NCI investment in nanotechnology: achievements and challenges for the future.
Nanotechnology offers an exceptional and unique opportunity for developing a new generation of tools addressing persistent challenges to progress in cancer research and clinical care. The National Cancer Institute (NCI) recognizes this potential, which is why it invests roughly $150 M per year in nanobiotechnology training, research and development. By exploiting the various capacities of nanomaterials, the range of nanoscale vectors and probes potentially available suggests much is possible for precisely investigating, manipulating, and targeting the mechanisms of cancer across the full spectrum of research and clinical care. NCI has played a key role among federal R&D agencies in recognizing early the value of nanobiotechnology in medicine and committing to its development as well as providing training support for new investigators in the field. These investments have allowed many in the research community to pursue breakthrough capabilities that have already yielded broad benefits. Presented here is an overview of how NCI has made these investments with some consideration of how it will continue to work with this research community to pursue paradigm-changing innovations that offer relief from the burdens of cancer.


Modulation of unloading-induced bone loss in mice with altered ERK signaling.
Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44(-/-) had impaired properties while FGF2(-/-) showed enhanced indices. NOD2(-/-) and Fas(-/-) did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44(-/-) and NOD2(-/-) mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44(-/-) mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.


Cognition and behavior in a prevalent cohort of children with epilepsy in rural northern Tanzania: A three-year follow-up study.
Eighty-five percent of the 33 million children with epilepsy (CWE) worldwide live in low- and middle-income countries (LMICs). There is limited research into epilepsy-related comorbidities in LMICs, and there are no studies of the long-term progression of behavioral and intellectual difficulties in childhood epilepsy in sub-Saharan Africa. We aimed to assess behavior and cognition at three-year follow-up in CWE in rural Tanzania. In 2010, a cross-sectional study identified 112 CWE 6 to 14years of age and 113 age- and sex-matched controls in the Hai district of northern Tanzania. From March to June 2013, cases and controls (now 10 to 18years of age) were followed up. At baseline, behavior was assessed using the Rutter A Questionnaire and cognition using the Goodenough-Harris Drawing Test. Details of current seizure frequency and antiepileptic drug (AED) use among CWE were collected. At follow-up, cases had significantly more behavioral difficulties compared with controls (48% of 108 cases versus 14% of 103 controls (p<0.001)). Additionally, 69% of the cases and 16% of the controls had cognitive impairment (p<0.001). In CWE with decreased seizure frequency from baseline to follow-up, behavior had improved significantly. At follow-up, there was no significant difference in behavior between CWE with decreased seizure frequency and those with good seizure control throughout. Behavioral difficulties and cognitive impairment are common among CWE in this population. Improved access to AED treatment and subsequent improved seizure control may reduce the frequency of behavioral difficulties seen in this population.


Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin.
Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of podocalyxin, we generated podocalyxin-deficient (podxl(-/)-) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl(-/)- mice, they fail to form foot processes and slit diaphragms and instead exhibit cell--cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl(-/)- mice frequently display herniation of the gut (omphalocele), suggesting that podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans.


Symbiotic soil fungi enhance ecosystem resilience to climate change.
Substantial amounts of nutrients are lost from soils through leaching. These losses can be environmentally damaging, causing groundwater eutrophication and also comprise an economic burden in terms of lost agricultural production. More intense precipitation events caused by climate change will likely aggravate this problem. So far it is unresolved to which extent soil biota can make ecosystems more resilient to climate change and reduce nutrient leaching losses when rainfall intensity increases. In this study, we focused on arbuscular mycorrhizal (AM) fungi, common soil fungi that form symbiotic associations with most land plants and which increase plant nutrient uptake. We hypothesized that AM fungi mitigate nutrient losses following intensive precipitation events (higher amount of precipitation and rain events frequency). To test this, we manipulated the presence of AM fungi in model grassland communities subjected to two rainfall scenarios: moderate and high rainfall intensity. The total amount of nutrients lost through leaching increased substantially with higher rainfall intensity. The presence of AM fungi reduced phosphorus losses by 50% under both rainfall scenarios and nitrogen losses by 40% under high rainfall intensity. Thus, the presence of AM fungi enhanced the nutrient interception ability of soils, and AM fungi reduced the nutrient leaching risk when rainfall intensity increases. These findings are especially relevant in areas with high rainfall intensity (e.g., such as the tropics) and for ecosystems that will experience increased rainfall due to climate change. Overall, this work demonstrates that soil biota such as AM fungi can enhance ecosystem resilience and reduce the negative impact of increased precipitation on nutrient losses.


Aortic valve replacement for patients with severe aortic stenosis: risk factors and their impact on 30-month mortality.
Few studies have reported population-based outcomes for aortic valve replacement patients. Patients with severe aortic valve stenosis who underwent aortic valve replacement with or without concomitant coronary artery bypass graft surgery from January 1, 2003, to December 31, 2005, were included in the study. Statistical models were developed to identify significant risk factors for mortality, to compare survival for patients with and without selected risk factors, and to compare survival to an age- and sex-matched group from US life tables. There was total of 6,369 patients in the study. The in-hospital and 30-day mortality rates were 3.97% for aortic valve replacement and 5.69% for aortic valve replacement with concomitant coronary artery bypass graft surgery. Significant risk factors for 30-month mortality included concomitant coronary artery bypass graft surgery, advancing age, lower body surface area, emergency status, low ejection fraction, congestive heart failure, previous heart surgery, and several comorbidities. The 64.3% of patients with isolated aortic valve replacement who had neither congestive heart failure, ejection fraction less than 0.40, acute myocardial infarction less than 24 hours, nor hemodynamic instability had a risk-adjusted survival of 89.9% compared with the 90.0% survival rate of the age- and sex-matched general population (p = 0.28). For the large number of patients without high-risk conditions, the 30-month survival is essentially as high as that of an age- and sex-matched group of the US population.


A pseudouridine synthase module is essential for mitochondrial protein synthesis and cell viability.
Pseudouridylation is a common post-transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity-biotinylation assay, we identified a protein module in mitochondrial RNA granules, platforms for post-transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine-Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival.


The establishment of murine blood outgrowth endothelial cells and observations relevant to gene therapy.
Endothelial cells are an attractive vehicle for gene therapy because they may be used in an autologous fashion and may allow for direct exposure of the gene product into the intravascular space. To explore this future potential, a reproducible system was developed for the culture of murine blood outgrowth endothelial cells. These cells demonstrated acetylated low-density lipoprotein (LDL) incorporation, matrigel tube formation, and specific endothelial staining characteristics, namely P1H12, VeCAD, vascular cell adhesion molecule (VCAM), vWF, platelet endothelial cell adhesion molecule (PECAM-1), and vascular endothelial growth factor receptor-2 (VEGFR2). They were also negative for smooth muscle actin and monocytic markers CD11b, CD14, and CD16. Moreover, these cells were amendable to gene transfer with red fluorescent and green fluorescent expression vectors as well as human Factor VIII (hFVIII) while maintaining endothelial characteristics. Both source- and gene-introduced cells also manifested excellent proliferative potential. Furthermore, murine blood outgrowth endothelial cells (BOECs) demonstrated persistent in vivo seeding in the liver, lung, spleen, and bone morrow of recipient mice.


Tri-layered elastomeric scaffolds for engineering heart valve leaflets.
Tissue engineered heart valves (TEHVs) that can grow and remodel have the potential to serve as permanent replacements of the current non-viable prosthetic valves particularly for pediatric patients. A major challenge in designing functional TEHVs is to mimic both structural and anisotropic mechanical characteristics of the native valve leaflets. To establish a more biomimetic model of TEHV, we fabricated tri-layered scaffolds by combining electrospinning and microfabrication techniques. These constructs were fabricated by assembling microfabricated poly(glycerol sebacate) (PGS) and fibrous PGS/poly(caprolactone) (PCL) electrospun sheets to develop elastic scaffolds with tunable anisotropic mechanical properties similar to the mechanical characteristics of the native heart valves. The engineered scaffolds supported the growth of valvular interstitial cells (VICs) and mesenchymal stem cells (MSCs) within the 3D structure and promoted the deposition of heart valve extracellular matrix (ECM). MSCs were also organized and aligned along the anisotropic axes of the engineered tri-layered scaffolds. In addition, the fabricated constructs opened and closed properly in an ex vivo model of porcine heart valve leaflet tissue replacement. The engineered tri-layered scaffolds have the potential for successful translation towards TEHV replacements.


Circulating matrix metalloproteinase-9 level is associated with cerebral white matter hyperintensities in non-stroke individuals.
The pathogenesis of cerebral white matter hyperintensities (WMH) has been poorly understood. Our aim was to investigate the association of circulating proteins, the biomarkers of inflammation, blood-brain barrier (BBB) dysfunction, and thrombosis with WMH in non-stroke individuals. Demographic, laboratory, and brain magnetic resonance imaging parameters were prospectively analyzed in 137 subjects. The relationship between plasma interleukin-6, tumor necrosis factor-α, matrx-metalloproteinase-9 (MMP-9), plasminogen activator inhibitor-1 and overt WMH (Fazekas grading score ≥2) was analyzed. In univariate analysis, old age, high blood pressure, history of hypertension, and elevated plasma MMP-9 level were associated with overt WMH. In multivariate analysis, plasma MMP-9 still maintained a significant association with WMH. Plasma MMP-9 level was weakly but significantly associated with WMH volume (r = 0.232, p = 0.006). All the other circulating proteins examined failed to demonstrate a significant relationship with WMH. Plasma MMP-9 is associated with pathophysiology of WMH development.


Are menthol cigarettes a starter product for youth?
This study assessed the relationship between menthol use and nicotine dependence. Data from the National Youth Tobacco Survey indicated that menthol cigarette use was significantly more common among newer, younger smokers. Additionally, youth who smoked menthol cigarettes had significantly higher scores on a scale of nicotine dependence compared with nonmenthol smokers, controlling for demographic background and the length, frequency, and level of smoking. The study suggests that menthol cigarettes are a starter product that may be associated with smoking uptake by youth.


Non-mass lesions on screening breast ultrasound.
The purpose of this study was to determine the significance of a non-mass lesion (NML) which is recognized during screening breast ultrasound (US). We included patients with a NML on screening breast US and no suspicious finding on mammography between March 2008 and June 2012. The final diagnoses were based on pathology results and a clinical or sonographic follow-up for more than 12 months. We calculated the incidence, likelihood of malignancy, and positive predictive value (PPV) of biopsy with a review of imaging and histopathological findings. A total of 17868 screening breast US were performed in 8856 asymptomatic patients. Ninety-five NMLs were detected in 88 patients (1.0%). Among the 93 lesions that were followed or confirmed histopathologically, 2 (2.2%) were malignant, 89 (95.6%) were benign, and 2 (2.2%) were high risk lesions. The likelihood of malignancy in a NML on screening breast US was 2.2% and the PPV of biopsy was 6.3% (2 of 32). The likelihood of malignancy for a NML on screening breast US was greater than 2%. It could be classified as a BI-RADS category 4a lesion and tissue diagnosis is warranted. This provides the potential management guidelines for a NML in screening patients and further prospective studies in a large, multicenter screening population are required.


Thyroid hormone controls the onset of androgen sensitivity in the developing larynx of Xenopus laevis.
Gonadal differentiation, the onset of androgen-stimulated laryngeal growth and the genesis of a sex difference in laryngeal innervation, all temporally coincide with thyroid hormone (TH)-induced metamorphosis in Xenopus laevis. To explore the role TH plays in the ontogeny of the Xenopus androgen-sensitive vocal neuromuscular system, we examined gonadal and laryngeal development in tadpoles in which metamorphosis had been blocked by treatment with the thyroxine synthesis inhibitor propylthiouracil (PTU). PTU treatment did not arrest gonadal differentiation. Testes from PTU-treated male tadpoles had seminiferous tubules and advanced stage male germ cells, while in females stage 1 oocytes were present. In contrast to the gonads, PTU did block morphological development of the larynx. Tadpoles treated with PTU for 50 or 100 days had larynges which structurally resembled those of stage 54 control tadpoles. PTU-treated animals did not exhibit the extensive development of the laryngeal cartilage seen in untreated animals. Laryngeal cartilages of hypothyroid tadpoles exhibited low density and minimal patterning of chondrocytes; the complex lumen and marked expansion of the dilator muscles characteristic of 50- and 100-day untreated animals were absent. Laryngeal growth evoked by exposure to exogenous androgen (dihydrotestosterone) was entirely prevented by PTU treatment. Hypothyroid tadpoles did not exhibit the decline in laryngeal nerve axon number characteristic of age-matched controls, nor were laryngeal nerve axon numbers sexually dimorphic. PTU treatment also interfered with the myelination of laryngeal axons. We conclude that while gonadal differentiation is independent of TH, androgen sensitive laryngeal development and sexually dimorphic laryngeal innervation require exposure to secreted TH.


Theranostic Biomarkers for Schizophrenia.
Schizophrenia is a highly heritable, chronic, severe, disabling neurodevelopmental brain disorder with a heterogeneous genetic and neurobiological background, which is still poorly understood. To allow better diagnostic procedures and therapeutic strategies in schizophrenia patients, use of easy accessible biomarkers is suggested. The most frequently used biomarkers in schizophrenia are those associated with the neuroimmune and neuroendocrine system, metabolism, different neurotransmitter systems and neurotrophic factors. However, there are still no validated and reliable biomarkers in clinical use for schizophrenia. This review will address potential biomarkers in schizophrenia. It will discuss biomarkers in schizophrenia and propose the use of specific blood-based panels that will include a set of markers associated with immune processes, metabolic disorders, and neuroendocrine/neurotrophin/neurotransmitter alterations. The combination of different markers, or complex multi-marker panels, might help in the discrimination of patients with different underlying pathologies and in the better classification of the more homogenous groups. Therefore, the development of the diagnostic, prognostic and theranostic biomarkers is an urgent and an unmet need in psychiatry, with the aim of improving diagnosis, therapy monitoring, prediction of treatment outcome and focus on the personal medicine approach in order to improve the quality of life in patients with schizophrenia and decrease health costs worldwide.


FZD10 regulates cell proliferation and mediates Wnt1 induced neurogenesis in the developing spinal cord.
Wnt/FZD signalling activity is required for spinal cord development, including the dorsal-ventral patterning of the neural tube, where it affects proliferation and specification of neurons. Wnt ligands initiate canonical, β -catenin-dependent, signaling by binding to Frizzled receptors. However, in many developmental contexts the cognate FZD receptor for a particular Wnt ligand remains to be identified. Here, we characterized FZD10 expression in the dorsal neural tube where it overlaps with both Wnt1 and Wnt3a, as well as markers of dorsal progenitors and interneurons. We show FZD10 expression is sensitive to Wnt1, but not Wnt3a expression, and FZD10 plays a role in neural tube patterning. Knockdown approaches show that Wnt1 induced ventral expansion of dorsal neural markes, Pax6 and Pax7, requires FZD10. In contrast, Wnt3a induced dorsalization of the neural tube is not affected by FZD10 knockdown. Gain of function experiments show that FZD10 is not sufficient on its own to mediate Wnt1 activity in vivo. Indeed excess FZD10 inhibits the dorsalizing activity of Wnt1. However, addition of the Lrp6 co-receptor dramatically enhances the Wnt1/FZD10 mediated activation of dorsal markers. This suggests that the mechanism by which Wnt1 regulates proliferation and patterning in the neural tube requires both FZD10 and Lrp6.


Genetically induced adult oligodendrocyte cell death is associated with poor myelin clearance, reduced remyelination, and axonal damage.
Loss of oligodendrocytes is a feature of many demyelinating diseases including multiple sclerosis. Here, we have established and characterized a novel model of genetically induced adult oligodendrocyte death. Specific primary loss of adult oligodendrocytes leads to a well defined and highly reproducible course of disease development that can be followed longitudinally by magnetic resonance imaging. Histological and ultrastructural analyses revealed progressive myelin vacuolation, in parallel to disease development that includes motor deficits, tremor, and ataxia. Myelin damage and clearance were associated with induction of oligodendrocyte precursor cell proliferation, albeit with some regional differences. Remyelination was present in the mildly affected corpus callosum. Consequences of acutely induced cell death of adult oligodendrocytes included secondary axonal damage. Microglia were activated in affected areas but without significant influx of B-cells, T-helper cells, or T-cytotoxic cells. Analysis of the model on a RAG-1 (recombination activating gene-1)-deficient background, lacking functional lymphocytes, did not change the observed disease and pathology compared with immune-competent mice. We conclude that this model provides the opportunity to study the consequences of adult oligodendrocyte death in the absence of primary axonal injury and reactive cells of the adaptive immune system. Our results indicate that if the blood-brain barrier is not disrupted, myelin debris is not removed efficiently, remyelination is impaired, and axonal integrity is compromised, likely as the result of myelin detachment. This model will allow the evaluation of strategies aimed at improving remyelination to foster axon protection.


Modeling the altered expression levels of genes on signaling pathways in tumors as causal bayesian networks.
This paper concerns a study indicating that the expression levels of genes in signaling pathways can be modeled using a causal Bayesian network (BN) that is altered in tumorous tissue. These results open up promising areas of future research that can help identify driver genes and therapeutic targets. So, it is most appropriate for the cancer informatics community. Our central hypothesis is that the expression levels of genes that code for proteins on a signal transduction network (STP) are causally related and that this causal structure is altered when the STP is involved in cancer. To test this hypothesis, we analyzed 5 STPs associated with breast cancer, 7 STPs associated with other cancers, and 10 randomly chosen pathways, using a breast cancer gene expression level dataset containing 529 cases and 61 controls. We identified all the genes related to each of the 22 pathways and developed separate gene expression datasets for each pathway. We obtained significant results indicating that the causal structure of the expression levels of genes coding for proteins on STPs, which are believed to be implicated in both breast cancer and in all cancers, is more altered in the cases relative to the controls than the causal structure of the randomly chosen pathways.


Comparing surgeon outcomes in endoscopic sinus surgery for chronic rhinosinusitis.
The objective of this investigation was to evaluate endoscopic sinus surgery (ESS) outcomes for chronic rhinosinusitis (CRS) between medical centers to determine if differences in quality-of-life outcomes were detectable. In addition, we sought to identify significant, independent cofactors toward the development of an ESS-specific risk-adjustment model so that ESS outcomes may be appropriately compared between institutions and healthcare providers. Prospective, multicenter, observational cohort. Study participants electing ESS for CRS were enrolled and randomly selected in equal numbers from three academic clinical practices in North America between April 2011 and May 2015. The magnitude of average 6-month postoperative improvement in patient-related outcome measures (PROMs) was compared between enrollment sites using multivariate linear regression modeling. A total of 228 participants met inclusion criteria and were included for final analyses (n = 76 per site). The prevalence of septal deviation/septoplasty and oral corticosteroid-dependent conditions was significantly different between enrollment sites (P ≤ 0.004). Each enrollment site generated significant within-subject improvement across all PROMs after ESS (P < 0.001); however, average unadjusted magnitudes of improvement were significantly different between sites for the primary outcome measure. After controlling for baseline PROMs, septal deviation, steroid-dependent conditions, and medication use variables, enrollment site was no longer associated with significant outcome differences (P = 0.535). Comparison of surgeon outcomes of ESS is feasible and must take into account a number of baseline patient characteristics. Further studies will be critical toward developing an ESS-specific risk-adjustment model and enabling a robust comparison of surgeon outcomes. 2c. Laryngoscope, 127:14-21, 2017.


The nature of caregiving in children of a parent with multiple sclerosis from multiple sources and the associations between caregiving activities and youth adjustment overtime.
This study explored youth caregiving for a parent with multiple sclerosis (MS) from multiple perspectives, and examined associations between caregiving and child negative (behavioural emotional difficulties, somatisation) and positive (life satisfaction, positive affect, prosocial behaviour) adjustment outcomes overtime. A total of 88 families participated; 85 parents with MS, 55 partners and 130 children completed questionnaires at Time 1. Child caregiving was assessed by the Youth Activities of Caregiving Scale (YACS). Child and parent questionnaire data were collected at Time 1 and child data were collected 12 months later (Time 2). Factor analysis of the child and parent YACS data replicated the four factors (instrumental, social-emotional, personal-intimate, domestic-household care), all of which were psychometrically sound. The YACS factors were related to parental illness and caregiving context variables that reflected increased caregiving demands. The Time 1 instrumental and social-emotional care domains were associated with poorer Time 2 adjustment, whereas personal-intimate was related to better adjustment and domestic-household care was unrelated to adjustment. Children and their parents exhibited highest agreement on personal-intimate, instrumental and total caregiving, and least on domestic-household and social-emotional care. Findings delineate the key dimensions of young caregiving in MS and the differential links between caregiving activities and youth adjustment.


Improving diagnostic accuracy of anaphylaxis in the acute care setting.
The identification and appropriate management of those at highest risk for life-threatening anaphylaxis remains a clinical enigma. The most widely used criteria for such patients were developed in a symposium convened by National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network. In this paper we review the current literature on the diagnosis of acute allergic reactions as well as atypical presentations that clinicians should recognize. Review of case series reveals significant variability in definition and approach to this common and potentially life-threatening condition. Series on fatal cases of anaphylaxis indicate that mucocutaneous signs and symptoms occur less frequently than in milder cases. Of biomarkers studied to aid in the work-up of possible anaphylaxis, drawing blood during the initial six hours of an acute reaction for analysis of serum tryptase has been recommended in atypical cases. This can provide valuable information when a definitive diagnosis cannot be made by history and physical exam.


Predator responses to fire: A global systematic review and meta-analysis.
Knowledge of how disturbances such as fire shape habitat structure and composition, and affect animal interactions, is fundamental to ecology and ecosystem management. Predators also exert strong effects on ecological communities, through top-down regulation of prey and competitors, which can result in trophic cascades. Despite their ubiquity, ecological importance and potential to interact with fire, our general understanding of how predators respond to fire remains poor, hampering ecosystem management. To address this important knowledge gap, we conducted a systematic review and meta-analysis of the effects of fire on terrestrial, vertebrate predators world-wide. We found 160 studies spanning 1978-2018. There were 36 studies with sufficient information for meta-analysis, from which we extracted 96 effect sizes (Hedges' g) for 67 predator species relating to changes in abundance indices, occupancy or resource selection in burned and unburned areas, or before and after fire. Studies spanned geographic locations, taxonomic families and study designs, but most were located in North America and Oceania (59% and 24%, respectively), and largely focussed on felids (24%) and canids (25%). Half (50%) of the studies reported responses to wildfire, and nearly one third concerned prescribed (management) fires. There were no clear, general responses of predators to fire, nor relationships with geographic area, biome or life-history traits (e.g. body mass, hunting strategy and diet). Responses varied considerably between species. Analysis of species for which at least three effect sizes had been reported in the literature revealed that red foxes Vulpes vulpes mostly responded positively to fire (e.g. higher abundance in burned compared to unburned areas) and eastern racers Coluber constrictor negatively, with variances overlapping zero only slightly for both species. Our systematic review and meta-analysis revealed strong variation in predator responses to fire, and major geographic and taxonomic knowledge gaps. Varied responses of predator species to fire likely depend on ecosystem context. Consistent reporting of ongoing monitoring and management experiments is required to improve understanding of the mechanisms driving predator responses to fire, and any broader effects (e.g. trophic interactions). The divergent responses of species in our study suggest that adaptive, context-specific management of predator-fire relationships is required.


Omega-3 fatty acid supplementation appears to attenuate particulate air pollution-induced cardiac effects and lipid changes in healthy middle-aged adults.
Air pollution exposure has been associated with adverse cardiovascular health effects. Findings of a recent epidemiological study suggested that omega-3 fatty acid (fish oil) supplementation blunted cardiac responses to air pollution exposure. We conducted a randomized, controlled exposure study to evaluate the efficacy of fish oil supplements in attenuating adverse cardiac effects of exposure to concentrated ambient fine and ultrafine particulate matter (CAP). Twenty-nine healthy middle-aged participants (mean, 58 ± 1 years of age) were supplemented in a randomized, double-blinded manner with 3 g/day of either fish oil or olive oil for 4 weeks before sequential chamber exposure to filtered air and CAP (mean mass concentration 278 ± 19 µg/m3) for 2 hr. Cardiac responses were assessed by comparing time and frequency domain changes in heart rate variability (HRV) and electrocardiographic repolarization changes measured before, immediately after, and 20 hr after exposure. Changes in plasma lipids were also evaluated at these time points. Fish oil supplementation appeared to attenuate CAP-induced reductions in high-frequency/low-frequency ratio, as well as elevations in normalized low-frequency HRV and prolongation of the QT interval corrected for heart rate (QTc). Very low-density lipoprotein and triglyceride concentrations increased significantly immediately after exposure to CAP in participants supplemented with olive oil, but not in those supplemented with fish oil. Exposure of healthy middle-aged adults to CAP for 2 hr induced acute cardiac and lipid changes after supplementation with olive oil, but not fish oil. Our findings suggest that omega-3 fatty acid supplements offer protection against the adverse cardiac and lipid effects associated with air pollution exposure.


Effect of NG-monomethyl-L-arginine on endothelium-dependent relaxation of human subcutaneous resistance arteries.
1. Using a myograph to measure isometric tension, we have looked at the action of NG-monomethyl-L-arginine on the endothelium-dependent relaxation of human subcutaneous resistance arteries. 2. NG-Monomethyl-L-arginine, the novel inhibitor of endothelium-derived relaxing factor synthesis, caused concentration-dependent but only partial inhibition of maximal relaxation induced by acetylcholine in human subcutaneous resistance arteries. 3. The inhibitory action of NG-monomethyl-L-arginine on acetylcholine-induced maximal relaxation was partially reversed by incubation of the arteries in equimolar concentrations of L-arginine and NG-monomethyl-L-arginine. Subsequent incubation in L-arginine led to further reversal, but this was no greater than with incubation in physiological saline. 4. A component of acetylcholine-induced relaxation was sensitive to indomethacin, suggesting that this response is mediated by prostanoids as well as by endothelium-derived relaxing factor. 5. NG-Monomethyl-L-arginine did not increase the tension of resting human subcutaneous resistance arteries. NG-Monomethyl-L-arginine did enhance the contractile response to noradrenaline, possibly due to inhibition of release of endothelium-derived relaxing factor resulting from stimulation of alpha 2-adrenoreceptors on the endothelial cells.


The Key Role of Mitochondrial Apoptotic Pathway in the Cytotoxic Effect of Mushroom Extracts on Cancer Cells.
Mushroom extracts have been extensively studied for their medicinal effects. They can stimulate immune responses and thus have been explored in cancer treatment. Recently, it has also been shown that some mushroom extracts can produce direct cytotoxic effect on cancer cells. In this review, we summarize the cytotoxic effect of mushroom extracts in cancer treatment revealed by both in vitro and in vivo studies. We also summarize the current understanding of the mechanisms associated with such an effect with an emphasis on the mitochondrial apoptotic pathway. The recent finding that mushroom extracts have direct cytotoxic effects supplements their known immune stimulating effects. Thus, novel anticancer agents based on new findings from mushroom extracts may soon be added to the present pool of anticancer drugs. Specifically, we propose that nanodelivery of the bioactive compounds of mushroom extracts to mitochondria will further increase their potential treatment efficacy.


Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality.
There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.). Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.


Leukocyte antimicrobial function in patients with leprosy.
Patients with lepromatous leprosy are unresponsive to lepromin skin-test material and possess defective lymphocyte function in vitro, including impaired mitogenesis in response to antigens of Mycobacterium leprae. It has been claimed that their macrophages cannot digest M. leprae in vitro; such a defect could explain both lepromin nonreactivity and impaired lymphocyte function on the basis of failure of the afferent limb of the immune response (i.e., defective macrophage "processing" of M. leprae). The present studies indicate that macrophages from patients with lepromatous and tuberculoid leprosy and from normal donors do not differ in their ability to digest heat-killed M. leprae in vitro, or in their ability to sustain the viability of M. leprae in tissue culture; that monocytes, macrophages, and polymorphonuclear leukocytes of leprosy patients and controls possess equivalent microbicidal activity against Listeria monocytogenes, Escherichia coli, Proteus vulgaris, Staphylococcus aureus, and Candida albicans; and that polymorphonuclear leukocytes from patients with lepromatous leprosy iodinate ingested bacteria normally. Whether the basic immune defect leading to the development of lepromatous leprosy resides in the lymphocyte or in the macrophage remains to be determined. However, the present study shows that phagocytic cells from patients with either principal form of leprosy function normally in a variety of sophisticated tests of antimicrobial function.


Slow-Wave Sleep and MRI Markers of Brain Aging in a Community-Based Sample.
To test the hypothesis that reduced slow-wave sleep, or N3 sleep, which is thought to underlie the restorative functions of sleep, is associated with MRI markers of brain aging, we evaluated this relationship in the community-based Framingham Heart Study Offspring cohort using polysomnography and brain MRI. We studied 492 participants (age 58.8 ± 8.8 years, 49.4% male) free of neurological diseases who completed a brain MRI scan and in-home overnight polysomnography to assess slow-wave sleep (absolute duration and percentage of total sleep). Volumes of total brain, total cortical, frontal cortical, subcortical gray matter, hippocampus, and white matter hyperintensities were investigated as a percentage of intracranial volume, and the presence of covert brain infarcts was evaluated. Linear and logistic regression models were adjusted for age, age squared, sex, time interval between polysomnography and MRI (3.3 ± 1.0 years), APOE ε4 carrier status, stroke risk factors, sleeping pill use, body mass index, and depression. Less slow-wave sleep was associated with lower cortical brain volume (absolute duration, β [standard error] = 0.20 [0.08], p = 0.015; percentage, 0.16 [0.08], p = 0.044), lower subcortical brain volume (percentage, 0.03 [0.02], p = 0.034), and higher white matter hyperintensities volume (absolute duration, -0.12 [0.05], p = 0.010; percentage, -0.10 [0.04], p = 0.033). Slow-wave sleep duration was not associated with hippocampal volume or the presence of covert brain infarcts. Loss of slow-wave sleep might facilitate accelerated brain aging, as evidence by its association with MRI markers suggestive of brain atrophy and injury. Alternatively, subtle injuries and accelerated aging might reduce the ability of the brain to produce slow-wave sleep.


pH-dependent electron transfer from re-bipyridyl complexes to metal oxide nanocrystalline thin films.
Photoinduced interfacial electron transfer (ET) from molecular adsorbates to semiconductor nanoparticles has been a subject of intense recent interest. Unlike intramolecular ET, the existence of a quasicontinuum of electronic states in the solid leads to a dependence of ET rate on the density of accepting states in the semiconductor, which varies with the position of the adsorbate excited-state oxidation potential relative to the conduction band edge. For metal oxide semiconductors, their conduction band edge position varies with the pH of the solution, leading to pH-dependent interfacial ET rates in these materials. In this work we examine this dependence in Re(L(P))(CO)3Cl (or ReC1P) [L(P) = 2,2'-bipyridine-4,4'-bis-CH2PO(OH)2] and Re(L(A))(CO)3Cl (or ReC1A) [L(A) = 2,2'-bipyridine-4,4'-bis-CH2COOH] sensitized TiO2 and ReC1P sensitized SnO2 nanocrystalline thin films using femtosecond transient IR spectroscopy. ET rates are measured as a function of pH by monitoring the CO stretching modes of the adsorbates and mid-IR absorption of the injected electrons. The injection rate to TiO2 was found to decrease by 1000-fold from pH 0-9, while it reduced by only a factor of a few to SnO2 over a similar pH range. Comparison with the theoretical predictions based on Marcus' theory of nonadiabatic interfacial ET suggests that the observed pH-dependent ET rate can be qualitatively accounted for by considering the change of density of electron-accepting states caused by the pH-dependent conduction band edge position.


Perceived control in health care: a conceptual model based on experiences of frail older adults.
Frail older adults are increasingly encouraged to be in control of their health care, in Western societies. However, little is known about how they themselves perceive control in health care. Therefore, this study aims to investigate the concept of health care-related perceived control from the viewpoint of frail older adults. A qualitative interview study was conducted following a Grounded Theory approach. Thirty-two Dutch frail older adults, aged 65 and over, participated in 20 in-depth interviews (n=20) and three focus group discussions (n=12). Data were analysed according to techniques of coding and constant comparison. From this analysis constituting factors of perceived control emerged, providing elements of a conceptual model. Perceived control reflects the feeling or belief that health care is under control, which is constituted by five, either internal or external, factors: (I) self-confidence in organising professional and/or informal care, (II) self-confidence in health management in the home setting, (III) perceived support from people in the social network, (IV) perceived support from health care professionals and organisations, and (V) perceived support from (health care) infrastructure and services. Therefore, the concept does not only consist of people's own perceived efforts, but also includes the influence of external sources. Our conceptual model points out what external factors should be taken into consideration by health care professionals and policy makers when enhancing older people's perceived control. Moreover, it can serve as the basis for the development of a measurement instrument, to enable future quantitative research on health care-related perceived control among older adults.


Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting.
Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.


Validity of indirect comparison for estimating efficacy of competing interventions: empirical evidence from published meta-analyses.
To determine the validity of adjusted indirect comparisons by using data from published meta-analyses of randomised trials. Direct comparison of different interventions in randomised trials and adjusted indirect comparison in which two interventions were compared through their relative effect versus a common comparator. The discrepancy between the direct and adjusted indirect comparison was measured by the difference between the two estimates. Database of abstracts of reviews of effectiveness (1994-8), the Cochrane database of systematic reviews, Medline, and references of retrieved articles. 44 published meta-analyses (from 28 systematic reviews) provided sufficient data. In most cases, results of adjusted indirect comparisons were not significantly different from those of direct comparisons. A significant discrepancy (P<0.05) was observed in three of the 44 comparisons between the direct and the adjusted indirect estimates. There was a moderate agreement between the statistical conclusions from the direct and adjusted indirect comparisons (kappa 0.51). The direction of discrepancy between the two estimates was inconsistent. Adjusted indirect comparisons usually but not always agree with the results of head to head randomised trials. When there is no or insufficient direct evidence from randomised trials, the adjusted indirect comparison may provide useful or supplementary information on the relative efficacy of competing interventions. The validity of the adjusted indirect comparisons depends on the internal validity and similarity of the included trials.


A Phase Ib/II Study of Ivosidenib with Venetoclax ± Azacitidine in IDH1-Mutated Myeloid Malignancies.
The safety and efficacy of combining the isocitrate dehydrogenase-1 (IDH1) inhibitor ivosidenib (IVO) with the BCL2 inhibitor venetoclax (VEN; IVO + VEN) ± azacitidine (AZA; IVO + VEN + AZA) were evaluated in four cohorts of patients with IDH1-mutated myeloid malignancies (n = 31). Most (91%) adverse events were grade 1 or 2. The maximal tolerated dose was not reached. Composite complete remission with IVO + VEN + AZA versus IVO + VEN was 90% versus 83%. Among measurable residual disease (MRD)-evaluable patients (N = 16), 63% attained MRD--negative remissions; IDH1 mutation clearance occurred in 64% of patients receiving ≥5 treatment cycles (N = 14). Median event-free survival and overall survival were 36 [94% CI, 23-not reached (NR)] and 42 (95% CI, 42-NR) months. Patients with signaling gene mutations appeared to particularly benefit from the triplet regimen. Longitudinal single-cell proteogenomic analyses linked cooccurring mutations, antiapoptotic protein expression, and cell maturation to therapeutic sensitivity of IDH1-mutated clones. No IDH isoform switching or second-site IDH1 mutations were observed, indicating combination therapy may overcome established resistance pathways to single-agent IVO. IVO + VEN + AZA is safe and active in patients with IDH1-mutated myeloid malignancies. Combination therapy appears to overcome resistance mechanisms observed with single-agent IDH-inhibitor use, with high MRD-negative remission rates. Single-cell DNA ± protein and time-of-flight mass-cytometry analysis revealed complex resistance mechanisms at relapse, highlighting key pathways for future therapeutic intervention. This article is highlighted in the In This Issue feature, p. 247.


Cerium oxide nanoparticles inhibit oxidative stress and nuclear factor-κB activation in H9c2 cardiomyocytes exposed to cigarette smoke extract.
Cigarette smoke contains and generates a large amount of reactive oxygen species (ROS) that affect normal cellular function and have pathogenic consequences in the cardiovascular system. Increased oxidative stress and inflammation are considered to be an important mechanism of cardiovascular injury induced by cigarette smoke. Antioxidants may serve as effective therapeutic agents against smoke-related cardiovascular disease. Because of the presence of oxygen vacancies on its surface and self-regenerative cycle of its dual oxidation states, Ce(3+) and Ce(4+), cerium oxide (CeO(2)) nanoparticles offer a potential to quench ROS in biological systems. In this study, we determined the ability of CeO(2) nanoparticles to protect against cigarette smoke extract (CSE)-induced oxidative stress and inflammation in cultured rat H9c2 cardiomyocytes. CeO(2) nanoparticles pretreatment of H9c2 cells resulted in significant inhibition of CSE-induced ROS production and cell death. Pretreatment of H9c2 cells with CeO(2) nanoparticles suppressed CSE-induced phosphorylation of IκBα, nuclear translocation of p65 subunit of nuclear factor-κB (NF-κB), and NF-κB reporter activity in H9c2 cells. CeO(2) nanoparticles pretreatment also resulted in a significant down-regulation of NF-κB-regulated inflammatory genes tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and inducible nitric-oxide synthase and further inhibited CSE-induced depletion of antioxidant enzymes, such as copper zinc superoxide dismutase, manganese superoxide dismutase, and intracellular glutathione content. These results indicate that CeO(2) nanoparticles can inhibit CSE-induced cell damage via inhibition of ROS generation, NF-κB activation, inflammatory gene expression, and antioxidant depletion and may have a great potential for treatment of smoking-related diseases.


U/Pb zircon geochronology and tempo of the end-permian mass extinction
The mass extinction at the end of the Permian was the most profound in the history of life. Fundamental to understanding its cause is determining the tempo and duration of the extinction. Uranium/lead zircon data from Late Permian and Early Triassic rocks from south China place the Permian-Triassic boundary at 251.4 +/- 0.3 million years ago. Biostratigraphic controls from strata intercalated with ash beds below the boundary indicate that the Changhsingian pulse of the end-Permian extinction, corresponding to the disappearance of about 85 percent of marine species, lasted less than 1 million years. At Meishan, a negative excursion in delta13C at the boundary had a duration of 165,000 years or less, suggesting a catastrophic addition of light carbon.


Colorectal cancer screening compliance by medicine residents: perceived and actual.
Implementation of colorectal cancer (CRC) screening with widely available techniques can result in a significant reduction in CRC-related mortality. Clinical practice paradigms are often ingrained in physicians during residency. We, therefore, investigated both compliance and perceived obstacles to CRC screening in the practices of physicians-in-training. We conducted a retrospective analysis of medical records of patients who were receiving their primary care in the internal medicine resident clinics at the University of Nebraska Medical Center and were at average risk for CRC. In addition to demographics, data on the use of screening mammography, Pap smear, cholesterol, fecal occult blood testing (FOBT), and flexible sigmoidoscopy (FS) were collected. A questionnaire was also distributed to all internal medicine residents to assess their CRC screening knowledge and perceived screening compliance. One hundred eight patient charts were reviewed. The percentage of patients appropriately screened for each test was as follows: mammography 66%, Pap smear 65%, cholesterol 53%, FOBT 13%, and FS 16%. Residents dramatically overestimated their perceived FS and FOBT screening rates, 78% and 88%, respectively. Most residents identified barriers to FS screening. Although rudimentary CRC screening knowledge appeared adequate, a number of knowledge-based deficiencies were identified. Internal medicine residents at our institution demonstrate poor CRC screening compliance especially when compared with other health care maintenance interventions. This cannot be entirely accounted for by inadequate knowledge; discrepancy between the perceived and actual implementation of CRC screening may be important. Efforts to improve screening compliance should include a focus on physicians-in-training.


Simplified method for quantifying theoretical underestimation of chamber-based trace gas fluxes.
Closed chambers used to measure soil-atmosphere exchange of trace gases including nitrous oxide (N(2)O) and carbon dioxide (CO(2)) generate errors due to suppression of the gas concentration gradient at the soil-atmosphere interface. A method is described here for estimating the magnitude of flux underestimation arising from chamber deployment. The technique is based on previously established gas transport theory and has been simplified to facilitate application while preserving the fundamental physical relationships. The method avoids the use of nonlinear regression but requires knowledge of soil properties including texture, bulk density, water content, temperature, and pH. Two options are presented: a numerical technique which is easily adapted to spreadsheet application, and a graphical method requiring minimal calculation. In both cases, the magnitude of theoretical flux underestimation (TFU) is determined, taking into account effects of chamber geometry and deployment time, the flux-calculation scheme, and properties of the soil and gas under consideration. Application to actual data and recent studies confirmed that TFU can vary widely within and across sites. The analysis also revealed a highly linear correlation between soil water content and TFU, suggesting that previously observed relationships between water content and trace gas flux may in part reflect artifacts of chamber methodology. The method described here provides a practical means of improving the absolute accuracy of flux estimates and normalizing data obtained using different chamber designs in different soils.


Regulation of the antibody response to type III pneumococcal polysaccharide by contrasuppressor T cells.
A soluble membrane component of type III pneumococcal polysaccharide-coupled spleen cells (S3-SCSM) induces S3-specific suppressor T cells (Ts) in mice. These Ts can be detected only if mice are pretreated with cyclophosphamide (Cy) or if cells adherent to the lectin Vicia villosa are removed from the spleen cell population prior to transfer. The V. villosa-adherent spleen cells from mice injected with S3-SCSM could abrogate suppression mediated by Ts induced by S3-SCSM in Cy-treated mice. The V. villosa-adherent contrasuppressor cells were shown to be T cells that were I-J+ and of the Lyt-1 phenotype. Contrasuppressor T cells (Tcs) were not present in V. villosa-adherent spleen cell fractions obtained from normal mice, from mice injected with polyvinylpyrrolidone-coupled spleen cells, or from Cy-treated mice injected with S3-SCSM, i.e., mice in which Ts activity is dominant. The V. villosa-adherent cells that abrogated the activity of Ts induced by S3-SCSM in Cy-treated mice did not abrogate suppression mediated by a different subset of S3-specific Ts, suggesting that the Tcs described here do not have activity against all Ts subsets. The ability of S3-SCSM to activate Tcs in normal mice provides an explanation for the inability to detect S3-specific Ts in several previous studies.


Ankle Arthrodesis using Ilizarov Ring Fixator: A Primary or Salvage Procedure? An Analysis of Twenty Cases.
Introduction: Ankle arthrodesis using the Ilizarov technique provides high union rate with the added benefits of early weight-bearing, and the unique advantage of its ability to promote regeneration of soft tissue around the bone, including skin, muscle and neuro-vascular structures, and its versatility to allow correction of the position of the foot by adjusting the frame post-operatively as needed. We describe our experience with this technique and the functional outcomes in our patients. Materials and Methods: This retrospective study was conducted in 20 ankle fusion cases using the Ilizarov method between the years 2007 and 2017. We defined success in treatment by loss of preoperative symptoms and radiological union on plain radiographs of the ankle. Results: Fusion was achieved in all patients (100%). Immediate post-operative ambulation was with full weight bearing (FWB) in 16 (83%) of the participants and non-weight bearing (NWB) in 3 patients (17%). Post-procedure 11 patients (67%) of the participants who were full weight bearing required some form of support for walking for 2-3 weeks. Post-operatively three patients had pin tract infection requiring intravenous antibiotics. Radiological union took range of 6-12 weeks, mean union time was 8 weeks. Only one patient required bone grafting due to bone loss. Average follow-up period was 10-45 months. Conclusion: The Ilizarov technique has a high union rate and leads to general favourable clinical outcome and may be considered for any ankle arthrodesis but is especially useful in complex cases such as for revisions, soft-tissue compromise, infection and in patients with risk for non-union. Early weight bearing is an extra benefit.


Management of children in the deployed intensive care unit at Camp Bastion, Afghanistan.
The deployed Intensive Therapy Unit (ITU) in the British military field hospital in Camp Bastion, Afghanistan, admits both adults and children. The purpose of this paper is to review the paediatric workload in the deployed ITU and to describe how the unit copes with the challenge of looking after critically injured and ill children. Retrospective review of patients <16 years of age admitted to the ITU in the British military field hospital in Camp Bastion, Afghanistan, over a 1-year period from April 2011 to April 2012. 112/811 (14%) admissions to the ITU were paediatric (median age 8 years, IQR 6-12, range 1-16). 80/112 were trauma admissions, 13 were burns, four were non-trauma admissions and 15 were readmissions. Mechanism of injury in trauma was blunt in 12, blast (improvised explosive device) in 45, blast (indirect fire) in seven and gunshot wound in 16. Median length of stay was 0.92 days (IQR 0.45-2.65). 82/112 admissions (73%) were mechanically ventilated, 16/112 (14%) required inotropic support. 12/112 (11%) died before unit discharge. Trauma scoring was available in 65 of the 80 trauma admissions. Eight had Injury Severity Score or New Injury Severity Score >60, none of whom survived. However, of the 16 patients with predicted mortality >50% by Trauma Injury Severity Score, seven survived. Seven cases required specialist advice and were discussed with the Birmingham Children's Hospital paediatric intensive care retrieval service. The mechanisms by which the Defence Medical Services support children admitted to the deployed adult ITU are described, including staff training in clinical, ethical and child protection issues, equipment, guidelines and clinical governance and rapid access to specialist advice in the UK. With appropriate support, it is possible to provide intensive care to children in a deployed military ITU.


Yarrowia lipolytica causes sporadic cases and local outbreaks of infections and colonisation.
Yarrowia lipolytica belongs to the normal human microbiota but is also found in substrates with high contents in lipids and used in biotechnological processes. It is sometimes reported as human pathogen and especially in catheter-related candidaemia. Two apparently grouped cases of infections and/or contamination were reported involving 3 and 9 patients, respectively, in two hospitals. The goal of this study was to design a molecular tool to study the genetic diversity of Y lipolytica and confirm or not the common source of contamination during these grouped cases. Given that there is no genotyping method, we used genomic markers assessed on environmental isolates to determine intra-species relationship. We selected five highly polymorphic intergenic regions, totalling more than 3200 bp and sequenced them for clinical (n = 20) and environmental (n = 14) isolates. Antifungal susceptibility was determined by EUCAST broth microdilution method. Multiple alignment of the five sequences revealed divergence of 0%-5.8% between isolates as compared to approximately 0.2%-0.25% after alignment of whole genomes, suggesting their potential usefulness to establish genetic relatedness. The analysis showed the multiple origins of the isolates. It uncovered two grouped case of fungaemia involving 3 and 2 patients, respectively. It also revealed several unrelated sporadic cases despite their temporal relationship and one probable laboratory contamination by a common yet uncovered source, explaining several consecutive positive cultures without infection. All isolates had high minimal inhibitory concentration (MIC) for flucytosine, the majority (14/34) was susceptible to fluconazole, and all to the other antifungal agents tested. This method could help elucidate cases related to the opportunistic pathogen Y lipolytica.


HIV, sexual risk, and ethnicity among men in England who have sex with men.
To examine ethnic group differences in HIV testing history and sexual HIV risk behaviours that may account for such differences, among men in England who have sex with men (MSM), in order to inform HIV prevention planning priorities. A self completion survey in the summer of 2001 was carried out in collaboration with community based health promoters. Three recruitment methods were used: "gay pride" festivals, health promoter distributed leaflets, internet version advertised with gay service providers. The leaflet was produced with an alternative cover for targeted recruitment of black men. In a sample of 13,369 MSM living in England, 17.0% were from minority ethnic groups and 5.4% had tested HIV positive. Compared to the white British majority, Asian men were 0.32 times as likely to be living with diagnosed HIV infection, while black men were 2.06 times as likely to be doing so. Among men who had not tested HIV positive, Asian men were less likely to have sex with a known HIV positive partner, while black men were more likely to have insertive unprotected anal intercourse both with a partner they knew to be HIV positive and with a partner whose HIV status they did not know. Among MSM in England, HIV prevalence is higher among black men and lower among Asian men compared with the white British majority. Increased sexual HIV risk behaviour, especially exposure during insertive anal intercourse, accounts for some of this difference. HIV prevention programmes for MSM and African people should both prioritise black MSM.


Diet and blood pressure in South Africa: Intake of foods containing sodium, potassium, calcium, and magnesium in three ethnic groups.
We investigated whether habitual intakes of sodium (Na), potassium, magnesium, and calcium differ across South African ethnic groups, assessed the proportion of Na intake, which is discretionary, and identified which food sources were the major contributors to Na intake. This was a cross-sectional study of 325 black, white, and mixed ancestry hypertensive and normotensive subjects. Three repeated 24-h urine samples were collected for assessment of urinary Na, and three corresponding 24-h dietary recalls were administered by trained fieldworkers. Blood pressure and weight were measured at each visit. Secondary analyses were performed on existing dietary databases obtained from four regional surveys undertaken in South African adults. Mean urinary Na excretion values equated to daily salt (NaCl) intakes of 7.8, 8.5, and 9.5 g in black, mixed ancestry, and white subjects, respectively (P < 0.05). Between 33% and 46% of total Na intake was discretionary, and, of the non-discretionary sources, bread was the single greatest contributor to Na intake in all groups. Ethnic differences in calcium intake were evident, with black subjects having particularly low intakes. Urban versus rural differences existed with respect to sources of dietary Na, with greater than 70% of total non-discretionary Na being provided by bread and cereals in rural black South Africans compared with 49% to 54% in urban dwellers. White South Africans have higher habitual intakes of Na, but also higher calcium intakes, than their black and mixed ancestry counterparts. All ethnic groups had Na intakes in excess of 6 g/d of salt, whereas potassium intakes in all groups were below the recommended level of 90 mM/d. Dietary differences may contribute to ethnically related differences in blood pressure.


Identification, prioritization, and evaluation of glycoproteins for aggressive prostate cancer using quantitative glycoproteomics and antibody-based assays on tissue specimens.
Prostate cancer is highly heterogeneous in nature; while the majority of cases are clinically insignificant, some cases are lethal. Currently, there are no reliable screening methods for aggressive prostate cancer. Since most established serum and urine biomarkers are glycoproteins secreted or leaked from the diseased tissue, the current study seeks to identify glycoprotein markers specific to aggressive prostate cancer using tissue specimens. With LC-MS/MS glycoproteomic analysis, we identified 350 glycopeptides with 17 being altered in aggressive prostate cancer. ELISA assays were developed/purchased to evaluate four candidates, that is, cartilage oligomeric matrix protein (COMP), periostin, membrane primary amine oxidase (VAP-1), and cathepsin L, in independent tissue sets. In agreement with the proteomic analysis, we found that COMP and periostin expressions were significantly increased in aggressive prostate tumors while VAP-1 expression was significantly decreased in aggressive tumor. In addition, the expression of these proteins in prostate metastases also follows the same pattern observed in the proteomic analysis. This study provides a workflow for biomarker discovery, prioritization, and evaluation of aggressive prostate cancer markers using tissue specimens. Our data suggest that increase in COMP and periostin and decrease in VAP-1 expression in the prostate may be associated with aggressive prostate cancer.


Limitation of immune tolerance-inducing thymic epithelial cell development by Spi-B-mediated negative feedback regulation.
Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B-mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage-specific negative feedback regulation. OPG-mediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL-Spi-B-OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.


Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.
Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.


Stopping light by an air waveguide with anisotropic metamaterial cladding.
We present a detailed study of oscillating modes in a slab waveguide with air core and anisotropic metamaterial cladding. It is shown that, under specific dielectric configurations, slow and even stopped electromagnetic wave can be supported by such an air waveguide. We propose a linearly tapped waveguide structure that could lead the propagating light to a complete standstill. Both the theoretical analysis and the proposed waveguide have been validated by full-wave simulation based on finite-difference time-domain method.


Crystal structures of two transcriptional regulators from Bacillus cereus define the conserved structural features of a PadR subfamily.
PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206-BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206-BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.


Photon beam characterization and modelling for Monte Carlo treatment planning.
Photon beams of 4, 6 and 15 MV from Varian Clinac 2100C and 2300C/D accelerators were simulated using the EGS4/BEAM code system. The accelerators were modelled as a combination of component modules (CMs) consisting of a target, primary collimator, exit window, flattening filter, monitor chamber, secondary collimator, ring collimator, photon jaws and protection window. A full phase space file was scored directly above the upper photon jaws and analysed using beam data processing software, BEAMDP, to derive the beam characteristics, such as planar fluence, angular distribution, energy spectrum and the fractional contributions of each individual CM. A multiple-source model has been further developed to reconstruct the original phase space. Separate sources were created with accurate source intensity, energy, fluence and angular distributions for the target, primary collimator and flattening filter. Good agreement (within 2%) between the Monte Carlo calculations with the source model and those with the original phase space was achieved in the dose distributions for field sizes of 4 cm x 4 cm to 40 cm x 40 cm at source surface distances (SSDs) of 80-120 cm. The dose distributions in lung and bone heterogeneous phantoms have also been found to be in good agreement (within 2%) for 4, 6 and 15 MV photon beams for various field sizes between the Monte Carlo calculations with the source model and those with the original phase space.


A framework for the development of targeted gas chromatography mass spectrometry (GC-MS) methods: Synthetic cannabinoids.
With the increased presence of novel psychoactive substances (NPSs) in casework, drug analysis has become more challenging. To address these challenges, new screening technologies with improved specificity are being implemented, allowing for the creation and adoption of targeted confirmatory analyses that produce more conclusive results. This paper outlines a six-step, data-driven, framework to develop and evaluate gas chromatography mass spectrometry (GC-MS) methods for targeted classes of drugs. The process emphasizes maximizing retention time differences (to minimize the potential for retention time acceptance windows to overlap) and understanding the trade-offs between sensitivity and reproducibility using a test solution containing pairs of compounds that are difficult to distinguish. The method is then evaluated by expanding the panel of compounds analyzed, identifying limitations in compound discrimination, comparing to current methods, and analyzing representative casework to establish usability. To demonstrate this framework, a method for synthetic cannabinoids was created. The developed method utilizes a DB-200 column and an isothermal temperature program. It was found that sensitivity could be adjusted, without compromising reproducibility, by altering the split ratio and injection volume. The targeted method successfully differentiated 50 cannabinoids based on either retention time differences or mass spectral dissimilarity - determined using a newly developed spectral comparison test. Compared to a general method used for casework, the targeted method was an order of magnitude more sensitive, a minute shorter, and provided major increases in retention time differences. This framework can be implemented and adapted to develop targeted methods for other applications or compound classes.


Combined effects of lipid transfers and lipolysis on gradient gel patterns of human plasma LDL.
The triglyceride content of the plasma very-low-density lipoprotein (VLDL) fraction is the most important factor affecting the size of low-density lipoprotein (LDL) in humans. Because cholesteryl ester transfer protein (CETP) can influence the size distribution of LDL particles in human plasma, the implication of lipid transfers in the formation of small-sized LDL patterns, which have been associated with elevated plasma triglyceride levels, was investigated. The size distribution of LDL particles in 15 plasma samples was determined by electrophoresis of the plasma LDL fraction on 20 to 160 g/L polyacrylamide gradient gels. The apparent diameter of the major LDL subfraction was shown to correlate negatively with triglyceride concentrations (r = -.706, P < .005) and positively with both high-density lipoprotein cholesterol levels (r = .637, P < .02) and the high-density lipoprotein/VLDL + LDL cholesterol ratio (r = .768, P < .001). In addition, LDL size correlated negatively with both the ability of plasma LDL to donate cholesteryl esters (r = -.79, P < .001) and its ability to acquire triglycerides (r = -.72, P < .005). Whereas these observations indicated that CETP-mediated alterations of the triglyceride/cholesteryl ester ratio of the LDL core would contribute to changes in LDL diameter, they suggested that the formation of small-sized gradient gel LDL patterns would require another biochemical event, such as lipolysis, in addition to neutral lipid transfers. To test this hypothesis, total plasma samples with or without added VLDL (added triglyceride concentration, 2.0 g/L) were preincubated for 24 hours at 37 degrees C. Preincubation mainly induced the replacement of cholesteryl esters by triglycerides in the LDL core, and changes in LDL composition were greater when total plasma was supplemented with VLDL. Subsequently, isolated LDL was incubated in the presence of bovine milk lipoprotein lipase as a source of triglyceride hydrolysis activity. Lipolysis tended to reduce the size of the major LDL subpopulation, and the mean change in LDL diameter was significantly greater when plasma was preincubated with VLDL supplementation than when it was not (-0.6 +/- 0.3 versus -0.2 +/- 0.2 nm, respectively; (P < .01). Moreover, sequential effects of lipid transfer and lipolysis activities induced dramatic changes in the general shape of gradient gel LDL patterns. The largest plasma LDL subpopulations tended to disappear, and the formation of new, small LDL particles could be observed. The combined effects of neutral lipid transfers and triglyceride hydrolysis could account for variations of gradient gel LDL profiles in human plasma.(ABSTRACT TRUNCATED AT 400 WORDS)


Phase II randomized trial of cisplatin chemotherapy regimens in the treatment of recurrent or metastatic squamous cell cancer of the cervix: a Southwest Oncology Group Study.
Cisplatin has proven to be the most active single agent in the treatment of metastatic and recurrent squamous cell cancer of the cervix. In a previous southwest Oncology Group (SWOG) pilot study, the addition of cisplatin to a mitomycin-C, vincristine, and bleomycin (MVB) regimen resulted in a relatively high percentage of durable complete responses. To gain more experience with cisplatin-based chemotherapy regimens, the SWOG initiated a phase II randomized trial of cisplatin, mitomycin-C plus cisplatin (MC), and MVB plus cisplatin (MVBC) in 119 patients with advanced squamous cell cancer of the cervix and no prior chemotherapy exposure. Because of slow patient accrual early in the trial, the cisplatin arm was discontinued. Five patients were declared ineligible according to protocol criteria. The three treatment groups were relatively well matched for age, prior radiation exposure, and sites of measurable disease. The overall objective response rates for cisplatin, MC, and MVBC treated patients were 33%, 25%, and 22%, respectively. Median response durations were greater than 6 months. Median survival durations associated with cisplatin, MC, and MVBC treatment were 17.0, 7.0, and 6.9 months, respectively. There were no drug-related deaths. Severe or life-threatening leukopenia and thrombocytopenia were observed in 18% to 24% of patients treated with MVBC and MC, but in none of those receiving cisplatin alone. We conclude that the low response rates and short durations of both response and survival observed in patients randomized to the two chemotherapy combinations suggest that only enhanced toxicity was gained through the addition of mitomycin-C or MVB to cisplatin in patients with advanced cervix cancer.


Dental development and life history in living African and Asian apes.
Life-history inference is an important aim of paleoprimatology, but life histories cannot be discerned directly from the fossil record. Among extant primates, the timing of many life-history attributes is correlated with the age at emergence of the first permanent molar (M1), which can therefore serve as a means to directly compare the life histories of fossil and extant species. To date, M1 emergence ages exist for only a small fraction of extant primate species and consist primarily of data from captive individuals, which may show accelerated dental eruption compared with free-living individuals. Data on M1 emergence ages in wild great apes exist for only a single chimpanzee individual, with data for gorillas and orangutans being anecdotal. This paucity of information limits our ability to make life-history inferences using the M1 emergence ages of extinct ape and hominin species. Here we report reliable ages at M1 emergence for the orangutan, Pongo pygmaeus (4.6 y), and the gorilla, Gorilla gorilla (3.8 y), obtained from the dental histology of wild-shot individuals in museum collections. These ages and the one reported age at M1 emergence in a free-living chimpanzee of approximately 4.0 y are highly concordant with the comparative life histories of these great apes. They are also consistent with the average age at M1 emergence in relation to the timing of life-history events in modern humans, thus confirming the utility of M1 emergence ages for life-history inference and providing a basis for making reliable life-history inferences for extinct apes and hominins.


Characteristics impacting on session rating of perceived exertion training load in Australian footballers.
The relationship between external training load and session rating of perceived exertion (s-RPE) training load and the impact that playing experience, playing position and 2-km time-trial performance had on s-RPE training load were explored. From 39 Australian Football players, 6.9 ± 4.6 training sessions were analysed, resulting in 270 samples. Microtechnology devices provided external training load (distance, average speed, high-speed running distance, player load (PL) and player loadslow (PLslow)). The external training load measures had moderate to very large associations (r, 95% CI) with s-RPE training load, average speed (0.45, 0.35-0.54), high-speed running distance (0.51, 0.42-0.59), PLslow (0.80, 0.75-0.84), PL (0.86, 0.83-0.89) and distance (0.88, 0.85-0.90). Differences were described using effect sizes (d ±95% CL). When controlling for external training load, the 4- to 5-year players had higher s-RPE training load than the 0- to 1- (0.44 ± 0.33) and 2- to 3-year players (0.51 ± 0.30), ruckmen had moderately higher s-RPE training load than midfielders (0.82 ± 0.58), and there was a 0.2% increase in s-RPE training load per 1 s increase in time-trial (95% CI: 0.07-0.34). Experience, position and time-trial performance impacted the relationship between external training load and s-RPE training load. This suggests that a given external training load may result in different internal responses between athletes, potentially leaving individuals at risk of overtraining or failing to elicit positive adaptation. It is therefore vital that coaches and trainers give consideration to these mediators of s-RPE training load.


Use of chemical ablation with trichloroacetic acid to treat eyelid apocrine hidrocystomas in a cat.
A 7-year-old Persian cat was evaluated for recurrence of multiple cystic periocular masses. A number of cyst-like lesions had been resected from the left eyelids 18 months earlier, with lesions recurring within 6 months after surgery. The cat had blepharospasm and signs of discomfort following rupture of the largest cyst the day prior to examination. Previous histologic examination of the cysts had revealed apocrine hidrocystomas. Multiple pigmented nodules were seen around the skin of the upper and lower left eyelids. The nodules were brownish to black, round, soft, and fluid-filled. Signs of pain were not evident during palpation of the nodules. The largest cyst on the upper eyelid was removed by means of a V-shaped full-thickness excision. Histologic and immunohistochemical examination of the excised tissue confirmed the diagnosis of apocrine hidrocystoma. The remaining periocular cysts were surgically debrided and then treated topically with 20% trichloroacetic acid. All lesions healed rapidly without any signs of discomfort. During a recheck examination 12 months later, the upper and lower left eyelids appeared morphologically normal, and there was no evidence of recurrence. Findings suggested that chemical ablation with trichloroacetic acid may be a useful treatment for apocrine hidrocystomas in cats.


Kinks, loops, and protein folding, with protein A as an example.
The dynamics and energetics of formation of loops in the 46-residue N-terminal fragment of the B-domain of staphylococcal protein A has been studied. Numerical simulations have been performed using coarse-grained molecular dynamics with the united-residue (UNRES) force field. The results have been analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger (DNLS) equation. In the case of proteins, the DNLS equation arises from a C(α)-trace-based energy function. Three individual kink profiles were identified in the experimental three-α-helix structure of protein A, in the range of the Glu16-Asn29, Leu20-Asn29, and Gln33-Asn44 residues, respectively; these correspond to two loops in the native structure. UNRES simulations were started from the full right-handed α-helix to obtain a clear picture of kink formation, which would otherwise be blurred by helix formation. All three kinks emerged during coarse-grained simulations. It was found that the formation of each is accompanied by a local free energy increase; this is expressed as the change of UNRES energy which has the physical sense of the potential of mean force of a polypeptide chain. The increase is about 7 kcal/mol. This value can thus be considered as the free energy barrier to kink formation in full α-helical segments of polypeptide chains. During the simulations, the kinks emerge, disappear, propagate, and annihilate each other many times. It was found that the formation of a kink is initiated by an abrupt change in the orientation of a pair of consecutive side chains in the loop region. This resembles the formation of a Bloch wall along a spin chain, where the C(α) backbone corresponds to the chain, and the amino acid side chains are interpreted as the spin variables. This observation suggests that nearest-neighbor side chain-side chain interactions are responsible for initiation of loop formation. It was also found that the individual kinks are reflected as clear peaks in the principal modes of the analyzed trajectory of protein A, the shapes of which resemble the directional derivatives of the kinks along the chain. These observations suggest that the kinks of the DNLS equation determine the functionally important motions of proteins.


Hematocrit, blood pressure, and hypertension. The Gubbio Population Study.
Baseline data from the Gubbio Population Study in north central Italy were used to investigate the relation of hematocrit to blood pressure and hypertension among 2,809 men and women aged 25-74 years. Independent of gender, age, and other confounders, the hypertensive group had a higher hematocrit than the nonhypertensive group (p less than 0.001). In comparison with the untreated hypertensive group, the hypertensive group being treated with diuretics or with other drugs only had similar mean hematocrit levels despite significantly lower blood pressures. Hematocrit was positively correlated with systolic pressure (r = 0.085, p less than 0.01 and r = 0.264, p less than 0.001 for men and women, respectively) and diastolic pressure (r = 0.214, p less than 0.001 and r = 0.266, p less than 0.001). In both sexes, whether or not the treated hypertensive group was included, age-adjusted prevalence of hypertension and average blood pressure were higher for persons in higher quintiles of hematocrit (p less than 0.001). The association of hematocrit with blood pressure and hypertension was significant and independent of several confounders. The regression coefficient of blood pressure on hematocrit ranged between 0.410 and 0.620 mm Hg per unit of hematocrit for systolic pressure and between 0.371 and 0.581 for diastolic pressure, depending on gender and whether the treated hypertensive group was included in multiple regression analysis. Based on exponentiation of the multiple logistic coefficient, prevalence of hypertension was at least two times greater for persons whose hematocrit levels were higher by 10 units.


Conformation of the mineralocorticoid receptor N-terminal domain: evidence for induced and stable structure.
The mineralocorticoid receptor (MR) binds the steroid hormones aldosterone and cortisol and has an important physiological role in the control of salt homeostasis. Regions of the protein important for gene regulation have been mapped to the amino-terminal domain (NTD) and termed activation function (AF)1a, AF1b, and middle domain (MD). In the present study, we used a combination of biophysical and biochemical techniques to investigate the folding and function of the MR-NTD transactivation functions. We demonstrate that MR-AF1a and MR-MD have relatively little stable secondary structure but have the propensity to form α-helical conformation. Induced folding of the MR-MD enhanced protein-protein binding with a number of coregulatory proteins, including the coactivator cAMP response element-binding protein-binding protein and the corepressors SMRT and RIP140. By contrast, the MR-AF1b domain appeared to have a more stable conformation consisting predominantly of β-secondary structure. Furthermore, MR-AF1b specifically interacted with the TATA-binding protein, via an LxxLL-like motif, in the absence of induced folding. Together, these data suggest that the MR-NTD contains a complex transactivation system made up of distinct structural and functional domains. The results are discussed in the context of the induced folding paradigm for steroid receptor NTDs.


Activity of descending propriospinal axons in the turtle hindlimb enlargement during two forms of fictive scratching: phase analyses.
In the preceding companion article (Berkowitz and Stein, 1994b), we showed that many descending propriospinal neurons in the turtle were rhythmically activated during two different motor patterns, fictive rostral scratching and fictive pocket scratching. In this article, we present phase analyses of the activity of each such neuron during fictive scratching. Each neuron's activity was concentrated in a particular phase of the ipsilateral hip flexor muscle nerve (VP-HP) activity cycle; each had a distinct "preferred phase." Each neuron's preferred phase during fictive rostral scratching was similar to its preferred phase during fictive pocket scratching. This result is consistent with the idea that some descending propriospinal neurons may contribute to the generation of both rostral scratching and pocket scratching. Many descending propriospinal neurons were rhythmically activated during fictive scratching evoked on either side of the body. This activity may contribute to production of bilateral hindlimb movements during scratching. It is also possible that synaptic interactions between the two sides of the spinal cord may be important in generating the motor patterns for movement of a single hindlimb. In addition, we present a model which illustrates that a population of propriospinal neurons, each of which is broadly tuned to a region of the body surface and is rhythmically activated in a constant phase of the hip control cycle, could mediate the selection and generation of rostral scratching and pocket scratching. Thus, the selection of an appropriate motor pattern and the production of the required knee-hip synergy may each be distributed over a diverse population of spinal cord neurons. This model requires that each such neuron project to both knee muscle and hip muscle motoneurons. According to this model, the process of selecting a motor pattern would not be completed until knee muscle motoneurons integrate overlapping excitatory and inhibitory inputs.


Venetoclax combines synergistically with FLT3 inhibition to effectively target leukemic cells in FLT3-ITD+ acute myeloid leukemia models.
FLT3 internal tandem duplication (FLT3-ITD) mutations account for ~25% of adult acute myeloid leukemia cases and are associated with poor prognosis. Venetoclax, a selective BCL-2 inhibitor, has limited monotherapy activity in relapsed/refractory acute myeloid leukemia with no responses observed in a small subset of FLT3-ITD+ patients. Further, FLT3-ITD mutations emerged at relapse following venetoclax monotherapy and combination therapy suggesting a potential mechanism of resistance. Therefore, we investigated the convergence of FLT3-ITD signaling on the BCL-2 family proteins and determined combination activity of venetoclax and FLT3-ITD inhibition in preclinical models. In vivo, venetoclax combined with quizartinib, a potent FLT3 inhibitor, showed greater anti-tumor efficacy and prolonged survival compared to monotherapies. In a patient-derived FLT3-ITD+ xenograft model, cotreatment with venetoclax and quizartinib at clinically relevant doses had greater anti-tumor activity in the tumor microenvironment compared to quizartinib or venetoclax alone. Use of selective BCL-2 family inhibitors further identified a role for BCL-2, BCL-XL and MCL-1 in mediating survival in FLT3-ITD+ cells in vivo and highlighted the need to target all three proteins for greatest anti-tumor activity. Assessment of these combinations in vitro revealed synergistic combination activity for quizartinib and venetoclax but not for quizartinib combined with BCL-XL or MCL-1 inhibition. FLT3-ITD inhibition was shown to indirectly target both BCL-XL and MCL-1 through modulation of protein expression, thereby priming cells toward BCL-2 dependence for survival. These data demonstrate that FLT3-ITD inhibition combined with venetoclax has impressive anti-tumor activity in FLT3-ITD+ acute myeloid leukemia preclinical models and provides strong mechanistic rational for clinical studies.