orthopaedic surgeons), followed by intra-articular injections (43%) and physical therapy (54%). No differences were found between patients receiving and not receiving all recommended non-surgical treatments in any of the patient characteristics tested. Similarly, no differences were found between orthopaedic surgeons prescribing and not prescribing all recommended non-surgical treatments in any of their characteristics. Patients with knee OA received acetaminophen more often than patients with hip OA (80% vs 63% respectively ( p=0.01)), but no differences were found in the use of other treatments. Furthermore, comparing patients and orthopaedic surgeons, no differences were found in the proportion using education about OA ( p=0.09), acetaminophen ( p=0.18), NSAIDs ( p=0.39), and the percentage using all recommended non-surgical treatments ( p=0.22). A smaller percentage of patients compared to orthopaedic surgeons reported having received/prescribed education about different treatment options ( p<0.001), lifestyle advice ( p<0.001) and dietary therapy ( p=0.03). The use of physical therapy on the other hand was reported to have been received by more patients than being prescribed by orthopaedic surgeons (73% vs 54% ( p<0.001)).
## Use of other non-surgical treatments
Glucosamine sulfate was the most frequently used other non-surgical treatment, reported by 33% of patients, and mostly (79%) used on their own initiative. Multidisciplinary care (7%) and TENS (6%) were the least often reported other treatments. Thirty-three per cent of the patients who received multidisciplinary care were referred by their orthopaedic surgeon, and none of the patients who used TENS was referred by their orthopaedic surgeon. Overall, orthopaedic surgeons rarely prescribed any of these treatments not recommended by published OA guidelines, the highest percentage (8%) was for the recommendation of glucosamine sulfate.
# Discussion
Our study showed that although most recommended non-surgical treatments seem to be frequently used as a single option in OA patients who receive(d) a THA or TKA, only a small percentage of patients received all recommended non-surgical treatments. For that matter, only 6% of patients and 10% of orthopaedic surgeons reported using all recommended non-surgical treatments in step 1 and 2 of the SCS. [bib_ref] Beating osteoARThritis": development of a stepped care strategy to optimize utilization and..., Smink [/bib_ref] Given the known effectiveness of each of these treatments individually, use of the full range of available modalities may improve patient outcomes. [bib_ref] OARSI recommendations for the management of hip and knee osteoarthritis: part III:..., Zhang [/bib_ref] [bib_ref] American College of Rheumatology 2012 recommendations for the use of nonpharmacologic and..., Hochberg [/bib_ref] [bib_ref] Care and management of osteoarthritis in adults: summary of NICE guidance, Conaghan [/bib_ref] [bib_ref] EULAR recommendations for the non-pharmacological core management of hip and knee osteoarthritis, Fernandes [/bib_ref] Dietary therapy was the least frequently used recommended non-surgical treatment for OA. Only 11% of overweight patients and 30% of obese patients reported having received dietary therapy, and only 28% of orthopaedic surgeons reported they would prescribe dietary therapy. Another study in the Netherlands showed that only 14% of overweight and obese patients with OA reported receiving dietary therapy. [bib_ref] Health care use of patients with osteoarthritis of the hip or knee..., Smink [/bib_ref] This is even lower than reported in our study, but these patients were recruited by general practitioners, and thus may have subsequently received dietary therapy later on in the care trajectory for example, after referral to an orthopaedic surgeon. In our study, patients had visited multiple healthcare providers, potentially increasing the likelihood of being offered dietary therapy when indicated. In other countries, dietary therapy seems to be more commonly used, for example, 59% of physicians prescribed 'weight reduction' in a study performed in France, [bib_ref] First line treatment of knee osteoarthritis in outpatients in France: adherence to..., Denoeud [/bib_ref] and 31% of patients with OA in a study in Canada. [bib_ref] Use of mainstream nonpharmacologic treatment by patients with arthritis, Li [/bib_ref] Although the numbers are higher, the overall low rates across studies suggest that there is room for improvement. Similarly, a considerable number of patients were not prescribed physical therapy. The use of physical therapy as a non-surgical treatment could even be overestimated, because orthopaedic surgeons Conditional percentage of patients receiving recommended non-surgical treatments in stepped care strategy as reported by patients
Recommended non-surgical treatments in stepped care strategy Conditional n (%)
Step 1: education about OA+education about different treatment options+lifestyle advice+acetaminophen 57 (33)
Step 1+2: step 1+(Referral to) dietary therapy, when indicated+physical therapy+NSAIDs 11Step 1, 2+3: step 1+2+intra-articular injections (for knee OA n=94) n/c n/c: not calculated, because many patients did not know which type of injection they received. NSAIDs, non-steroidal anti-inflammatory drugs; OA, osteoarthritis. sometimes prescribe physical therapy as preparation before surgery instead of a non-surgical treatment to delay surgery. Dietary therapy and physical therapy are the only two recommended non-surgical treatments that an orthopaedic surgeon cannot provide himself, but for which referral is needed. Improving the use of these two treatments in orthopaedic care, may result in better quality of patient care as the combination of weight loss plus exercise is shown to provide better overall improvements in function, pain and mobility among older overweight and obese adults with knee OA compared with either intervention alone. [bib_ref] Exercise and dietary weight loss in overweight and obese older adults with..., Messier [/bib_ref] This study has some limitations. First, because of the retrospective nature of our study and the reliance on self-reported data, it is susceptible to recall bias. In an attempt to reduce this bias, we limited inclusion to patients who had a TKA or THA no longer than 12 months ago, or were scheduled for surgery within the next 3 months. Second, the use of an internet-based survey could induce selection bias. It is known that the majority of THA and TKA patients prefer pen-and-paper questionnaires, and that patients who prefer electronic questionnaires differ from patients who prefer pen-and-paper questionnaires. [bib_ref] Hip and knee replacement patients prefer pen-and-paper questionnaires: Implications for future patient-reported..., Keurentjes [/bib_ref] It is possible that more elderly persons do not have internet or an email address compared to younger persons, which could have led to a selection of younger persons. The average age of patients with OA is 68.2 years 29 and our population is slightly younger, on average 64 (SD 7.7) years. However, age was not associated with the use of all recommended non-surgical treatments. Third, the use of a sample of patients responding to an advertisement may have introduced sampling bias. However, as our responding patients were distributed across different regions in the Netherlands, and had an age and gender distribution comparable to OA patients, we think that any bias that may have occurred is likely to be small. Similarly, selection bias may have occurred as a result of the low response rate (38%) among orthopaedic surgeons. However, such a response rate is comparable or higher than found in other online surveys among orthopaedic surgeons in the Netherlands. It is possible that orthopaedic surgeons who are not interested in nonsurgical treatment were less likely to complete the questionnaire or that orthopaedic surgeons overestimate their use of non-surgical treatments. This would only lead to an overestimation of non-surgical treatment use and the use may be even lower. Furthermore, the use of acetaminophen, NSAIDs and tramadol could have been overestimated, as we were not able to define a minimum for the use of these treatments (eg, at least 1 tablet per day) due to differences between recommendations. Therefore, we simply reported whether patients took acetaminophen or NSAIDs (yes/ no) without any minimum dose. However, in some cases the use was less than multiple days per month (4% for acetaminophen and 5% for NSAIDs, results not shown). In addition, 57 patients (33%) suffered from hypertension and 12 (7%) from cardiovascular diseases, both of which are contraindications to NSAIDs use.This may have resulted in underestimating the use of NSAIDs or Tramadol, as these patients should be excluded from these estimates.
To our knowledge this is the first study that evaluated the full range of combinations of non-surgical treatments for OA, both from the perspective of orthopaedic surgeons and patients. While most recommended nonsurgical treatments for OA were used frequently as single therapy, the combination is used in only a small percentage of OA patients who receive(d) a THA or TKA. Despite their potential for reducing symptoms of knee and hip OA, dietary therapy and physical therapy appear to be least frequently used. By increasing the use of these two non-surgical treatments, primary care physicians and orthopaedic surgeons may be able to help patients better manage their symptoms, thereby improving quality of care and potentially postpone the need for joint arthroplasty, resulting in improved long-term patient outcomes. Future studies should focus on evaluating the reasons (barriers) why some orthopaedic surgeons do not use recommended non-surgical treatments. Such findings may be helpful in developing targeted strategies to improve the use of these treatments in orthopaedic practice and thereby to improve quality of care. Although the recommended non-surgical treatment options have been proven to be effective individually or in combination (eg, physical therapy with dietary therapy [bib_ref] Exercise and dietary weight loss in overweight and obese older adults with..., Messier [/bib_ref] , there are no published studies that investigated the combined effect of all of these treatments. Nevertheless, it has been hypothesised that optimised non-surgical treatment could result in significantly Conditional percentage of orthopaedic surgeons who prescribe recommended non-surgical treatments in stepped care strategy
Recommended non-surgical treatments in stepped care strategy Conditional n (%)
Step 1: education about OA+education about different treatment options+lifestyle advice +acetaminophen 96 (56)
Step 1+2: step 1+(referral to) dietary therapy, when indicated+physical therapy+NSAIDs 17 [bib_ref] Quality of osteoarthritis care for community-dwelling older adults, Hunter [/bib_ref] Step 1, 2+3: step 1+2+intra-articular injections (for knee OA n=94) 10 (6) NSAIDs, non-steroidal anti-inflammatory drugs; OA, osteoarthritis.
greater pain reduction, functional improvement and increase in quality of life than usual care in knee OA. [bib_ref] Efficacy of multimodal, systematic non-surgical treatment of knee osteoarthritis for patients not..., Skou [/bib_ref] The results from the present study suggest that such better outcomes may be achieved in a considerable part of OA patients.
[table] Table 1: Characteristics of included patients with hip or knee OA [/table]
[table] Table 2: Characteristics of orthopaedic surgeons who treated patients with hip or knee OA [/table]
[table] Table 3: Received recommended and non-recommended non-surgical treatments by patients as reported by patients [/table]
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Subsets and Splits