Effect of dietary protein restriction on renal purines and purine-metabolizing enzymes in adriamycin nephrosis in rats: a mechanism for protection against acute proteinuria involving xanthine oxidase inhibition.
1. A low protein diet prevents the development of proteinuria and glomerular damage in adriamycin experimental nephrosis without affecting renal haemodynamics. In this study the hypothesis was tested as to whether protein restriction is able to modulate the purine metabolic cycle and related enzymes such as xanthine oxidase, one of the putative effectors of adriamycin nephrotoxicity. 2. Renal activities of xanthine oxidase and purine nucleoside phosphorylase were markedly depressed in adriamycin-treated rats fed a 9% casein (low protein) diet compared with the group fed a 22% casein (normal protein) diet both 1 day after adriamycin administration and at the time of appearance of heavy proteinuria (day 15), whereas the activity of renal adenosine deaminase was unchanged. 3. The concentrations of the metabolic substrates of xanthine oxidase, i.e. hypoxanthine and xanthine, were constantly lower in renal homogenates of rats fed a low protein diet compared with those on a normal protein diet. In urine, uric acid, the product of hypoxanthine-xanthine transformation, was lower 1 day after adriamycin injection in protein-restricted rats compared with the group on a normal protein diet which showed a marked increase in its excretion. At the same time, the urinary efflux of adenosine 5'-monophosphate, which is the precursor nucleotide of the above-mentioned nucleosides and bases, was very high in rats fed a low protein diet, whereas it was absent in the group on a normal protein diet. 4. The progressive increment in proteinuria of glomerular origin (i.e. increased excretion of albumin and transferrin) typical of adriamycin-treated rats fed a normal protein diet was inhibited in the protein-restricted animals, which were normoproteinuric on day 10 and were only slightly proteinuric on day 15. 5. Like protein restriction, the pharmacological suppression of renal xanthine oxidase by dietary tungstate and the scavenging by dimethylthiourea of the putative free radical deriving from the action of xanthine oxidase, were associated with a similar (quantitative and qualitative) inhibition of glomerular proteinuria. 6. These data demonstrate that dietary protein restriction is associated with a block in purine metabolism within the kidney due to a marked reduction in the activities of two main enzymes of the cycle, i.e. purine nucleoside phosphorylase and xanthine oxidase, the latter being a putative effector of adriamycin nephrotoxicity. The partial reduction of proteinuria induced by a low protein diet is quantitatively and qualitatively comparable with the reduction induced by the specific block of renal xanthine oxidase or by the scavenging of OH.deriving from hypoxanthine and xanthine transformation.(ABSTRACT TRUNCATED AT 400 WORDS)


Sleep and circadian phenotype in people without cone-mediated vision: a case series of five CNGB3 and two CNGA3 patients.
Light exposure entrains the circadian clock through the intrinsically photosensitive retinal ganglion cells, which sense light in addition to the cone and rod photoreceptors. In congenital achromatopsia (prevalence 1:30-50 000), the cone system is non-functional, resulting in severe light avoidance and photophobia at daytime light levels. How this condition affects circadian and neuroendocrine responses to light is not known. In this case series of genetically confirmed congenital achromatopsia patients (n = 7; age 30-72 years; 6 women, 1 male), we examined survey-assessed sleep/circadian phenotype, self-reported visual function, sensitivity to light and use of spectral filters that modify chronic light exposure. In all but one patient, we measured rest-activity cycles using actigraphy over 3 weeks and measured the melatonin phase angle of entrainment using the dim-light melatonin onset. Owing to their light sensitivity, congenital achromatopsia patients used filters to reduce retinal illumination. Thus, congenital achromatopsia patients experienced severely attenuated light exposure. In aggregate, we found a tendency to a late chronotype. We found regular rest-activity patterns in all patients and normal phase angles of entrainment in participants with a measurable dim-light melatonin onset. Our results reveal that a functional cone system and exposure to daytime light intensities are not necessary for regular behavioural and hormonal entrainment, even when survey-assessed sleep and circadian phenotype indicated a tendency for a late chronotype and sleep problems in our congenital achromatopsia cohort.


Whose information is it anyway? Informing a 12-year-old patient of her terminal prognosis.
To examine students' attitudes and potential behaviour towards informing a 12-year-old patient of her terminal prognosis in a situation in which her parents do not wish her to be told, as they pass through a modern medical curriculum. A cohort study of students entering Glasgow University's new medical curriculum in October 1996. Students' responses obtained before year 1 and at the end of years 1, 3, and 5 to the "childhood leukaemia" vignette of the Ethics in Health Care Survey Instrument (EHCI) were examined quantitatively and qualitatively. Analysis of the students' multichoice answers enabled measurement of the movement towards professional consensus opinion. An analysis of their written justifications for their answers helped to determine whether their reasoning was consistent with professional consensus and enabled measurement of changes in knowledge content and recognition of the values inherent in the vignette. Themes on the students' reasoning behind their decision to tell the patient or not were also identified. Unlike other vignettes of the EHCI in which autonomy was a main theme, few students chose the consensus answer before year 1 and there was no significant movement towards consensus at any point during the course. In defence of their decision to withhold information, the students expressed strong paternalistic opinions. The patient's age was seen as a barrier to respecting her autonomy. It is important to identify students' perceptions on entry to medical school. Transformative learning theory may provide the basis for an approach to foster doctors who consider the rights of young people. Small-group teaching is most conducive to this approach. The importance of positive role modelling is also emphasised.


Frequency of K-ras mutations in pancreatic intraductal neoplasias associated with pancreatic ductal adenocarcinoma and chronic pancreatitis: a meta-analysis.
Molecular analyses have demonstrated mutations in the K-ras gene at codon 12 in the majority of pancreatic ductal adenocarcinomas (PDACs). In order to determine whether the K-ras mutation rate increases parallel to the grade of dysplasia in duct lesions, we performed a meta-analysis of the studies published between 1988 and 2003 that provide information on K-ras mutations in hyperplastic and dysplastic duct lesions in the pancreas. The described duct lesions were reclassified according to the nomenclature for pancreatic intraepithelial neoplasia (PanIN), and the molecular methods for detecting K-ras were reviewed. In PanIN lesions from pancreata of patients with PDAC, there was a stepwise increase in K-ras mutations that correlated with the grade of dysplasia of the PanIN lesion. K-ras mutations were found in 36%, 44%, and 87% of PanIN-1a, 1b, and 2-3 lesions, respectively (trend statistic P <.001). Mutation-enriched polymerase chain reaction (PCR) resulted in higher rates of K-ras mutations in PanIN than plain PCR did. The incidence of K-ras mutations in PanIN lesions associated with chronic pancreatitis (CP) or normal pancreas was low (around 10%). In CP, K-ras mutations were only found after a disease duration of 3 years. The correlation of the incidence of K-ras mutations with the grade of dysplasia in PanIN and the occurrence of these mutations in CP with a duration of more than 3 years underlines the importance of this genetic change for the development of PDAC.


Interaction of renal prostaglandins with the renin-angiotensin and renal adrenergic nervous systems in healthy subjects during dietary changes in sodium intake.
In six healthy subjects the role of renal prostaglandins (PG) in modulating the actions of the renin-angiotensin and renal adrenergic nervous systems on renal function was investigated. During high dietary sodium intake (350 mmol/day) for 4 days no changes in urinary excretion of PGE2, PGF2 alpha, noradrenaline or adrenaline were noted, whereas plasma renin activity (PRA) and urinary aldosterone excretion were suppressed. After 4 days of low sodium intake (35 mmol/day) urinary excretion of PGE2, aldosterone and noradrenaline, as well as PRA, had significantly increased. Inhibition of PG synthesis with indomethacin (2 mg/kg body weight) had no effects on renal function on day 5 of high sodium intake. Despite suppression of PRA and urinary aldosterone, indomethacin significantly reduced p-aminohippurate (PAH) clearance, glomerular filtration rate (GFR) and urinary sodium excretion on day 5 of low sodium intake, when urinary noradrenaline excretion remained high. The results point to the crucial role of the renal adrenergic nervous system in controlling renal vascular resistance and sodium conservation in healthy subjects during low sodium intake, which is unmasked when renal PG synthesis is blocked by indomethacin. Enhanced renal PG synthesis during sodium restriction therefore not only attenuates the vascular and tubular effects of the renin-angiotensin system but, more importantly, also those of the highly stimulated renal adrenergic nervous system.


Steroid hormone receptors and direct effects of steroid hormones on ram spermatozoa.
This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes during in vitro capacitation and the actions of progesterone (P4) and 17β-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40-45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERβ). ERα was located in the postacrosomal region of all the spermatozoa and ERβ on the apical region of 63.7% of the cells. The presence of ERβ was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848, P < 0.001). This significantly decreased after in vitro capacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 before in vitro capacitation resulted in a higher (P < 0.001) acrosome-reacted sperm rate compared with the control (13.0%), noticeably greater after 3 h and when added to a high-cAMP medium (37.3% and 47.0% with E2 and P4, respectively). In conclusion, the results of this study demonstrate for the first time that ovine spermatozoa have progesterone and estrogen receptors and that both steroid hormones are related with the induction of the acrosome reaction.


NEWS Scoring System: Use in Hematologic Malignancies and Cellular Therapeutics Patient Populations.
Systemic inflammatory response syndrome (SIRS) criteria can be overly sensitive in detecting clinical deterioration in hematologic malignancies and cellular therapeutics (HMCT) populations. This may create inconsistency in activation of a rapid response team (RRT) with patient decline. The aim is to evaluate the implementation and use of the National Early Warning Score (NEWS) scoring system in the HMCT population. Retrospective data collection from 2016 to 2017 was used. NEWS scores were calculated every 4 hours for the 24 hours prior to RRT activation. The difference between the time of the first NEWS score of 5 or greater and the RRT activation time mean and median values were calculated for the pre- and postimplementation time frames. A statistically significant decrease in the mean time between patients meeting a NEWS score of 5 or greater and RRT activation was noted in the postimplementation group. An increase in frequency of escalation of care to the intensive care unit for patients with multiple RRT activations during admission was statistically significant.


Alterations of the Host Microbiome Affect Behavioral Responses to Cocaine.
Addiction to cocaine and other psychostimulants represents a major public health crisis. The development and persistence of addictive behaviors comes from a complex interaction of genes and environment - the precise mechanisms of which remain elusive. In recent years a surge of evidence has suggested that the gut microbiome can have tremendous impact on behavioral via the microbiota-gut-brain axis. In this study we characterized the influence of the gut microbiota on cocaine-mediated behaviors. Groups of mice were treated with a prolonged course of non-absorbable antibiotics via the drinking water, which resulted in a substantial reduction of gut bacteria. Animals with reduced gut bacteria showed an enhanced sensitivity to cocaine reward and enhanced sensitivity to the locomotor-sensitizing effects of repeated cocaine administration. These behavioral changes were correlated with adaptations in multiple transcripts encoding important synaptic proteins in the brain's reward circuitry. This study represents the first evidence that alterations in the gut microbiota affect behavioral response to drugs of abuse.


Antiarrhythmic drugs inhibit the G-protein and K+ channels in the cultured thyroid cell line.
We examined the effects of antiarrhythmic drugs on the induction of cAMP by TSH (thyroid-stimulating hormone), using continuously cultured FRTL-5 rat thyroid cells. Group Ia antiarrhythmic drugs had no effect, but Group Ib antiarrhythmic drugs suppressed cAMP induction by TSH. These drugs suppressed cAMP induction in response to cholera toxin but did not inhibit TSH receptor binding or cAMP induction by forskolin. These results indicate that Group Ib antiarrhythmic drugs inhibit thyroid G-protein, resulting in a decrease in cAMP induction by TSH. We also examined the effect of antiarrhythmic drugs on K+ channels. Group Ia antiarrhythmic drugs had no effect on K+ channel activation by TSH and cAMP. On the other hand, Group Ib antiarrhythmic drugs suppressed K+ channel activation by TSH and cAMP. This indicates that the mechanism of suppression is not inhibition of TSH receptors or G-proteins but the direct suppression of K+ channels. Group Ib antiarrhythmic drugs inhibited thyroid Gs-protein and thyroid K+ channels. Considering the close relationship between G-protein and ion channels in the cardiac cell membrane, these different effects of Group Ia and Group Ib antiarrhythmic drugs on G-proteins and K+ channels are of interest. Further investigation is necessary to clarify the relationship between thyroid G-proteins and thyroid K+ channels.


Impaired DNA double-strand break repair contributes to chemoresistance in HIF-1 alpha-deficient mouse embryonic fibroblasts.
A mismatch between metabolic demand and oxygen delivery leads to microenvironmental changes in solid tumors. The resulting tumor hypoxia is associated with malignant progression, therapy resistance and poor prognosis. However, the molecular mechanisms underlying therapy resistance in hypoxic tumors are not fully understood. The hypoxia-inducible factor (HIF) is a master transcriptional activator of oxygen-regulated gene expression. Transformed mouse embryonic fibroblasts (MEFs) derived from HIF-1alpha-deficient mice are a popular model to study HIF function in tumor progression. We previously found increased chemotherapy and irradiation susceptibility in the absence of HIF-1alpha. Here, we show by single-cell electrophoresis, histone 2AX phosphorylation and nuclear foci formation of gammaH2AX and 53BP1, that the number of DNA double-strand breaks (DSB) is increased in untreated and etoposide-treated HIF-deficient MEFs. In etoposide-treated cells, cell cycle control and p53-dependent gene expression were not affected by the absence of HIF-1alpha. Using a candidate gene approach to screen 17 genes involved in DNA repair, messenger RNA (mRNA) and protein of three members of the DNA-dependent protein kinase complex were found to be decreased in HIF-deficient MEFs. Of note, residual HIF-1alpha protein in cancer cells with a partial HIF-1alpha mRNA knockdown was sufficient to confer chemoresistance. In summary, these data establish a novel molecular link between HIF and DNA DSB repair. We suggest that selection of early, non-hypoxic tumor cells expressing low levels of HIF-1alpha might contribute to HIF-dependent tumor therapy resistance.


Storage of spatial information by the maintenance mechanism of LTP.
Analogous to learning and memory storage, long-term potentiation (LTP) is divided into induction and maintenance phases. Testing the hypothesis that the mechanism of LTP maintenance stores information requires reversing this mechanism in vivo and finding out whether long-term stored information is lost. This was not previously possible. Recently however, persistent phosphorylation by the atypical protein kinase C isoform, protein kinase Mzeta (PKMz), has been found to maintain late LTP in hippocampal slices. Here we show that a cell-permeable PKMz inhibitor, injected in the rat hippocampus, both reverses LTP maintenance in vivo and produces persistent loss of 1-day-old spatial information. Thus, the mechanism maintaining LTP sustains spatial memory.


Fourier domain Pump-Probe Optical Coherence Tomography imaging of melanin.
We report the development of a two-color Fourier domain Pump-Probe Optical Coherence Tomography (PPOCT) system. Tissue phantom experiments to characterize the system performance demonstrated imaging depths in excess of 725 microm, nearly comparable to the base Optical Coherence Tomography system. PPOCT A-line rates were also demonstrated in excess of 1 kHz. The physical origin of the PPOCT signal was investigated with a series of experiments which revealed that the signal is a mixture of short and long lifetime component signals. The short lifetime component was attributed to transient absorption while the long lifetime component may be due to a mixture of transient absorption and thermal effects. Ex vivo images of porcine iris demonstrated the potential for imaging melanin in the eye, where cancer of the melanocytes is the most common form of eye cancer in adults.


Structural comparison of two esterases from Drosophila mojavensis isolated by immunoaffinity chromatography.
Antibodies raised against esterase-4 and esterase-5 from Drosophila mojavensis were coupled to Protein A-Sepharose CL-4B to prepare high-efficiency immunomatrices used for their purification. Final purification was achieved by anion-exchange h.p.l.c., in the case of esterase-5 followed by gel-filtration h.p.l.c. The resultant esterase preparations were homogeneous, as judged by gel-filtration h.p.l.c., SDS/polyacrylamide-gel electrophoresis and non-denaturing gel electrophoresis. Esterase-4 and esterase-5 are the products of a duplicated gene. They are differently localized in the insect's body and expressed in different periods during development. Although both enzymes exhibit little immunological cross-reactivity, their amino acid compositions show few significant differences and their N-terminal sequences are largely identical, which clearly show their common origin.


Venom of the Coral Snake Micrurus clarki: Proteomic Profile, Toxicity, Immunological Cross-Neutralization, and Characterization of a Three-Finger Toxin.
Micrurus clarki is an uncommon coral snake distributed from the Southeastern Pacific of Costa Rica to Western Colombia, for which no information on its venom could be found in the literature. Using a 'venomics' approach, proteins of at least nine families were identified, with a moderate predominance of three-finger toxins (3FTx; 48.2%) over phospholipase A₂ (PLA₂; 36.5%). Comparison of this venom profile with those of other Micrurus species suggests that it may represent a more balanced, 'intermediate' type within the dichotomy between 3FTx- and PLA₂-predominant venoms. M. clarki venom was strongly cross-recognized and, accordingly, efficiently neutralized by an equine therapeutic antivenom against M. nigrocinctus, revealing their high antigenic similarity. Lethal activity for mice could be reproduced by a PLA₂ venom fraction, but, unexpectedly, not by fractions corresponding to 3FTxs. The most abundant venom component, hereby named clarkitoxin-I, was identified as a short-chain (type I) 3FTx, devoid of lethal effect in mice, whose target remains to be defined. Its amino acid sequence of 66 residues shows high similarity with predicted sequences of venom gland transcripts described for M. fulvius, M. browni, and M. diastema.


Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication.
As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity.


Artificial intelligence in retina.
Major advances in diagnostic technologies are offering unprecedented insight into the condition of the retina and beyond ocular disease. Digital images providing millions of morphological datasets can fast and non-invasively be analyzed in a comprehensive manner using artificial intelligence (AI). Methods based on machine learning (ML) and particularly deep learning (DL) are able to identify, localize and quantify pathological features in almost every macular and retinal disease. Convolutional neural networks thereby mimic the path of the human brain for object recognition through learning of pathological features from training sets, supervised ML, or even extrapolation from patterns recognized independently, unsupervised ML. The methods of AI-based retinal analyses are diverse and differ widely in their applicability, interpretability and reliability in different datasets and diseases. Fully automated AI-based systems have recently been approved for screening of diabetic retinopathy (DR). The overall potential of ML/DL includes screening, diagnostic grading as well as guidance of therapy with automated detection of disease activity, recurrences, quantification of therapeutic effects and identification of relevant targets for novel therapeutic approaches. Prediction and prognostic conclusions further expand the potential benefit of AI in retina which will enable personalized health care as well as large scale management and will empower the ophthalmologist to provide high quality diagnosis/therapy and successfully deal with the complexity of 21st century ophthalmology.


Applying microscopy to the analysis of nuclear structure and function.
One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.


Arrhythmogenesis toxicity of aconitine is related to intracellular ca(2+) signals.
Aconitine is a well-known arrhythmogenic toxin and induces triggered activities through cardiac voltage-gated Na(+) channels. However, the effects of aconitine on intracellular Ca(2+) signals were previously unknown. We investigated the effects of aconitine on intracellular Ca(2+) signals in rat ventricular myocytes and explored the possible mechanism of arrhythmogenic toxicity induced by aconitine. Ca(2+) signals were evaluated by measuring L-type Ca(2+) currents, caffeine-induced Ca(2+) release and the expression of NCX and SERCA2a. Action potential and triggered activities were recorded by whole-cell patch-clamp techniques. In rat ventricular myocytes, the action potential duration was significantly prolonged by 1 µM aconitine. At higher concentrations (5 µM and 10 µM), aconitine induced triggered activities and delayed after-depolarizations (6 of 8 cases), which were inhibited by verapamil. Aconitine (1 µM) significantly increased the ICa-L density from 12.77 ± 3.12 pA/pF to 18.98 ± 3.89 pA/pF (n=10, p<0.01). The activation curve was shifted towards more negative potential, while the inactivation curve was shifted towards more positive potential by 1 μM aconitine. The level of Ca(2+) release induced by 10 mM caffeine was markedly increased. Aconitine (1 µM) increased the expression of NCX, while SERCA2a expression was reduced. In conclusion, aconitine increased the cytosolic [Ca(2+)]i by accelerating ICa-L and changing the expression of NCX and SERCA2a. Then, the elevation of cytosolic [Ca(2+)]i induced triggered activities and delayed after-depolarizations. Arrhythmogenesis toxicity of aconitine is related to intracellular Ca(2+) signals.


Vitamin D genes influence MS relapses in children.
The aim of this study was to determine whether a vitamin D genetic risk score (vitDGRS) is associated with 25-hydroxyvitamin D (25(OH)D) level and multiple sclerosis (MS) relapses in children. DNA samples were typed for single nucleotide polymorphisms (SNPs) from four genes previously identified to be associated with 25(OH)D levels. SNPs with strong associations with 25(OH)D after multiple comparison correction were used to create a genetic risk score (vitDGRS). Cox regression models tested associations of vitDGRS with relapse hazard. Two independent SNPs within or near GC and NADSYN1/DHCR7 genes were strongly associated with 25(OH)D levels in the discovery cohort (n = 182) after Bonferroni correction. The vitDGRS of these SNPs explained 4.5% of the variance of 25(OH)D level after adjustment for genetic ancestry. Having the highest versus lowest vitDGRS was associated with 11 ng/mL lower 25(OH)D level (95% confidence interval (CI) = -17.5, -4.5, p = 0.001) in the discovery cohort. Adjusting for ancestry, sex, disease-modifying therapy (DMT), and HLA-DRB1*15 carrier status, the highest versus lowest vitDGRS was associated with 2.6-fold (95% CI = 1.37, 5.03, p = 0.004) and 2.0-fold (95% CI = 0.75, 5.20, p = 0.16) higher relapse hazard in the discovery and replication cohorts, respectively. The vitDGRS identifies children at greater risk of relapse. These findings support a causal role for vitamin D in MS course.


EpiCO (epirubicin, cyclophosphamide and vincristine) as treatment for extrapulmonary high-grade neuroendocrine neoplasms.
High-grade neuroendocrine neoplasms (NEN) comprise a rare entity. Due to the lack of randomized controlled trials, therapy recommendations were mainly extrapolated from its pulmonary analogue, small cell lung cancer and mostly validated in small retrospective case series. The multicentric Nordic NEC Study of gastro-entero-pancreatic (GEP) and cancer of unknown primary (CUP) high-grade neuroendocrine neoplasms showed a significant disease control upon treatment with etoposide and platinum-based chemotherapies 1. Such a combination with etoposide and a platinum (CE) compound is currently considered standard first-line treatment for high-grade GEP/CUP NEN. High-grade mixed-neuroendocrine-non-neuroendocrine neoplasms (MiNEN) formerly termed mixed adeno-neuroendocrine carcinomas (MANEC) also have a poor prognosis and are generally treated like other high-grade NEN. The CE protocol has significant activity in high-grade NEN and MiNEN, but the response is short-lived in most cases with response rates around 50-60 %. Second-line treatment alternatives are not established so far. The need for additional treatment options is evident.Combination chemotherapy with doxorubicin, cyclophosphamide and vincristine (CAV) showed efficacy in small cell lung carcinoma (SCLC) and was considered standard first-line therapy before the era of etoposide and platinum combinations. Due to a better toxicity profile, doxorubicin was replaced by epirubicin, resulting in the combination of epirubicin, cyclophosphamide and vincristine (abbreviated as EpiCO or CEV).In analogy to SCLC, selected patients with high-grade NEN were treated with the EpiCO regimen in second line (or in one patient first line) at our center. In this report we present the retrospective series of 5 cases with metastatic high-grade GEP/CUP NEN/MiNEN who received chemotherapy according to this protocol.


Effects of chlorine level on the growth of biofilm in water pipes.
Three analogous laboratory-scale water pipe systems were constructed to study the effects of three chlorine levels of finished water on the growth of biofilm. The experimental results indicated that the heterotrophic plate counts (HPCs) of biofilm for chlorine-free water were approximately 2 and 3 orders of magnitude higher than those for low-chlorine water (0.3-0.5mg Cl2/L) and high-chlorine water (1.2-1.5mg Cl2/L). The difference in HPC levels between low-chlorine water and high-chlorine water was below 1 order. Average biofilm formation rates are 325 ATP pg/cm2-day for chlorine-free water, 159 ATP pg/cm2-day for low-chlorine water and 118 ATP pg/cm2-day for high-chlorine water. The scanning electron microscope (SEM) observation showed that bacterial species separated from the biofilm were more complex than from the finished water. The Gram-negative bacteria were dominant in these species.


A Comparison of Different Training Methods in the Successful Learning of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration.
Standardized training of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS- TBNA) is available only in highly developed countries. To compare the skill differences in the performance of EBUS-TBNA by pulmonologists trained under different learning methods. We performed a retrospective study of 145 EBUS-TBNA cases. We set up 3 comparative training groups: a standardized training group, a teaching group, and a self-study group. The main clinical outcomes were sensitivity, specificity and accuracy of EBUS-TBNA. Cumulative sum analysis was used to track EBUS-TBNA proficiency of the 3 groups. The sensitivities of EBUS-TBNA in the different groups were 90, 88, and 67%, respectively. The accuracies of the different groups were 91, 90, and 71%, respectively. With respect to the sensitivity and accuracy, there was no significant difference between the standardized training group and teaching group. However, the self-study group was inferior to the other 2 groups. By comparing the learning curve among the groups, we found that the minimum number of operations needed to reach a 90% accuracy for the standardized training group, the teaching group and the self-study group was 17, 32 and 40 cases, respectively. Compared with the teaching and self-study groups, the standardized training group resulted in practitioners achieving high skill levels following the performance of a lower number of cases. Pulmonologists trained in the teaching group achieved a similar level of skills, but required more experience to reach this level. Practitioners in the self-study group did not achieve a satisfactory level of skills, despite performing a high number of cases.


Patients with iron deficiency anemia have an increased prevalence of gallstones.
We determined the frequency of gallstones (GS) in iron deficiency anemia (IDA) patients and evaluated factors that could affect GS formation-like lipid levels and gallbladder (GB) motilities of the patients. One hundred and eleven IDA patients (88 females, 23 males; median age, 42) and 81 healthy controls (68 females, 13 males; median age, 42) were included into our study. The clinical findings of all IDA patients were recorded down; biochemical values and body mass index (BMI) were determined; and abdominal ultrasonography was performed. In addition, GB emptying was monitored by ultrasound at 30-min intervals for 2 h after a mixed meal in randomly chosen, age-matched 25 IDA patients and 26 controls. Fasting volume (FV), residual volume (RV), and ejection fraction (EF) for all GBs were determined. The frequency of GS plus cholecystectomy was significantly higher in IDA patients (15 cases, 13.5%) than in the control group (five cases, 6.2%, p = 0.048). IDA patients with GS plus cholecystectomy were older than those without GS plus cholecystectomy (p < 0.001). FV and EF did not differ between IDA and control groups (p > 0.05). On the other hand, RV was significantly higher in IDA group than in controls (p = 0.035). The frequency of GS in IDA patients was significantly higher than in controls. The increased prevalence of GS in IDA might be explained with impaired GB motility.


Ethnic differences in quality of life in persons with heart failure.
Chronic illness burdens some groups more than others. In studies of ethnic/racial groups with chronic illness, some investigators have found differences in health-related quality of life (HRQL), whereas others have not. Few such comparisons have been performed in persons with heart failure. The purpose of this study was to compare HRQL in non-Hispanic white, black, and Hispanic adults with heart failure. Data for this longitudinal comparative study were obtained from eight sites in the Southwest, Southeast, Northwest, Northeast, and Midwest United States. Enrollment and 3- and 6-month data on 1212 patients were used in this analysis. Propensity scores were used to adjust for sociodemographic and clinical differences among the ethnic/racial groups. Health-related quality of life was measured using the Minnesota Living with Heart Failure Questionnaire. Significant ethnic/racial effects were demonstrated, with more favorable Minnesota Living with Heart Failure Questionnaire total scores post-baseline for Hispanic patients compared with both black and white patients, even after adjusting for baseline scores, age, gender, education, severity of illness, and care setting (acute vs. chronic), and estimating the treatment effect (intervention vs. usual care). The models based on the physical and emotional subscale scores were similar, with post hoc comparisons indicating more positive outcomes for Hispanic patients than non-Hispanic white patients. Cultural differences in the interpretation of and response to chronic illness may explain why HRQL improves more over time in Hispanic patients with heart failure compared with white and black patients.


Anticoagulation Type and Early Recurrence in Cardioembolic Stroke: The IAC Study.
In patients with acute ischemic stroke and atrial fibrillation, treatment with low molecular weight heparin increases early hemorrhagic risk without reducing early recurrence, and there is limited data comparing warfarin to direct oral anticoagulant (DOAC) therapy. We aim to compare the effects of the treatments above on the risk of 90-day recurrent ischemic events and delayed symptomatic intracranial hemorrhage. We included consecutive patients with acute ischemic stroke and atrial fibrillation from the IAC (Initiation of Anticoagulation after Cardioembolic) stroke study pooling data from stroke registries of 8 comprehensive stroke centers across the United States. We compared recurrent ischemic events and delayed symptomatic intracranial hemorrhage between each of the following groups in separate Cox-regression analyses: (1) DOAC versus warfarin and (2) bridging with heparin/low molecular weight heparin versus no bridging, adjusting for pertinent confounders to test these associations. We identified 1289 patients who met the bridging versus no bridging analysis inclusion criteria and 1251 patients who met the DOAC versus warfarin analysis inclusion criteria. In adjusted Cox-regression models, bridging (versus no bridging) treatment was associated with a high risk of delayed symptomatic intracranial hemorrhage (hazard ratio, 2.74 [95% CI, 1.01-7.42]) but a similar rate of recurrent ischemic events (hazard ratio, 1.23 [95% CI, 0.63-2.40]). Furthermore, DOAC (versus warfarin) treatment was associated with a lower risk of recurrent ischemic events (hazard ratio, 0.51 [95% CI, 0.29-0.87]) but not delayed symptomatic intracranial hemorrhage (hazard ratio, 0.57 [95% CI, 0.22-1.48]). Our study suggests that patients with ischemic stroke and atrial fibrillation would benefit from the initiation of a DOAC without bridging therapy. Due to our study limitations, these findings should be interpreted with caution pending confirmation from large prospective studies.


Dual-wavelength operation diffractive phase elements for pattern formation.
We report on the design and fabrication of novel diffractive phase elements that reconstruct distinct intensity patterns in the far-field on illumination with two specific wavelengths. The elements contain deep surface-relief structures that represent phase-delays of greater than 2p radians. The design process incorporates a modified version of the iterative Fourier transform algorithm. A 16 phase-level element for dual wavelength (blue and red) operation, with high diffraction efficiency, is demonstrated experimentally.


Deep sequencing of the vaginal microbiota of women with HIV.
Women living with HIV and co-infected with bacterial vaginosis (BV) are at higher risk for transmitting HIV to a partner or newborn. It is poorly understood which bacterial communities constitute BV or the normal vaginal microbiota among this population and how the microbiota associated with BV responds to antibiotic treatment. The vaginal microbiota of 132 HIV positive Tanzanian women, including 39 who received metronidazole treatment for BV, were profiled using Illumina to sequence the V6 region of the 16S rRNA gene. Of note, Gardnerella vaginalis and Lactobacillus iners were detected in each sample constituting core members of the vaginal microbiota. Eight major clusters were detected with relatively uniform microbiota compositions. Two clusters dominated by L. iners or L. crispatus were strongly associated with a normal microbiota. The L. crispatus dominated microbiota were associated with low pH, but when L. crispatus was not present, a large fraction of L. iners was required to predict a low pH. Four clusters were strongly associated with BV, and were dominated by Prevotella bivia, Lachnospiraceae, or a mixture of different species. Metronidazole treatment reduced the microbial diversity and perturbed the BV-associated microbiota, but rarely resulted in the establishment of a lactobacilli-dominated microbiota. Illumina based microbial profiling enabled high though-put analyses of microbial samples at a high phylogenetic resolution. The vaginal microbiota among women living with HIV in Sub-Saharan Africa constitutes several profiles associated with a normal microbiota or BV. Recurrence of BV frequently constitutes a different BV-associated profile than before antibiotic treatment.


Possibility of an optical focal shift with polarization masks.
The polarization phase shift (PPS) has emerged as an important analytical tool in optical metrology. The present study utilizes the concept of controlling the polarization phase in applications such as focal shift and automatic focusing. When elliptically polarized light, in general, is incident upon a circularly symmetric polarization mask consisting of circular and annular zones with each zone having a unique linear polarizability, the polarization-phase difference introduced between the polarization-masked zones is also circularly symmetric. With the mask at the lens aperture, the polarization phase introduced is multiplicative with the lens function and is shown to result in a shift of the Gaussian focus plane. Because the polarization phase can be controlled by variation of the polarization parameters, the effective focal length of the imaging system can be varied within a small range. A study of the point-spread functions at the shifted focal planes has shown that the quality of the focal patch in these planes is comparable with that produced by a diffraction-limited imaging system at Gaussian focus. The shift of focus can be achieved by control of the polarization of the input beam. It is anticipated that this technique may find application in areas for which dynamic focusing within a small range is required.


Levels of evidence in research published in The Journal of Bone and Joint Surgery (American Volume) over the last thirty years.
The recent emphasis on evidence-based medicine has led to increasing levels of evidence being published in surgical journals. The purpose of the present study was to review the levels of evidence in reports published in The Journal of Bone and Joint Surgery (American Volume) over the last thirty years. We reviewed all of the articles published in The Journal in the years 1975, 1985, 1995, and 2005. Cadaver studies, animal studies, basic-science studies, review articles, Instructional Course Lectures, and correspondence were excluded. Articles were scored according to The Journal's levels of evidence for a primary research question. A total of 1058 articles were reviewed. Of these, 134, 123, 120, and 174 articles met the inclusion criteria for the years 1975, 1985, 1995, and 2005, respectively, and were ranked according to level of evidence. The number of articles for each level of evidence rating was then expressed as a percentage of the total number of articles meeting the inclusion criteria for that year. There was a significant trend toward higher levels of evidence, with the combined percentage of Level-I, II, and III studies increasing from 17% to 52% (p < 0.01). The percentage of Level-I studies increased from 4% in 1975 to 21% in 2005. The average level of evidence rating improved from 3.72 to 2.90 during the study period. The level of evidence in The Journal has improved significantly over the last thirty years.


Progression of trunk imbalance in adolescent idiopathic scoliosis with a thoracolumbar/lumbar curve: is it predictable at the initial visit?
OBJECTIVE Progression of trunk imbalance is an important finding during follow-up of patients with adolescent idiopathic scoliosis (AIS). Nevertheless, no factors that predict progression of trunk imbalance have been identified. The purpose of this study was to identify parameters that predict progression of trunk imbalance in cases of AIS with a structural thoracolumbar/lumbar (TL/L) curve. METHODS This study included 105 patients with AIS and a structural TL/L curve who were followed up at an outpatient clinic. Patients with trunk imbalance (trunk shift ≥ 20 mm) at the initial visit were excluded. All patients were followed up for more than 2 years. Patients were divided into the following groups according to progression of trunk imbalance: 1) Group P, trunk shift ≥ 20 mm at the final visit and degree of progression ≥ 10 mm; and 2) Group NP, trunk shift < 20 mm at the final visit or degree of progression < 10 mm. Radiological parameters included Cobb angle, upper end vertebrae and lower end vertebrae (LEV), LEV tilt, disc wedge angle between LEV and LEV+1, trunk shift, apical vertebral translation, and apical vertebral rotation (AVR). Each parameter was compared between groups. Radiological parameters were assessed at every visit using whole-spine standing anteroposterior radiographs. RESULTS Among the 105 patients examined, 13 showed trunk imbalance with progression ≥ 10 mm at the final visit (Group P). Multivariate logistic regression analysis identified a lower Risser grade (p = 0.002) and a greater initial AVR (p = 0.020) as predictors of progressive trunk imbalance. A change in LEV tilt during follow-up was associated with trunk imbalance (p = 0.001). CONCLUSIONS Risser grade and AVR measured at the initial visit may predict progression of trunk imbalance. Surgeons should consider the risk of progressive trunk imbalance if patients show skeletal immaturity and a greater AVR at the initial visit.


Phylogenetic and Evolutionary Analyses of the Frizzled Gene Family in Common Carp (Cyprinus carpio) Provide Insights into Gene Expansion from Whole-Genome Duplications.
In humans, the frizzled (FZD) gene family encodes 10 homologous proteins that commonly localize to the plasma membrane. Besides being associated with three main signaling pathways for cell development, most FZDs have different physiological effects and are major determinants in the development process of vertebrates and. Here, we identified and annotated the FZD genes in the whole-genome of common carp (Cyprinus carpio), a teleost fish, and determined their phylogenetic relationships to FZDs in other vertebrates. Our analyses revealed extensive gene duplications in the common carp that have led to the 26 FZD genes that we detected in the common carp genome. All 26 FZD genes were assigned orthology to the 10 FZD genes of on-land vertebrates, with none of genes being specific to the fish lineage. We postulated that the expansion of the FZD gene family in common carp was the result of an additional whole genome duplication event and that the FZD gene family in other teleosts has been lost in their evolution history with the reason that the functions of genes are redundant and conservation. Through the expression profiling of FZD genes in common carp, we speculate that the ancestral gene was likely capable of performing all functions and was expressed broadly, while some descendant duplicate genes only performed partial functions and were specifically expressed at certain stages of development.


Metabolic consequences of muscle disuse atrophy.
In response to decreased usage, skeletal muscle undergoes an adaptive reductive remodeling. This adaptive response has been found with disuse during human spaceflight, rat spaceflight, rat hind-limb unloading, bed rest, and aging. The reductive remodeling of skeletal muscle with disuse is largely independent of the reason for the disuse. The process involves more than a transition from slow to fast myosin fiber types. There are associated metabolic changes including a fuel shift toward glycolysis, decreased capacity for fat oxidation, and energy substrate accumulation in the atrophied muscles. Glycolysis is very effective for high-intensity short-duration acute activities, but if sustained output is needed, an energy profile where fat use is favored rather than compromised is desirable. For astronauts, there is a need to maintain as much functional capacity as possible during spaceflight for extravehicular activities. The shift toward increased activity of the glycolytic enzymes in atrophied muscle is accommodated by an increase in gluconeogenic capacity in the liver.


Diagnosis and treatment of primary sclerosing cholangitis.
PSC is a progressive chronic hepatobiliary disorder of unknown etiology for which no effective medical or surgical therapy now exists. This syndrome occurs most commonly in young men and is frequently associated with CUC. The diagnosis is best made utilizing endoscopic retrogradecopic retrograde cholangiography. Although liver histologic findings alone are infrequently diagnostic of PSC, it remains important to exclude other causes of chronic cholestasis. Although the etiology remains unknown, preliminary studies suggest that PSC is related to immunologic damage. Although viral infections can induce obliterative cholangitis in children, their role in the cause of PSC remains undefined. PSC progresses slowly from an asymptomatic stage to cirrhosis, portal hypertension, and premature death secondary to liver failure. Bile duct cancer appears to be a frequent complication of long-standing PSC. Since no therapy is of established efficacy, controlled clinical trials of both medical and surgical therapy should be encouraged. Fat-soluble vitamin deficiencies commonly occur in the advanced stages of PSC, and therefore serum levels of vitamins A, D, E, and prothrombin time should be monitored on a regular basis to prevent complications associated with these deficiencies. Liver transplantation is now a therapeutic option for the treatment of end-stage PSC. Palliative surgical biliary drainage procedures, proctocolectomy, and surgical decompressive shunts that increase the risk of liver transplants, therefore, should be avoided, if possible, in young PSC patients. If a total proctocolectomy is surgically indicated, we would strongly recommend performing a ileoanal pull-through procedure, thus, avoiding the formation of an abdominal ileostoma and the risk of developing bleeding peristomal varices.


Challenges and Opportunities in Data Visualization Education: A Call to Action.
This paper is a call to action for research and discussion on data visualization education. As visualization evolves and spreads through our professional and personal lives, we need to understand how to support and empower a broad and diverse community of learners in visualization. Data Visualization is a diverse and dynamic discipline that combines knowledge from different fields, is tailored to suit diverse audiences and contexts, and frequently incorporates tacit knowledge. This complex nature leads to a series of interrelated challenges for data visualization education. Driven by a lack of consolidated knowledge, overview, and orientation for visualization education, the 21 authors of this paper-educators and researchers in data visualization-identify and describe 19 challenges informed by our collective practical experience. We organize these challenges around seven themes People, Goals & Assessment, Environment, Motivation, Methods, Materials, and Change. Across these themes, we formulate 43 research questions to address these challenges. As part of our call to action, we then conclude with 5 cross-cutting opportunities and respective action items: embrace DIVERSITY+INCLUSION, build COMMUNITIES, conduct RESEARCH, act AGILE, and relish RESPONSIBILITY. We aim to inspire researchers, educators and learners to drive visualization education forward and discuss why, how, who and where we educate, as we learn to use visualization to address challenges across many scales and many domains in a rapidly changing world: viseducationchallenges.github.io.


Expression of Ets-1 in human clear cell renal cell carcinomas: implications for angiogenesis.
Expression of vascular endothelial growth factor (VEGF) has been reported in renal cell carcinoma (RCC), a highly angiogenic carcinoma. However, little or no information is available on the expression of Ets-1, which is one of the target molecules of VEGF. In the present study, we examined the expression of Ets-1 and VEGF in RCC by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), and correlations between expression and the microvessel density (MVD) were evaluated. Ets-1 was immunolocalized to carcinoma cells and endothelial cells of the microvessels in clear cell RCC, but not in papillary RCC. Immunohistochemical Ets-1 expression and MVD were significantly higher in clear cell RCC than in papillary RCC. Predominant mRNA expression of Ets-1 in clear cell RCC was confirmed by RT-PCR. The expression of Ets-1 correlated directly with MVD in clear cell RCC. Hypoxic treatment upregulated the mRNA expression of Ets-1 and VEGF in cell lines derived from clear cell RCC, suggesting that hypoxia is a key regulator for these molecules. These results demonstrate the expression of Ets-1 in human clear cell RCC and suggest the possibility that Ets-1 is involved in angiogenesis in clear cell RCC.


Optical transport, lifting and trapping of micro-particles by planar waveguides.
Optical waveguides can be used to trap and transport micro-particles. The particles are held close to the waveguide surface by the evanescent field and propelled forward. We propose a new technique to lift and trap particles above the surface of the waveguides. This is made possible by a gap between two opposing, planar waveguides. The field emitted from each of the waveguide ends diverge fast, away from the substrate and into the cover-medium. By combining two fields propagating at an angle upwards and coming from opposite sides of a gap, particles can be stably lifted and trapped at the crossing of the two fields. Thus, particles are transported by waveguides leading to a gap, where they are lifted away from the substrate and trapped. The experiments are supported by numerical simulations of the forces on the micro-particles. Fluorescence imaging is used to track the particles in 3D with a precision of 50 nm.


Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells.
Circulating endothelial progenitor cells (EPCs) have been reported previously. In this study, we examined the hypothesis that overexpression of vasculoprotective gene endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) in EPCs enhances their ability to inhibit neointimal hyperplasia. EPCs were isolated from rabbit peripheral blood, expanded in culture, and transduced with pseudotyped retroviral vectors expressing human eNOS (eNOS-EPCs), HO-1 (HO-1-EPCs), or green fluorescent protein (GFP-EPCs). Transduction efficiency of EPCs ex vivo was >90%. Four groups of rabbits (n=5 to 6 per group) were subjected to balloon angioplasty of the common carotid artery. Immediately after injury, approximately 5x10(6) autologous eNOS-EPCs or HO-1-EPCs were transplanted into the injured vessel. Control animals received an equivalent number of GFP-EPCs or Ringer's saline. Two weeks after transplantation, eNOS and HO-1 transgene transcripts and proteins were detected in the transduced rabbit vessels. Endothelialization was enhanced in the EPC-transplanted vessels independently of gene transfer. Neointimal thickening was significantly reduced in the GFP-EPC-treated vessels relative to the saline control. Neointima size was further reduced in vessels treated with eNOS-EPCs. Surprisingly, no additional reduction was seen in vessels treated with HO-1-EPCs relative to GFP-EPCs. Thrombosis occurred in approximately 50% of the saline-treated vessels but was virtually absent in all EPC-transplanted vessels. We conclude that transplantation of autologous EPCs overexpressing eNOS in injured vessels enhances the vasculoprotective properties of the reconstituted endothelium, leading to inhibition of neointimal hyperplasia. This cell-based gene therapy strategy may be useful in treatment of vascular disease.


Up-regulation of the Yersinia enterocolitica yop regulon by deletion of the flagellum master operon flhDC.
The Yop virulon enables extracellularly located Yersinia, in close contact with a eukaryotic target cell, to inject bacterial toxic proteins directly into the cytosol of this cell. Several Ysc proteins, forming the Yop secretion apparatus, display homology with proteins of the flagellar basal body. To determine whether this relationship could extend to the regulatory pathways, we analyzed the influence of flhDC, the master regulatory operon of the flagellum, on the yop regulon. In an flhDC mutant, the yop regulon was up-regulated. The transcription of virF and the steady-state level of the transcriptional activator VirF were enhanced. yop transcription was increased at 37 degrees C and could also be detected at a low temperature. Yop secretion was increased at 37 degrees C and occurred even at a low temperature. The Ysc secretion machinery was thus functional at room temperature in the absence of flagella, implying that in wild-type bacteria, FlhD and/or FlhC, or the product of a gene downstream of flhDC, represses the yop regulon. In agreement with this notion, increased expression of flhDC in wild-type bacteria resulted in the oversecretion of flagellins at room temperature and in decreased Yop secretion at 37 degrees C.


Selection signatures in worldwide sheep populations.
The diversity of populations in domestic species offers great opportunities to study genome response to selection. The recently published Sheep HapMap dataset is a great example of characterization of the world wide genetic diversity in sheep. In this study, we re-analyzed the Sheep HapMap dataset to identify selection signatures in worldwide sheep populations. Compared to previous analyses, we made use of statistical methods that (i) take account of the hierarchical structure of sheep populations, (ii) make use of linkage disequilibrium information and (iii) focus specifically on either recent or older selection signatures. We show that this allows pinpointing several new selection signatures in the sheep genome and distinguishing those related to modern breeding objectives and to earlier post-domestication constraints. The newly identified regions, together with the ones previously identified, reveal the extensive genome response to selection on morphology, color and adaptation to new environments.


Markers of skeletal muscle mitochondrial function and lipid accumulation are moderately associated with the homeostasis model assessment index of insulin resistance in obese men.
Lower skeletal muscle mitochondrial oxidative phosphorylation capacity (OXPHOS) and intramyocellular lipid (IMCL) accumulation have been implicated in the etiology of insulin resistance (IR) in obesity. The purpose of this study was to examine the impact of endurance exercise on biochemical and morphological measures of IMCL and mitochondrial content, and their relationship to IR in obese individuals. We examined mitochondrial content (subunit protein abundance and maximal activity of electron transport chain enzymes), IMCL/mitochondrial morphology in both subsarcolemmal (SS) and intermyofibrillar (IMF) regions by transmission electron microscopy, and intracellular lipid metabolites (diacylglycerol and ceramide) in vastus lateralis biopsies, as well as, the homeostasis model assessment index of IR (HOMA-IR) prior to and following twelve weeks of an endurance exercise regimen in healthy age- and physical activity-matched lean and obese men. Obese men did not show evidence of mitochondrial OXPHOS dysfunction, disproportionate IMCL content in sub-cellular regions, or diacylglycerol/ceramide accretion despite marked IR vs. lean controls. Endurance exercise increased OXPHOS and mitochondrial size and density, but not number of individual mitochondrial fragments, with moderate improvements in HOMA-IR. Exercise reduced SS IMCL content (size, number and density), increased IMF IMCL content, while increasing IMCL/mitochondrial juxtaposition in both regions. HOMA-IR was inversely associated with SS (r = -0.34; P = 0.051) and IMF mitochondrial density (r = -0.29; P = 0.096), IMF IMCL/mitochondrial juxtaposition (r = -0.30; P = 0.086), and COXII (r = -0.32; P = 0.095) and COXIV protein abundance (r = -0.35; P = 0.052); while positively associated with SS IMCL size (r = 0.28; P = 0.119) and SS IMCL density (r = 0.25; P = 0.152). Our findings suggest that once physical activity and cardiorespiratory fitness have been controlled for, skeletal muscle mitochondrial and IMCL profile in obesity may only partially contribute to the development of IR.


Dysregulated autoantibodies targeting vaso- and immunoregulatory receptors in Post COVID Syndrome correlate with symptom severity.
Most patients with Post COVID Syndrome (PCS) present with a plethora of symptoms without clear evidence of organ dysfunction. A subset of them fulfills diagnostic criteria of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Symptom severity of ME/CFS correlates with natural regulatory autoantibody (AAB) levels targeting several G-protein coupled receptors (GPCR). In this exploratory study, we analyzed serum AAB levels against vaso- and immunoregulatory receptors, mostly GPCRs, in 80 PCS patients following mild-to-moderate COVID-19, with 40 of them fulfilling diagnostic criteria of ME/CFS. Healthy seronegative (n=38) and asymptomatic post COVID-19 controls (n=40) were also included in the study as control groups. We found lower levels for various AABs in PCS compared to at least one control group, accompanied by alterations in the correlations among AABs. Classification using random forest indicated AABs targeting ADRB2, STAB1, and ADRA2A as the strongest classifiers (AABs stratifying patients according to disease outcomes) of post COVID-19 outcomes. Several AABs correlated with symptom severity in PCS groups. Remarkably, severity of fatigue and vasomotor symptoms were associated with ADRB2 AAB levels in PCS/ME/CFS patients. Our study identified dysregulation of AAB against various receptors involved in the autonomous nervous system (ANS), vaso-, and immunoregulation and their correlation with symptom severity, pointing to their role in the pathogenesis of PCS.


RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells.
A fraction of cancer cells maintain telomeres through the telomerase-independent, 'Alternative Lengthening of Telomeres' (ALT) pathway. ALT relies on homologous recombination (HR) between telomeric sequences; yet, what makes ALT telomeres recombinogenic remains unclear. Here we show that the RNA endonuclease RNaseH1 regulates the levels of RNA-DNA hybrids between telomeric DNA and the long noncoding RNA TERRA, and is a key mediator of telomere maintenance in ALT cells. RNaseH1 associated to telomeres specifically in ALT cells and its depletion led to telomeric hybrid accumulation, exposure of single-stranded telomeric DNA, activation of replication protein A at telomeres and abrupt telomere excision. Conversely, overexpression of RNaseH1 weakened the recombinogenic nature of ALT telomeres and led to telomere shortening. Altering cellular RNaseH1 levels did not perturb telomere homoeostasis in telomerase-positive cells. RNaseH1 maintains regulated levels of telomeric RNA-DNA hybrids at ALT telomeres to trigger HR without compromising telomere integrity too severely.


Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth.
Whether micro-organisms can live in periapical endodontic lesions of asymptomatic teeth is under debate. The aim of the present study was to visualize and identify micro-organisms within periapical lesions directly, using fluorescence in situ hybridization (FISH) in combination with epifluorescence and confocal laser scanning microscopy (CLSM). Thirty-nine periapical lesions were surgically removed, fixed, embedded in cold polymerizing resin and sectioned. The probe EUB 338, specific for the domain Bacteria, was used together with a number of species-specific 16S rRNA-directed oligonucleotide probes to identify bacteria. To control non-specific binding of EUB 338, probe NON 338 was used. Alternatively, DAPI (4',6'-diamidino-2-phenylindole) staining was applied to record prokaryotic and eukaryotic DNA in the specimens. Hybridization with NON 338 gave no signals despite background fluorescence of the tissue. The eubacterial probe showed bacteria of different morphotypes in 50 % of the lesions. Rods, spirochaetes and cocci were spread out in areas of the tissue while other parts seemed bacteria-free. Bacteria were also seen to co-aggregate inside the tissue, forming microcolonies. Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis and treponemes of phylogenetic Group I were detected with specific probes. In addition, colonies with Streptococcus spp. were seen in some lesions. A number of morphotypes occurred that could not be identified with the specific probes used, indicating the presence of additional bacterial species. CLSM confirmed that bacteria were located in different layers of the tissue. Accordingly, the FISH technique demonstrated mixed consortia of bacteria consisting of rods, spirochaetes and cocci in asymptomatic periapical lesions of root-filled teeth.


Normal tissue complications after radiation therapy.
This paper describes the biological mechanisms of normal tissue reactions after radiation therapy, with reference to conventional treatments, new treatments, and treatments in developing countries. It also describes biological reasons for the latency period before tissue complications arise, the relationship of dose to incidence, the effect of increasing the size of the irradiated volume, early and late tissue reactions, effects of changes in dose fractionation and dose rate, and combined chemotherapy and radiotherapy responses. Examples are given of increases in knowledge of clinical radiobiology from trials of new protocols. Potential modification to treatments include the use of biological response modifiers. The introduction of "response prediction" modifications to treatments might also be available in the near future. Finally, the paper points out that in some radiotherapy centers, the biologically-effective doses prescribed for combined brachytherapy and teletherapy treatment of cervix cancer are lower than those prescribed in other centers. This issue needs to be addressed further. The wealth of preclinical and clinical data has led to a much greater understanding of the biological basis to radiotherapy. This understanding has underpinned a variety of new approaches in radiotherapy, including both physical and biological strategies. There is also the important issue of treatment of a large number of cancers in developing countries, for which efficacious resource-sparing protocols are being continuously developed. A unified scoring system should be widely accepted as the new standard in reporting the adverse effects of radiation therapy. Likewise, late toxicity should be reported on an actuarial basis as a mandatory endpoint.


Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease.
Nonalcoholic fatty liver disease (NAFLD) is a major worldwide health problem. Recent studies suggest that the gut microbiota influences NAFLD pathogenesis. Here, a murine model of high-fat diet-induced (HFD-induced) NAFLD was used, and the effects of alterations in the gut microbiota on NAFLD were determined. Mice treated with antibiotics or tempol exhibited altered bile acid composition, with a notable increase in conjugated bile acid metabolites that inhibited intestinal farnesoid X receptor (FXR) signaling. Compared with control mice, animals with intestine-specific Fxr disruption had reduced hepatic triglyceride accumulation in response to a HFD. The decrease in hepatic triglyceride accumulation was mainly due to fewer circulating ceramides, which was in part the result of lower expression of ceramide synthesis genes. The reduction of ceramide levels in the ileum and serum in tempol- or antibiotic-treated mice fed a HFD resulted in downregulation of hepatic SREBP1C and decreased de novo lipogenesis. Administration of C16:0 ceramide to antibiotic-treated mice fed a HFD reversed hepatic steatosis. These studies demonstrate that inhibition of an intestinal FXR/ceramide axis mediates gut microbiota-associated NAFLD development, linking the microbiome, nuclear receptor signaling, and NAFLD. This work suggests that inhibition of intestinal FXR is a potential therapeutic target for NAFLD treatment.


Loss analysis for a two wire optical waveguide for chip-to-chip communication.
We propose an optical interconnect system for chip-to-chip communication using gold bond wires as a two wire waveguide. Here the loss of such a waveguide is determined for near-IR wavelengths, for different wire sizes and configurations, and show that we can achieve transmission loss coefficients less than 0.4 mm(-1) (1.7 dB/mm) making chip-to-chip optical communication possible using two-wire transmission lines made of standard gold bond wires. Such an optical waveguide scheme would greatly simplify inter-chip optical communication compared with existing waveguide concepts.


Apoptosis governs the elimination of Schistosoma japonicum from the non-permissive host Microtus fortis.
The reed vole, Microtus fortis, is the only known mammalian host in which schistosomes of Schistosoma japonicum are unable to mature and cause significant pathogenesis. However, little is known about how Schistosoma japonicum maturation (and, therefore, the development of schistosomiasis) is prevented in M. fortis. In the present study, the ultrastructure of 10 days post infection schistosomula from BALB/c mice and M. fortis were first compared using scanning electron microscopy and transmission electron microscopy. Electron microscopic investigations showed growth retardation and ultrastructural differences in the tegument and sub-tegumental tissues as well as in the parenchymal cells of schistosomula from M. fortis compared with those in BALB/c mice. Then, microarray analysis revealed significant differential expression between the schistosomula from the two rodents, with 3,293 down-regulated (by ≥ 2-fold) and 71 up-regulated (≥ 2 fold) genes in schistosomula from the former. The up-regulated genes included a proliferation-related gene encoding granulin (Grn) and tropomyosin. Genes that were down-regulated in schistosomula from M. fortis included apoptosis-inhibited genes encoding a baculoviral IAP repeat-containing protein (SjIAP) and cytokine-induced apoptosis inhibitor (SjCIAP), genes encoding molecules involved in insulin metabolism, long-chain fatty acid metabolism, signal transduction, the transforming growth factor (TGF) pathway, the Wnt pathway and in development. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and PI/Annexin V-FITC assays, caspase 3/7 activity analysis, and flow cytometry revealed that the percentages of early apoptotic and late apoptotic and/or necrotic cells, as well as the level of caspase activity, in schistosomula from M. fortis were all significantly higher than in those from BALB/c mice.


Metabolic control of type I (insulin dependent) diabetes after pancreas transplantation.
A study was conducted of the circadian hormonal and metabolic patterns of 10 type I (insulin dependent) uraemic diabetic patients after pancreas and renal transplantation. A single 24 hour profile was obtained in each patient following as closely as possible his or her normal daily routine two to 15 months after transplantation. None of the patients were using insulin at the time of the study. Compared with a group of six normal subjects the transplant recipients had mildly raised blood glucose concentrations, hyperinsulinaemia between meals and at night, delayed postprandial insulin peaks, mild hyperketonaemia, and normal blood lactate and plasma glucagon concentrations. The findings showed that successful pancreas transplantation results in disappearance of the need for insulin and return to normal or near normal of the metabolic abnormalities of diabetes. The minor differences observed in comparison with normal hormonal and metabolic homoeostasis were probably due to intrinsic (reduced islet mass, denervation, peripheral hormone delivery) and environmental (immunosuppression, relatively impaired renal function) factors.


Provider Perspectives on Use of Medical Marijuana in Children With Cancer.
Although medical marijuana (MM) may have utility in the supportive care of children with serious illness, it remains controversial. We investigated interdisciplinary provider perspectives on legal MM use in children with cancer. We sent a 32-item, cross-sectional survey to 654 pediatric oncology providers in Illinois, Massachusetts, and Washington characterizing MM practices, knowledge, attitudes, and barriers. Forty-eight percent responded; 44% (n = 288) were included in analyses. Providers were stratified by status as legally eligible to certify (ETC) for MM. We used Fisher's exact and Wilcoxon rank tests and univariate and multivariate logistic regression models for group comparisons. The provider median age was 35 years (range 22-70 years); 33% were ETC (83 physicians; 13 Washington state advance practice providers). Thirty percent of providers received ≥1 request for MM in the previous month. Notably, only 5% of all providers knew state-specific regulations. ETC providers were more likely to know that MM is against federal laws (P < .0001). Whereas most providers (92%) reported willingness to help children with cancer access MM, in adjusted models, ETC providers were less likely to indicate approval of patient MM use by smoking, oral formulations, as cancer-directed therapy, or to manage symptoms (P < .005 for all). Forty-six percent of all providers cited the absence of standards around formulations, potency, or dosing to be the greatest barrier to recommending MM. Most pediatric oncology providers are willing to consider MM use in children with cancer and receive frequent inquiries. However, ETC providers endorse less favorable attitudes overall. The absence of standards is an important barrier to recommending MM.


Comparison of analgesic efficacy and safety of continuous epidural infusion versus local infiltration and systemic opioids in video-assisted thoracoscopic surgery decortication in pediatric empyema patients.
The stripping of the densely innervated and inflamed parietal pleura in empyema during video-assisted thoracoscopic surgery (VATS) decortication can lead to significant pain and major postoperative respiratory compromise. Hence, we compared the analgesic efficacy of continuous epidural infusion versus local infiltration and systemic opioids in children undergoing VATS decortications. Following ethics approval and informed consent, forty patients from 1 to 12 years of age were randomized into two groups, Group E (epidural) and Group L (local infiltration) after induction of anesthesia. In Group E, a thoracic epidural catheter was inserted between T4 and T8. A bolus dose of 0.5 ml/kg of 0.25% injection bupivacaine was given epidurally before incision. Postoperatively, the patients received epidural infusion with bupivacaine and fentanyl up to 48 h using an elastomeric balloon pump. In Group L, patients received local infiltration of bupivacaine (2 mg/kg) and lignocaine (5 mg/kg) at the port sites before incision and at the end of surgery. They also received injection tramadol 1 mg/kg intravenously TDS with thrice daily postoperatively. The pain scores (Face, Legs, Activity, Cry, Consolability/ Wong-Baker FACES scale) were assessed every 4 h on the 1st day and 6 h on the 2nd day. Injection diclofenac 1 mg/kg intravenous was used as a rescue analgesic for pain scores more than 4. Side effects such as nausea, vomiting, constipation, and motor blockade were noted. Quantitative and categorical data were assessed using t-test and Chi-square test, respectively. The pain scores were lower in the epidural group than in the local infiltration group at 0, 4, and 20 h postoperatively (P = 0.001, 0.01, and 0.038, respectively). Seventeen out of nineteen patients required rescue analgesia in the local infiltration group in the postoperative period as compared to five patients in the epidural group with a P value of 0.000081. Epidural analgesia can be considered as an effective modality of reducing pain in patients undergoing VATS decortication for empyema in pediatric patients.


Freehand thoracic pedicle screw technique using a uniform entry point and sagittal trajectory for all levels: preliminary clinical experience.
Experience with freehand thoracic pedicle screw placement is well described in the literature. Published techniques rely on various starting points and trajectories for each level or segment of the thoracic spine. Furthermore, few studies provide specific guidance on sagittal and axial trajectories. The goal of this study was to propose a uniform entry point and sagittal trajectory for all thoracic levels during freehand pedicle screw placement and determine the accuracy of this technique. The authors retrospectively reviewed postoperative CT scans of 33 consecutive patients who underwent open, freehand thoracic pedicle-screw fixation using a uniform entry point and sagittal trajectory for all levels. The same entry point for each level was defined as a point 3 mm caudal to the junction of the transverse process and the lateral margin of the superior articulating process, and the sagittal trajectory was always orthogonal to the dorsal curvature of the spine at that level. The medial angulation (axial trajectory) was approximately 30° at T-1 and T-2, and 20° from T-3 to T-12. Breach was defined as greater than 25% of the screw diameter residing outside of the pedicle or vertebral body. A total of 219 thoracic pedicle screws were placed with a 96% accuracy rate. There were no medial breaches and 9 minor lateral breaches (4.1%). None of the screws had to be repositioned postoperatively, and there were no neurovascular complications associated with the breaches. It is feasible to place freehand thoracic pedicle screws using a uniform entry point and sagittal trajectory for all levels. The entry point does not have to be adjusted for each level as reported in existing studies, although this technique was not tested in severe scoliotic spines. While other techniques are effective and widely used, this particular method provides more specific parameters and may be easier to learn, teach, and adopt.


Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe.
The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers.


flexsurv: A Platform for Parametric Survival Modeling in R.
flexsurv is an R package for fully-parametric modeling of survival data. Any parametric time-to-event distribution may be fitted if the user supplies a probability density or hazard function, and ideally also their cumulative versions. Standard survival distributions are built in, including the three and four-parameter generalized gamma and F distributions. Any parameter of any distribution can be modeled as a linear or log-linear function of covariates. The package also includes the spline model of Royston and Parmar (2002), in which both baseline survival and covariate effects can be arbitrarily flexible parametric functions of time. The main model-fitting function, flexsurvreg, uses the familiar syntax of survreg from the standard survival package (Therneau 2016). Censoring or left-truncation are specified in 'Surv' objects. The models are fitted by maximizing the full log-likelihood, and estimates and confidence intervals for any function of the model parameters can be printed or plotted. flexsurv also provides functions for fitting and predicting from fully-parametric multi-state models, and connects with the mstate package (de Wreede, Fiocco, and Putter 2011). This article explains the methods and design principles of the package, giving several worked examples of its use.


Establishment of an innovative staging system for extramedullary plasmacytoma.
Extramedullary plasmacytoma (EMP) is a rare malignant disease that lacks a unique clinical staging system to predict the survival of EMP patients and to design individualized treatment. Instead, clinicians have chosen to use the multiple myeloma (MM) staging system. Forty-eight EMP patients treated between 1996 and 2014 were included in this study. The new clinical stages were established according to independent survival factors using Cox regression model. Lymph node metastasis and a larger primary tumor (≥5 cm) were the only two independent poor prognostic factors for overall survival (OS) and disease-free survival (P < 0.05). Stage I was defined as the disease without those two poor prognostic factors. Stage II was defined as the presence of either factor, and Stage III was defined as the presence of both factors. OS was significantly different in each stage of the new staging system (P < 0.001), with a median follow-up time for Stage I, Stage II and Stage III of 68, 23 and 14 months. The new staging system had enhanced prognostic value compared to the MM staging system (the area under ROC 0.763 versus 0.520, P = 0.044). Although no difference was observed between treatments in Stage I, the combination treatment was associated with a significantly beneficial OS in the late stages (5-year OS: 15.3 % versus 79.5 %; P = 0.032). The new staging system exhibited a promising prognostic value for survival and could aid clinicians in choosing the most suitable treatment for EMP patients.


Measuring Alliance Toward Embodied Virtual Therapists in the Era of Automated Treatments With the Virtual Therapist Alliance Scale (VTAS): Development and Psychometric Evaluation.
Automated virtual reality exposure therapies (VRETs) are self-help treatments conducted by oneself and supported by a virtual therapist embodied visually and/or with audio feedback. This simulates many of the nonspecific relational elements and common factors present in face-to-face therapy and may be a means of improving adherence to and efficacy of self-guided treatments. However, little is known about alliance toward the virtual therapist, despite alliance being an important predictor of treatment outcome. In this study, we aimed to evaluate the first alliance instrument developed for use with embodied virtual therapists in an automated treatment format-the Virtual Therapist Alliance Scale (VTAS)-by (1) assessing its psychometric properties, (2) verifying the dimensionality of the scale, and (3) determining the predictive ability of the scale with treatment outcome. A psychometric evaluation and exploratory factor analysis of the VTAS was conducted using data from two samples of spider-fearful patients treated with VRET and the help of an embodied, voice-based virtual therapist (n=70). Multiple regression models and bivariate correlations were used to assess the VTAS relationship with treatment outcome, according to self-reported fear and convergence with presence and user-friendliness process measures. The VTAS showed a sound two-factor solution composed of a primary factor covering task, goal, and copresence; adequate internal consistency; and good convergent validity, including moderate correlation (r=.310, P=.01) with outcomes over follow-up. These preliminary results suggest that alliance toward a virtual therapist is a significant predictor of treatment outcome, favors the importance of a task-goal over bond-factor, and should be explored in studies with larger sample sizes and in additional forms of embodiment.


Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii.
Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages.


Blood brain barrier precludes the cerebral arteries to intravenously-injected antisense oligonucleotide.
Alternative splicing of the ryanodine receptor subtype 3 (RyR3) produces a short isoform (RyR3S) able to negatively regulate the ryanodine receptor subtype 2 (RyR2), as shown in cultured smooth muscle cells from mice. The RyR2 subtype has a crucial role in the control of vascular reactivity via the fine tuning of Ca(2+) signaling to regulate cerebral vascular tone. In this study, we have shown that the inhibition of RyR3S expression by a specific antisense oligonucleotide (asRyR3S) was able to increase the Ca(2+) signals implicating RyR2 in cerebral arteries ex vivo. Moreover, we tried to inhibit the expression of RyR3S in vivo. The asRyR3S was complexed with JetPEI and injected intravenously coupled with several methods known to induce a blood brain barrier disruption. We tested solutions to induce osmotic choc (mannitol), inflammation (bacteria lipopolysaccharide and pertussis toxin), vasoconstriction or dilatation (sumatriptan, phenylephrine, histamine), CD73 activation (NECA) and lipid instability (Tween80). All tested technics failed to target asRyR3 in the cerebral arteries wall, whereas the molecule was included in hepatocytes or cardiomyocytes. Our results showed that the RyR3 alternative splicing could have a function in cerebral arteries ex vivo; however, the disruption of the blood brain barrier could not induce the internalization of antisense oligonucleotides in the cerebral arteries, in order to prove the function of RYR3 short isoform in vivo.


Vesicoureteric reflux in children.
This study aimed to identify the differences between primary and secondary vesicoureteric reflux (VUR) and the effect of associated bladder abnormalities on kidney function. We retrospectively reviewed the medical records of children with VUR who were followed up at King Abdulaziz University Hospital from January 2005 to December 2010. The review included results of radiological investigations and kidney function tests. We used Chi-square test for statistical analysis and paired t-test to compare group means for initial and last creatinine levels. Ninety-nine children were included in this study. Twenty (20.2%) had primary VUR, 11 had high-grade VUR, while 9 had low-grade reflux. All children with low-grade VUR had normal dimercaptosuccinic acid (DMSA). Renal scars were present in 72% of the children with high-grade VUR. The mean creatinine levels (initial and last) for both groups were normal. Seventy-nine (79.8%) children had secondary VUR, which was due to posterior urethral valves (PUV) (46.8%), neurogenic bladder caused by meningomyelocele (25.3%), non-neurogenic neurogenic bladder (NNB) (21.5%), or neurogenic bladder associated with prune belly syndrome (6.3%). Children with NNB, meningomyelocele and PUV had high creatinine at presentation with no considerable worsening of their kidney functions during the last visit. Renal scars were present in 49.4% of the children with secondary VUR. Children with primary VUR and normal bladder had good-functioning kidneys, while those with secondary VUR associated with abnormal bladder caused by NNB, spina bifida or PUV had abnormal kidney functions. DMSA scans were useful in predicting higher grades of VUR in children with primary reflux.


Dysregulation of p53/Sp1 control leads to DNA methyltransferase-1 overexpression in lung cancer.
Overexpression of DNA 5'-cytosine-methyltransferases (DNMT), which are enzymes that methylate the cytosine residue of CpGs, is involved in many cancers. However, the mechanism of DNMT overexpression remains unclear. Here, we showed that wild-type p53 negatively regulated DNMT1 expression by forming a complex with specificity protein 1 (Sp1) protein and chromatin modifiers on the DNMT1 promoter. However, the stoichiometry between p53 and Sp1 determined whether Sp1 acts as a transcription activator or corepressor. Low level of exogenous Sp1 enhanced the repressive activity of endogenous p53 on the DNMT1 promoter whereas high level of Sp1 upregulated DNMT1 gene expression level in A549 (p53 wild-type) cells. In H1299 (p53 null) cells, exogenous Sp1 induced DNMT1 expression in a dose-dependent manner. We also discovered a new mechanism whereby high level of Sp1, via its COOH-terminal domain, induced interaction between p53 and MDM2, resulting in degradation of p53 by MDM2-mediated ubiquitination. Clinical data from 102 lung cancer patients indicated that overexpression of DNMT1 was associated with p53 mutation (P = 0.014) and high expression of Sp1 protein (P = 0.006). In addition, patients with overexpression of both DNMT1 and Sp1 proteins showed poor prognosis (P = 0.037). Our cell and clinical data provided compelling evidence that deregulation of DNMT1 is associated with gain of transcriptional activation of Sp1 and/or loss of repression of p53. DNMT1 overexpression results in epigenetic alteration of multiple tumor suppressor genes and ultimately leads to lung tumorigenesis and poor prognosis.


Structural basis for the second step of group II intron splicing.
The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3' exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3' splice site within the catalytic core of the group II intron. The 3'-OH of the 5' exon is positioned in close proximity to the 3' splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having different configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3' splice site capture.


Antiviral Treatment against Monkeypox: A Scoping Review.
During the COVID-19 pandemic, the increase in reports of human monkeypox virus infection cases spreading in many countries outside Africa is a major cause for concern. Therefore, this study aimed to explore the evidence of antiviral pharmacotherapy available for the treatment of adult patients with monkeypox. A scoping review of the literature was conducted using PubMed, Scopus, Web of Science, Embase, and CENTRAL databases until 12 September 2022. The key search terms used were "monkeypox" and "treatment". A total of 1927 articles were retrieved using the search strategy. After removing duplicates (n = 1007) and examining by title, abstract, and full text, 11 studies reporting case reports of monkeypox with antiviral treatment were included, detailing the number of monkeypox cases, clinical manifestations, number of participants with antiviral treatment, history of sexually transmitted diseases, method of diagnosis, location of skin lesions, drugs used in antiviral treatment, route of administration, and outcome. A total of 1281 confirmed cases of monkeypox have been reported, of which 65 monkeypox cases had antiviral treatment distributed most frequently in the United States (n = 30), the United Kingdom (n = 6), and Spain (n = 6). Of the total cases, 1269 (99.1%) were male with an age range of 18 to 76 years, and 1226 (95.7%) had a sexual behavior of being men who have sex with men. All confirmed cases of monkeypox were diagnosed by reverse transcriptase polymerase chain reaction (RT-PCR). The most frequent clinical manifestations were skin lesions, fever, lymphadenopathy, headache, fatigue, and myalgia. The most frequent locations of the lesions were perianal, genital, facial, and upper and lower extremities. The most commonly used drugs for antiviral treatment of monkeypox were: tecovirimat, cidofovir, and brincidofovir. All patients had a complete recovery. According to current evidence, the efficacy and safety of antiviral drugs against monkeypox is of low quality and scarce.


Right parietal cortex mediates recognition memory for melodies.
Functional brain imaging studies have highlighted the significance of right-lateralized temporal, frontal and parietal brain areas for memory for melodies. The present study investigated the involvement of bilateral posterior parietal cortices (PPCs) for the recognition memory of melodies using transcranial direct current stimulation (tDCS). Participants performed a recognition task before and after tDCS. The task included an encoding phase (12 melodies), a retention period, as well as a recognition phase (24 melodies). Experiment 1 revealed that anodal tDCS over the right PPC led to a deterioration of overall memory performance compared with sham. Experiment 2 confirmed the results of Experiment 1 and further showed that anodal tDCS over the left PPC did not show a modulatory effect on memory task performance, indicating a right lateralization for musical memory. Furthermore, both experiments revealed that the decline in memory for melodies can be traced back to an interference of anodal stimulation on the recollection process (remember judgements) rather than to familiarity judgements. Taken together, this study revealed a causal involvement of the right PPC for memory for melodies and demonstrated a key role for this brain region in the recollection process of the memory task.


Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.
The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to β-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.