SH3 domains of the adapter molecule Grb2 complex with two proteins in T cells: the guanine nucleotide exchange protein Sos and a 75-kDa protein that is a substrate for T cell antigen receptor-activated tyrosine kinases.
In T lymphocytes activated via the T cell antigen receptor (TCR), the SH2- and SH3-containing adapter molecule Grb2 forms a complex with the Ras guanine nucleotide exchange protein Sos and tyrosine phospho-proteins. The interaction of Sos with Grb2 is mediated via the Grb2 SH3 domains. In this study, it is shown that a 75-kDa protein is also complexed with the Grb2 SH3 domains in T cells, but not in Rat-1 fibroblasts. The identity of the p75 protein is not known, but immunoblot analysis with phosphotyrosine antibodies indicated that it is rapidly tyrosine-phosphorylated in TCR-activated T cells. This characteristic clearly distinguishes p75 from Sos since Sos is not a phosphotyrosine protein. In vitro binding studies indicated that the p75 phosphotyrosine protein binds to a glutathione S-transferase fusion protein of intact Grb2, but not to a Grb2 fusion protein mutated in its SH3 domains. p75 can also bind to the single COOH-terminal Grb2 SH3 domain, whereas Sos has an in vitro binding preference for the NH2-terminal Grb2 SH3 domain. Collectively, these data indicate that in T cells, two proteins can complex with the Grb2 SH3 domains: Sos and a p75 molecule that is tyrosine-phosphorylated in TCR-activated cells. The significance of p75 association with Grb2 is not clear, but by analogy with Sos, p75 is a potential candidate for a Grb2 effector protein. Data are presented showing that the interaction of the Grb2 SH2 domains with tyrosine phosphoproteins may be regulated by conformational restraints imposed by different molecules complexing with the Grb2 SH3 domains. It is thus possible to speculate that the interaction of either p75 or Sos with the Grb2 SH3 domain may influence the interaction of the Grb2 SH2 domain with tyrosine phosphoproteins.


Performance Characteristics of Sepsis Screening Tools During Delivery Admissions.
To evaluate the screening performance characteristics of existing tools for the diagnosis of sepsis during delivery admissions. This was a case-control study using electronic health record data, including vital signs and laboratory results, for all delivery admissions of patients with sepsis from 59 nationally distributed hospitals. Patients with sepsis were matched by gestational age at delivery in a 1:4 ratio with patients without sepsis to create a comparison group. Patients with chorioamnionitis and sepsis were compared with a complete cohort of patients with chorioamnionitis without sepsis. Multiple screening criteria for sepsis were evaluated: the CMQCC (California Maternal Quality Care Collaborative), SIRS (Systemic Inflammatory Response Syndrome), the MEWC (the Maternal Early Warning Criteria), UKOSS (United Kingdom Obstetric Surveillance System), and the MEWT (Maternal Early Warning Trigger Tool). Sensitivity, false-positive rates, and C-statistics were reported for each screening tool. Analyses were stratified into cohort 1, which excluded patients with chorioamnionitis-endometritis, and cohort 2, which included those patients. Delivery admissions at 59 hospitals were extracted for patients with sepsis. Cohort 1 comprised 647 patients with sepsis, including 228 with end-organ injury, matched with a control group of 2,588 patients without sepsis. Cohort 2 comprised 14,591 patients with chorioamnionitis-endometritis, of whom 1,049 had sepsis and 238 had end-organ injury. In cohort 1, the CMQCC and the UKOSS pregnancy-adjusted criteria had the lowest false-positive rates (6.9% and 9.6%, respectively) and the highest C-statistics (0.92 and 0.91, respectively). Although other screening criteria, such as SIRS and the MEWC, had similar sensitivities, it was at the cost of much higher false-positive rates (21.3% and 38.3%, respectively). In cohort 2, including all patients with chorioamnionitis-endometritis, the highest C-statistics were again for the CMQCC (0.67) and UKOSS (0.64). All screening tools had high false-positive rates, but the false-positive rates for the CMQCC and UKOSS were substantially lower than those for SIRS and the MEWC. During delivery admissions, the CMQCC and UKOSS pregnancy-adjusted screening criteria have the lowest false-positive results while maintaining greater than 90% sensitivity rates. Performance of all screening tools was degraded in the setting of chorioamnionitis-endometritis.


Cognitive effects of transcranial direct current stimulation treatment in patients with major depressive disorder: An individual patient data meta-analysis of randomised, sham-controlled trials.
Transcranial direct current stimulation (tDCS) has emerged as a promising new treatment for major depression. While recent randomised, sham-controlled studies found tDCS to have antidepressant effects, it remains to be determined whether a tDCS treatment course may also enhance cognitive function independent of mood effects in depressed patients. This systematic review and individual patient data (IPD) meta-analysis examined cognitive outcomes from randomised, sham-controlled trials of tDCS treatment for major depression. Seven randomised, sham-controlled trials (n = 478 participants, 260 in active and 218 in sham) of tDCS for major depression were included. Results showed no cognitive enhancement after active tDCS compared to sham for the 12 cognitive outcomes investigated. Active relative to sham tDCS treatment was associated with reduced performance gains on a measure of processing speed (β = -0.33, 95% CI -0.58; -0.08, p = 0.011). Active tDCS treatment for depression did not show cognitive benefits independent of mood effects. Rather, tDCS treatment relative to sham stimulation for major depression may instead be associated with a reduced practice effect for processing speed.


Unraveling the molecular pathophysiology of myelodysplastic syndromes.
Somatically acquired genetic abnormalities lead to the salient features that define myelodysplastic syndromes (MDS): clonal hematopoiesis, aberrant differentiation, peripheral cytopenias, and risk of progression to acute myeloid leukemia. Although specific karyotypic abnormalities have been linked to MDS for decades, more recent findings have demonstrated the importance of mutations within individual genes, focal alterations that are not apparent by standard cytogenetics, and aberrant epigenetic regulation of gene expression. The spectrum of genetic abnormalities in MDS implicates a wide range of molecular mechanisms in the pathogenesis of these disorders, including activation of tyrosine kinase signaling, genomic instability, impaired differentiation, altered ribosome function, and changes in the bone marrow microenvironment. Specific alterations present in individual patients with MDS may explain much of the heterogeneity in clinical phenotype associated with this disease and can predict prognosis and response to therapy. Elucidation of the full complement of genetic causes of MDS promises profound insight into the biology of the disease, improved classification and prognostic scoring schemes, and the potential for novel targeted therapies with molecular predictors of response.


Temperature dependent radiative and non-radiative recombination dynamics in CdSe-CdTe and CdTe-CdSe type II hetero nanoplatelets.
We investigate the temperature-dependent decay kinetics of type II CdSe-CdTe and CdTe-CdSe core-lateral shell nanoplatelets. From a kinetic analysis of the photoluminescence (PL) decay and a measurement of the temperature dependent quantum yield we deduce the temperature dependence of the non-radiative and radiative lifetimes of hetero nanoplates. In line with the predictions of the giant oscillator strength effect in 2D we observe a strong increase of the radiative lifetime with temperature. This is attributed to an increase of the homogeneous transition linewidth with temperature. Comparing core only and hetero platelets we observe a significant prolongation of the radiative lifetime in type II platelets by two orders in magnitude while the quantum yield is barely affected. In a careful analysis of the PL decay transients we compare different recombination models, including electron hole pairs and exciton decay, being relevant for the applicability of those structures in photonic applications like solar cells or lasers. We conclude that the observed biexponential PL decay behavior in hetero platelets is predominately due to spatially indirect excitons being present at the hetero junction and not ionized e-h pair recombination.


Trends in hospital discharges, management and in-hospital mortality from acute myocardial infarction in Switzerland between 1998 and 2008.
Since the late nineties, no study has assessed the trends in management and in-hospital outcome of acute myocardial infarction (AMI) in Switzerland. Our objective was to fill this gap. Swiss hospital discharge database for years 1998 to 2008. AMI was defined as a primary discharge diagnosis code I21 according to the ICD10 classification. Invasive treatments and overall in-hospital mortality were assessed. Overall, 102,729 hospital discharges with a diagnosis of AMI were analyzed. The percentage of hospitalizations with a stay in an Intensive Care Unit decreased from 38.0% in 1998 to 36.2% in 2008 (p for trend < 0.001). Percutaneous revascularizations increased from 6.0% to 39.9% (p for trend < 0.001). Bare stents rose from 1.3% to 16.6% (p for trend < 0.001). Drug eluting stents appeared in 2004 and increased to 23.5% in 2008 (p for trend < 0.001). Coronary artery bypass graft increased from 1.0% to 3.0% (p for trend < 0.001). Circulatory assistance increased from 0.2% to 1.7% (p for trend < 0.001). Among patients managed in a single hospital (not transferred), seven-day and total in-hospital mortality decreased from 8.0% to 7.0% (p for trend < 0.01) and from 11.2% to 10.1%, respectively. These changes were no longer significant after multivariate adjustment for age, gender, region, revascularization procedures and transfer type. After multivariate adjustment, differing trends in revascularization procedures and in in-hospital mortality were found according to the geographical region considered. In Switzerland, a steep rise in hospital discharges and in revascularization procedures for AMI occurred between 1998 and 2008. The increase in revascularization procedures could explain the decrease in in-hospital mortality rates.


Safety and immunogenicity of the SARS-CoV-2 ARCoV mRNA vaccine in Chinese adults: a randomised, double-blind, placebo-controlled, phase 1 trial.
Safe and effective vaccines are urgently needed to end the COVID-19 pandemic caused by SARS-CoV-2 infection. We aimed to assess the preliminary safety, tolerability, and immunogenicity of an mRNA vaccine ARCoV, which encodes the SARS-CoV-2 spike protein receptor-binding domain (RBD). This single centre, double-blind, randomised, placebo-controlled, dose-escalation, phase 1 trial of ARCoV was conducted at Shulan (Hangzhou) hospital in Hangzhou, Zhejiang province, China. Healthy adults aged 18-59 years negative for SARS-CoV-2 infection were enrolled and randomly assigned using block randomisation to receive an intramuscular injection of vaccine or placebo. Vaccine doses were 5 μg, 10 μg, 15 μg, 20 μg, and 25 μg. The first six participants in each block were sentinels and along with the remaining 18 participants, were randomly assigned to groups (5:1). In block 1 sentinels were given the lowest vaccine dose and after a 4-day observation with confirmed safety analyses, the remaining 18 participants in the same dose group proceeded and sentinels in block 2 were given their first administration on a two-dose schedule, 28 days apart. All participants, investigators, and staff doing laboratory analyses were masked to treatment allocation. Humoral responses were assessed by measuring anti-SARS-CoV-2 RBD IgG using a standardised ELISA and neutralising antibodies using pseudovirus-based and live SARS-CoV-2 neutralisation assays. SARS-CoV-2 RBD-specific T-cell responses, including IFN-γ and IL-2 production, were assessed using an enzyme-linked immunospot (ELISpot) assay. The primary outcome for safety was incidence of adverse events or adverse reactions within 60 min, and at days 7, 14, and 28 after each vaccine dose. The secondary safety outcome was abnormal changes detected by laboratory tests at days 1, 4, 7, and 28 after each vaccine dose. For immunogenicity, the secondary outcome was humoral immune responses: titres of neutralising antibodies to live SARS-CoV-2, neutralising antibodies to pseudovirus, and RBD-specific IgG at baseline and 28 days after first vaccination and at days 7, 15, and 28 after second vaccination. The exploratory outcome was SARS-CoV-2-specific T-cell responses at 7 days after the first vaccination and at days 7 and 15 after the second vaccination. This trial is registered with www.chictr.org.cn (ChiCTR2000039212). Between Oct 30 and Dec 2, 2020, 230 individuals were screened and 120 eligible participants were randomly assigned to receive five-dose levels of ARCoV or a placebo (20 per group). All participants received the first vaccination and 118 received the second dose. No serious adverse events were reported within 56 days after vaccination and the majority of adverse events were mild or moderate. Fever was the most common systemic adverse reaction (one [5%] of 20 in the 5 μg group, 13 [65%] of 20 in the 10 μg group, 17 [85%] of 20 in the 15 μg group, 19 [95%] of 20 in the 20 μg group, 16 [100%] of 16 in the 25 μg group; p<0·0001). The incidence of grade 3 systemic adverse events were none (0%) of 20 in the 5 μg group, three (15%) of 20 in the 10 μg group, six (30%) of 20 in the 15 μg group, seven (35%) of 20 in the 20 μg group, five (31%) of 16 in the 25 μg group, and none (0%) of 20 in the placebo group (p=0·0013). As expected, the majority of fever resolved in the first 2 days after vaccination for all groups. The incidence of solicited systemic adverse events was similar after administration of ARCoV as a first or second vaccination. Humoral immune responses including anti-RBD IgG and neutralising antibodies increased significantly 7 days after the second dose and peaked between 14 and 28 days thereafter. Specific T-cell response peaked between 7 and 14 days after full vaccination. 15 μg induced the highest titre of neutralising antibodies, which was about twofold more than the antibody titre of convalescent patients with COVID-19. ARCoV was safe and well tolerated at all five doses. The acceptable safety profile, together with the induction of strong humoral and cellular immune responses, support further clinical testing of ARCoV at a large scale. National Key Research and Development Project of China, Academy of Medical Sciences China, National Natural Science Foundation China, and Chinese Academy of Medical Sciences.


Correlation analysis a tool for comparing relaxation-type models to experimental data.
We describe a new technique for comparing mathematical models to the biological systems that are described. This technique is appropriate for systems that produce relaxation oscillations or bursting oscillations, and takes advantage of noise that is inherent to all biological systems. Both types of oscillations are composed of active phases of activity followed by silent phases, repeating periodically. The presence of noise adds variability to the durations of the different phases. The central idea of the technique is that the active phase duration may be correlated with either/both the previous or next silent phase duration, and the resulting correlation pattern provides information about the dynamic structure of the system. Correlation patterns can easily be determined by making scatter plots and applying correlation analysis to the cluster of data points. This could be done both with experimental data and with model simulation data. If the model correlation pattern is in general agreement with the experimental data, then this adds support for the validity of the model. Otherwise, the model must be corrected. While this tool is only one test of many required to validate a mathematical model, it is easy to implement and is noninvasive.


Transfer between pose and illumination training in face recognition.
The relationship between pose and illumination learning in face recognition was examined in a yes-no recognition paradigm. The authors assessed whether pose training can transfer to a new illumination or vice versa. Results show that an extensive level of pose training through a face-name association task was able to generalize to a new illumination (Experiments 1 and 3), but an equal level of illumination training failed to generalize to a new pose (Experiment 2). The transfer of pose training was likely to depend on a relatively extensive level of training because the same faces with reduced level of exposure (Experiment 4) were unable to reproduce the transfer effect. The findings suggest that generalization of pose training may be extended to different types of image variation, whereas generalization of illumination training may be confined within the trained type of variation.


Selective expression of Syntaxin-7 protein in benign melanocytes and malignant melanoma.
To search for proteins expressed in human melanocytes and melanoma, we employed an antibody-based proteomics strategy to screen for protein expression in tissue microarrays containing normal tissues, cancer tissues and cell lines. Syntaxin-7 (STX7) was identified as a novel protein, not previously characterized in cells of melanocytic lineage, displaying a cell type-specific protein expression pattern. In tumor tissues, STX7 was expressed in malignant melanoma and lymphoma. The protein was further characterized regarding subcellular localization, specificity, tissue distribution pattern and potential as a diagnostic and prognostic marker using cell lines and tissue microarrays containing normal skin, melanocytic nevi and primary and metastatic melanoma. STX7 was expressed in normal melanocytes, various benign melanocytic nevi, atypical nevi and malignant melanoma. Analysis in two independent melanoma cohorts demonstrated STX7 expression in nearly all investigated tumors, although at varying levels (> 90% positive tumors). The expression level of STX7 protein was inversely correlated to tumor stage, suggesting that decreased expression of STX7 is associated with more aggressive tumors. In conclusion, we present protein profiling data for a novel protein showing high sensitivity and specificity for cells of the melanocytic lineage. The presented antibody-based proteomics approach can be used as an effective strategy to identify novel tumor markers and evaluate their potential clinical relevance.


The clinical and pathological phenotype of C9ORF72 hexanucleotide repeat expansions.
There is increasing evidence that frontotemporal dementia and amyotrophic lateral sclerosis are part of a disease continuum. Recently, a hexanucleotide repeat expansion in C9orf72 was identified as a major cause of both sporadic and familial frontotemporal dementia and amyotrophic lateral sclerosis. The aim of this study was to investigate clinical and neuropathological characteristics of hexanucleotide repeat expansions in C9orf72 in a large cohort of Dutch patients with frontotemporal dementia. Repeat expansions were successfully determined in a cohort of 353 patients with sporadic or familial frontotemporal dementia with or without amyotrophic lateral sclerosis, and 522 neurologically normal controls. Immunohistochemistry was performed in a series of 10 brains from patients carrying expanded repeats using a panel of antibodies. In addition, the presence of RNA containing GGGGCC repeats in paraffin-embedded sections of post-mortem brain tissue was investigated using fluorescence in situ hybridization with a locked nucleic acid probe targeting the GGGGCC repeat. Hexanucleotide repeat expansions in C9orf72 were found in 37 patients with familial (28.7%) and five with sporadic frontotemporal dementia (2.2%). The mean age at onset was 56.9 ± 8.3 years (range 39-76), and disease duration 7.6 ± 4.6 years (range 1-22). The clinical phenotype of these patients varied between the behavioural variant of frontotemporal dementia (n = 34) and primary progressive aphasia (n = 8), with concomitant amyotrophic lateral sclerosis in seven patients. Predominant temporal atrophy on neuroimaging was present in 13 of 32 patients. Pathological examination of the 10 brains from patients carrying expanded repeats revealed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein-positive inclusions of variable type, size and morphology in all brains. Fluorescence in situ hybridization analysis of brain material from patients with the repeat expansion, a microtubule-associated protein tau or a progranulin mutation, and controls did not show RNA-positive inclusions specific for brains with the GGGGCC repeat expansion. The hexanucleotide repeat expansion in C9orf72 is an important cause of frontotemporal dementia with and without amyotrophic lateral sclerosis, and is sometimes associated with primary progressive aphasia. Neuropathological hallmarks include neuronal and glial inclusions, and dystrophic neurites containing transactive response DNA binding protein. Future studies are needed to explain the wide variation in clinical presentation.


Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex.
Proteolysis of mitotic cyclins depends on a multisubunit ubiquitin-protein ligase, the anaphase promoting complex (APC). Proteolysis commences during anaphase, persisting throughout G1 until it is terminated by cyclin-dependent kinases (CDKs) as cells enter S phase. Proteolysis of mitotic cyclins in yeast was shown to require association of the APC with the substrate-specific activator Hct1 (also called Cdh1). Phosphorylation of Hct1 by CDKs blocked the Hct1-APC interaction. The mutual inhibition between APC and CDKs explains how cells suppress mitotic CDK activity during G1 and then establish a period with elevated kinase activity from S phase until anaphase.


Health and social characteristics and children's cognitive functioning: results from a national cohort.
The purpose of this study was to examine the associations between cognitive functioning in children and sociodemographic, family, and health characteristics. Data from phase 1 of the third National Health and Nutrition Examination Survey were used to evaluate performance on standardized cognitive tests in a representative sample of 2531 children 6 to 16 years of age. Multivariate analyses were used to assess independent associations between covariates and test performance. Lower income, minority status, and lower education of an adult reference person (one of the persons in the household who owned or rented the home) were independently associated with poorer performance on all cognitive subtests. To a lesser degree, general health status, history of birth complications, and sex also were independent predictors of performance for some of the subtests. These findings illustrate disparities in cognitive functioning across sociodemographic and health characteristics of children in the US population. They suggest the need for public health policies to take a multifaceted approach to optimizing childhood environments in order to overcome the effects of socioeconomic and minority status.


When Ligand Exchange Leads to Ion Exchange: Nanocrystal Facets Dictate the Outcome.
This study demonstrates that ligand exchange of nanocrystals (NCs) is not always an innocuous process, but can lead to facile (room temperature) ion exchange, depending on the surface crystal faceting. Rock salt PbTe NCs prepared as cubes with neutral facets undergo room-temperature ligand exchange with sulfide ions, whereas cuboctahedron-shaped particles with neutral {100} and polar {111} facets are transformed to PbS, driven by ion exchange along the ⟨111⟩ direction. Likewise, cation exchange (with Ag+) occurs rapidly for cuboctahedra, whereas cubes remain inert. This dramatic difference is attributed to the relative surface area of {111} facets that promote rapid ion exchange and shows how facet engineering is a powerful knob for the control of reaction pathways in nanoparticles.


Mitochondrial genome of Ciona savignyi (Urochordata, Ascidiacea, Enterogona): comparison of gene arrangement and tRNA genes with Halocynthia roretzi mitochondrial genome.
The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNAGly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNAMet genes; anticodon CAT and anticodon TAT. The tRNACys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.


The contribution of parental and grandparental childhood social disadvantage to circulatory disease diagnosis in young Swedish men.
Men born out of wedlock in early twentieth century Sweden who never married have previously been shown to have a doubled mortality risk from ischaemic heart disease compared to the corresponding group of men born to married parents. This study further explores the question of childhood social disadvantage and its long-term consequences for cardiovascular health by examining the two subsequent generations. The question posed is whether the sons and grandsons of men and women born out of wedlock in early twentieth century Sweden have an increased risk of circulatory disease compared with the corresponding descendants of those born inside marriage. We examined this by use of military conscription data. The material used is the Uppsala Birth Cohort Multigenerational database consisting of individuals born at Uppsala University Hospital between 1915 and 1929 (UG1), their children (UG2) and grandchildren (UG3). Conscription data were available for UG2s born between 1950 and 1982 (n=5,231) and UG3s born between 1953 and 1985 (n=10,074) corresponding to 72.1% and 73.6%, respectively, of all males born in each time-period. Logistic regression showed that significant excess risk of circulatory disease diagnoses was present only among descendants of men born outside marriage, with sons and grandsons demonstrating odds ratios of 1.64 and 1.83, respectively, when BMI and height at the time of conscription, father's social class in mid-life and father's or grandfather's history of circulatory disease had been adjusted for. Separate analyses showed that the effect of the maternal and paternal grandfather was of approximately the same magnitude. Further analyses revealed an interaction between the father's social class and the grandfather's legitimacy status at birth on UG3-men's likelihood of having a circulatory disease, with elevated odds only among those whose fathers were either manual workers or self-employed. The results of this study suggest that social disadvantage in one generation can be linked to health disadvantage in the subsequent two generations.


Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates.
Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.


Increases in intracellular calcium perturb blood-brain barrier via protein kinase C-alpha and apoptosis.
An increase in intracellular calcium represents one of the early events during an ischaemic stroke. It triggers many downstream processes which promote the formation of brain oedema, the leading cause of death after an ischaemic stroke. As impairment of blood-brain barrier (BBB) accounts for much of oedema formation, the current study explored the impact of intracellular calcium on barrier integrity in relation to protein kinase C, caspase-3/7, plasminogen activators and the pro-oxidant enzyme NADPH oxidase. Human brain microvascular endothelial cells alone or in co-culture with human astrocytes were subjected to 4h of oxygen-glucose deprivation alone or followed by 20h of reperfusion (OGD±R) in the absence or presence of inhibitors for urokinase plasminogen activator (amiloride), NADPH oxidase (apocynin), intracellular calcium (BAPTA-AM) and protein kinase C-α (RO-32-0432). Endothelial cells with protein kinase C-α knockdown, achieved by siRNA, were also exposed to the above conditions. BBB permeability was measured by transendothelial electrical resistance and Evan's blue-albumin and sodium fluorescein flux. Intracellular calcium and total superoxide anion levels, caspase-3/7, NADPH oxidase, plasminogen activator and protein kinase C activities, stress fibre formation, the rate of apoptosis and BBB permeability were increased by OGD±R. Treatment with the specific inhibitors or knockdown of protein kinase C-α attenuated them. This study reveals successive increases in intracellular calcium levels and protein kinase C-α activity are key mechanisms in OGD±R-mediated impairment of BBB. Furthermore inhibition of protein kinase C-α may be therapeutic in restoring BBB function by reducing the rate of cytoskeletal reorganisation, oxidative stress and apoptosis.


Dissecting nematode resistance regions in soybean revealed pleiotropic effect of soybean cyst and reniform nematode resistance genes.
Reniform nematode (RN, Rotylenchulus reniformis Linford & Oliveira) has emerged as one of the most important plant parasitic nematodes of soybean [Glycine max (L.) Merr.]. Planting resistant varieties is the most effective strategy for nematode management. The objective of this study was to identify quantitative trait loci (QTL) for RN resistance in an exotic soybean line, PI 438489B, using two linkage maps constructed from the Universal Soybean Linkage Panel (USLP 1.0) and next-generation whole-genome resequencing (WGRS) technology. Two QTL controlling RN resistance were identified-the soybean cyst nematode (SCN, Heterodera glycines) resistance gene GmSNAP18 at the rhg1 locus and its paralog GmSNAP11. Strong association between resistant phenotype and haplotypes of the GmSNAP11 and GmSNAP18 was observed. The results indicated that GmSNAP11 possibly could have epistatic effect on GmSNAP18, or vice versa, with the presence of a significant correlation in RN resistance of rhg1-a GmSNAP18 vs. rhg1-b GmSNAP18. Most importantly, our preliminary data suggested that GmSNAP18 and GmSNAP11 proteins physically interact in planta, suggesting that they belong to the same pathway for resistance. Unlike GmSNAP18, no indication of GmSNAP11 copy number variation was found. Moreover, gene-based single nucleotide polymorphism (SNP) markers were developed for rapid detection of RN or SCN resistance at these loci. Our analysis substantiates synergic interaction between GmSNAP11 and GmSNAP18 genes and confirms their roles in RN as well as SCN resistance. These results could contribute to a better understanding of evolution and subfunctionalization of genes conferring resistance to multiple nematode species and provide a framework for further investigations.


Relaxation of arterial smooth muscle by calcium sparks.
Local increases in intracellular calcium ion concentration ([Ca2+]i) resulting from activation of the ryanodine-sensitive calcium-release channel in the sarcoplasmic reticulum (SR) of smooth muscle cause arterial dilation. Ryanodine-sensitive, spontaneous local increases in [Ca2+]i (Ca2+ sparks) from the SR were observed just under the surface membrane of single smooth muscle cells from myogenic cerebral arteries. Ryanodine and thapsigargin inhibited Ca2+ sparks and Ca(2+)-dependent potassium (KCa) currents, suggesting that Ca2+ sparks activate KCa channels. Furthermore, KCa channels activated by Ca2+ sparks appeared to hyperpolarize and dilate pressurized myogenic arteries because ryanodine and thapsigargin depolarized and constricted these arteries to an extent similar to that produced by blockers of KCa channels. Ca2+ sparks indirectly cause vasodilation through activation of KCa channels, but have little direct effect on spatially averaged [Ca2+]i, which regulates contraction.


Community-based rehabilitation (CBR) monitoring and evaluation methods and tools: a literature review.
To identify and analyse tools and methods that have been reported in the literature for the monitoring and evaluation of community-based rehabilitation (CBR) programmes. A literature review and descriptive analysis were carried out to scope CBR monitoring and evaluation methods and tools. A search was conducted using PubMed and Google Scholar databases, hand searches and reference lists. Reports were retrieved, screened and information was extracted and analysed against research questions. There were 34 reports which met the inclusion criteria. Analysis of the 34 reports showed that most reports used demographic and programme data. A range of methods were used: interviews, focus groups and questionnaires being the most common. Apart from this, no common standardised procedures or tools were identified and there was not a standard approach to the inclusion of people with disabilities or other CBR stakeholders. The findings suggest that there would be value in creating resources such as guidelines, common processes and checklists for monitoring and evaluation of CBR, to facilitate efficient and comparable practices and more comparable data. This needs to be done in partnership with people with disabilities, CBR providers, partners and researchers to ensure that all stakeholders' needs are understood and met. Implications for Rehabilitation While there is broad scope and complexity of CBR programmes, there needs to be consistency and a valid approach in the monitoring and evaluation methods and tools used by CBR programmes. The principles of CBR and CRPD require that monitoring and evaluation involve people with disabilities, CBR managers and staff not only as informants but also in the design and execution of monitoring and evaluation activities. The consistent use of appropriate and valid monitoring and evaluation methods and tools will contribute to developing a stronger evidence base on the efficacy and effectiveness of CBR.


Relationship between early-life nutrition and ages at menarche and first pregnancy, and childbirth rates of young adults: Evidence from APCAPS in India.
India's Integrated Child Development Services (ICDS) provides daily supplementary nutrition and other public health services to women and children. We estimated associations between exposure to early-childhood ICDS nutrition and adult reproductive outcomes. During 1987-1990, a balanced protein-calorie supplement called "upma"-made from locally available corn-soya ingredients-was rolled out by subdistricts near Hyderabad and offered to pregnant women and children under age 6 years. In a controlled trial, 15 villages received the supplement and 14 did not. We used data from a 2010-2012 resurvey of adults born during the trial (n = 715 in intervention and n = 645 in control arms). We used propensity score matching methods to estimate the associations between birth in an intervention village and menarcheal age, age at first pregnancy, and fertility of adults. We found that women born in the intervention group during the trial, as compared with the control group, had menarche 0.45 (95% confidence interval [CI: 0.22, 0.68]; p < .001) years later and first pregnancy 0.53 (95% CI [0.04, 1.02]; p < .05) years later. Married women from the intervention group had menarche 0.36 (95% CI [0.09, 0.64]; p < .01) years later, first cohabitation with partner 0.8 (95% CI [0.27, 1.33]; p < .01) years later, and first pregnancy 0.53 (95% CI [0.04, 1.02]; p < .05) years later than married women in the control group. There was no significant difference between intervention and control group women regarding whether they had at least one childbirth or the total number of children born. The findings were similar when we employed inverse propensity score weighted regression models.


Progress in matrix metalloproteinase research.
Matrix metalloproteinases (MMPs) are now acknowledged as key players in the regulation of both cell-cell and cell-extracellular matrix interactions. They are involved in modifying matrix structure, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis. They play central roles in morphogenesis, wound healing, tissue repair and remodelling in response to injury and in the progression of diseases such as arthritis, cancer and cardiovascular disease. Because of their wide spectrum of activities and expression sites, the elucidation of their potential as drug targets in disease or as important features of the repair process will be dependent upon careful analysis of their role in different cellular locations and at different disease stages. Novel approaches to the specific regulation of individual MMPs in different contexts are also being developed.


Physicians' stated trade-off preferences for chronic hepatitis B treatment outcomes in Germany, France, Spain, Turkey, and Italy.
To quantify physicians' preferences among possible outcomes associated with chronic hepatitis B treatments and to determine which outcomes are most important to physicians in making treatment decisions. Physicians in five countries who treat chronic hepatitis B patients completed a web-enabled, choice-format, conjoint-analysis survey. The survey presented physicians with four treatment-choice questions for three different patient types. Each treatment-choice question included a pair of hypothetical medication profiles. Medication outcomes included how long the medication has been studied (weight of evidence); the probability that a patient's viral load remains undetectable for 5 years, with a possible histological improvement or reversal of disease progression (long-term efficacy); the 5-year treatment-related risk of fracture; the 5-year treatment-related risk of renal dysfunction; and patient cost. Treatment-choice questions were derived from a predetermined experimental design with known statistical properties. For each country, the random-parameters logit was used to estimate preference weights for all outcome levels and the mean relative importance of each outcome. Long-term efficacy and risk of renal dysfunction were the most important outcomes for the 788 physicians completing the survey, whereas weight of evidence was the least important. However, physicians perceived significant differences in weight of evidence timeframes. Physicians in Germany and France ranked efficacy above side-effect risk, whereas physicians in Spain, Italy, and Turkey ranked side-effect risk above efficacy in importance. Physician preferences among treatment profiles indicate systematic differences in the relative importance of treatment outcomes. Physicians require higher efficacy for treatments with higher side-effect risk but somewhat less efficacy for treatments with longer evidence.


Plasmodium falciparum asparagine and aspartate rich protein 2 is an evolutionary conserved protein whose repeats identify a new family of parasite antigens.
We describe here a new Plasmodium falciparum antigen, asparagine and aspartate rich protein 2 (PfAARP2) of 150 kDa, which is encoded by a unique gene on chromosome 1. PfAARP2 is first expressed 12 h post-invasion and accumulates in trophozoites and schizonts. Immunofluorescence studies indicate that PfAARP2 is translocated into the red blood cell cytoplasm. The central region of Pfaarp2 contains blocks of repetitions encoding asparagine and aspartate residues, which define a new family of related genes dispersed on different chromosomes, and two members of this family have also been identified. Interestingly, the non-repeated N- and C-termini of PfAARP2 display significant similarity to two yeast and human predicted proteins, and its possible function is discussed.


Coupling of the 99mtechnetium-nitrido group to monoclonal antibody and use of the complexes for the detection of tumors in mice.
The in vivo detection of tumors by immunoscintigraphy with the use of radiolabeled monoclonal antibodies (MoAb) is a new diagnostic procedure currently undergoing clinical evaluation. In the present study the use of 99mtechnetium (99mTc) for this purpose was explored. A simple method for the labeling of microgram quantities of MoAb with 99mTc based on the substitution reaction of MoAb and tetrachloronitridotechnetate ion (99mTcNCl4-) is described. The selective activity of the 99mtechnetium-nitrido-MoAb (99mTcN-MoAb) complexes was proved in vitro by a binding assay with different target cells. The 99mTcN-MoAb complexes were shown to bind reactive cells up to 20 times more avidly than nonreactive cells. The specificity of the 99mTcN-MoAb complexes was shown in vivo. (C57BL/6 X BALB/c)F1 mice bearing palpable tumors (0.3-1.5 cm in diameter) were given an iv injection of 1 of 2 MoAb (one reactive and the other nonreactive) identically labeled with 99mTcNCl4- and then scanned with a gamma camera, and/or the tissues were removed and the localization of 99mTc-nitrido group-labeled MoAb was measured. Tumor localization of the reactive MoAb (1.8-2.2% of the injected dose) was four times greater than that of the nonreactive 99mTcN-MoAb (0.3-0.4% of the injected dose). The localization of specific 99mTcN-MoAb to a murine thymoma was observed in the gamma camera image at just 2 hours after injection. At 27 hours, tumors could readily be detected by 99mTcN-MoAb without the need for background subtraction. Nonreactive 99mTcN-MoAb did not image the tumors. The use of 99mTcN-MoAb offers substantial improvement over radioiodinated (125I or 131I) MoAb for the detection of tumors. The use of 99mTcNCl4- as a labeling agent results in 99mTc-labeled MoAb with high specific activity and specificity when compared with the specific activity and specificity of the 99mTc-MoAb prepared by using the conventional SnCl2 reduction of pertechnetate.


Effect of caffeine on upper-body anaerobic performance in wrestlers in simulated competition-day conditions.
Peak power (PP) and mean power (MP) attained in upper body sprint performance test are considered important factors for competitive success in wrestling. This study aimed to determine whether acute caffeine ingestion would better maintain PP and MP across a simulated competition day in wrestling. In a double-blind, counterbalanced, crossover study, 14 trained wrestlers ingested either placebo or 5 mg/kg caffeine and completed four 6-min upper body intermittent sprint performance tests with 30-min recovery periods between consecutive tests. PP and MP were recorded during and blood lactate concentration was measured before and after each test. Ratings of perceived fatigue (RPF) and exertion (RPE) were recorded before and after each test, respectively. Heart rate (HR) was monitored across the whole testing period. Mean power decreased across four tests in both trials (p < .05), but the reduction in PP (from 277.2 ± 34.6 W to 257.3 ± 45.1 W; p < .05) only occurred in caffeine trial. Both pretest blood lactate concentration and HR were higher in caffeine than in placebo trial (p < .05) in the third and fourth tests. No between-trial differences occurred in RPF or RPE. Under simulated competition day conditions mimicking four consecutive wrestling matches, acute caffeine ingestion has a partially detrimental effect on upper body intermittent sprint performance in trained wrestlers. Elevated HR and blood lactate levels observed between tests after caffeine ingestion suggest that caffeine may impair recovery between consecutive maximal efforts.


The role of feedback in learning a vernier discrimination task.
We compare improvement through training in vernier acuity under different feedback conditions in order to clarify the role of feedback during learning of a perceptual task and to test different (neural network) models of perceptual learning. Improvement of performance is measured in 49 observers under feedback, no feedback, uncorrelated feedback, partial feedback, and block feedback conditions. Correct feedback conditions yield a larger improvement of performance than manipulated and no feedback conditions. Providing feedback that is uncorrelated to the observers' responses prevents learning, while the effect of block feedback does not differ significantly from complete feedback. Our results cannot be explained by learning rules that depend exclusively on an external teacher or by models that propose learning in an exposure-dependent way with unsupervised learning rules but without top-down influences.


Reconstitution studies of amino acid transport system L in rat erythrocytes.
In many cell types, including human erythrocytes, membrane transport of hydrophobic amino acids such as leucine and phenylalanine is mediated primarily by Na(+)-independent system L. In this paper we demonstrate that erythrocytes from the rat have a 400-fold higher system L transport capacity than human erythrocytes. We have exploited this high transport activity to achieve the first successful reconstitution of an erythrocyte amino acid transporter into phospholipid vesicles. Rat erythrocyte membranes were depleted of extrinsic membrane proteins, solubilized in 50 mM n-octyl glucoside and reconstituted into egg-yolk phospholipid vesicles by a gel filtration freeze-thaw protocol. Optimal reconstitution of transport activity occurred at lipid/protein ratios of 25-35:1. At a lipid/protein ratio of 25:1, one-half of the total uptake of L-[14C]leucine (0.2 mM, 25 degrees C) was inhibited by 2 mM phloretin and thus judged to be carrier-mediated. This component of L-leucine uptake was inhibited by non-radioactive L-phenylalanine and L-leucine, and only to a very much weaker extent by glycine and L-alanine. Two other inhibitors of system L in intact cells, MK196 and PCMBS (p-chloromercuriphenylsulphonate), were also effective inhibitors of phloretin-sensitive L-leucine transport in reconstituted proteoliposomes. Phloretin-insensitive uptake of L-leucine in proteoliposomes occurred by simple diffusion across the lipid bilayer.


A proposed time-resolved X-ray scattering approach to track local and global conformational changes in membrane transport proteins.
Time-resolved X-ray scattering has emerged as a powerful technique for studying the rapid structural dynamics of small molecules in solution. Membrane-protein-catalyzed transport processes frequently couple large-scale conformational changes of the transporter with local structural changes perturbing the uptake and release of the transported substrate. Using light-driven halide ion transport catalyzed by halorhodopsin as a model system, we combine molecular dynamics simulations with X-ray scattering calculations to demonstrate how small-molecule time-resolved X-ray scattering can be extended to the study of membrane transport processes. In particular, by introducing strongly scattering atoms to label specific positions within the protein and substrate, the technique of time-resolved wide-angle X-ray scattering can reveal both local and global conformational changes. This approach simultaneously enables the direct visualization of global rearrangements and substrate movement, crucial concepts that underpin the alternating access paradigm for membrane transport proteins.


A neural-specific GAP-43 core promoter located between unusual DNA elements that interact to regulate its activity.
In an effort to identify cis-acting elements that respond to signals controlling different stages of neural differentiation, we have analyzed the promoter and surrounding regulatory sequences of the rat GAP-43 gene. Expression of this gene is both neural specific and, within neurons, strongly modulated by signals related to axon integrity. Expression analysis in cell lines and primary rat cortical cultures demonstrates that neural-selective gene expression can be directed by a 386 base pair GAP-43 promoter fragment that contains canonical TATA and CCAAT box consensus sequences. A short region of homology with other neural-specific genes, identified upstream of the core promoter, is not essential for selective expression in neuronal cells. Within cortical cell cultures, expression is strongly modulated by two interacting elements on either side of the promoter, each of which contains a sequence with the potential to adopt an unusual DNA conformation. While each of these flanking elements reduces expression when added alone to the core promoter, each counteracts the negative influence of the other when both elements are present.


Cardiovascular Autonomic Neuropathy and Cardiovascular Outcomes in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) Study.
To examine whether cardiovascular autonomic neuropathy (CAN) is an independent risk factor of cardiovascular disease (CVD) events during DCCT/EDIC. Standardized cardiovascular autonomic reflex tests (R-R response to paced breathing, Valsalva maneuver, postural changes in blood pressure) were performed at DCCT baseline, every 2 years throughout DCCT, and at two time points in EDIC. CVD events were ascertained throughout the study and adjudicated by a review committee. Cox proportional hazards models were used to estimate the effect of CAN at DCCT closeout on subsequent CVD risk. There were 299 adjudicated CVD events in 165 participants following the DCCT closeout assessment: 132 of 1,262 subjects (10%) without CAN at DCCT closeout who experienced 244 CVD events versus 33 of 131 subjects (25%) with CAN at DCCT closeout who experienced 55 events (hazard ratio 2.79, 95% CI 1.91-4.09 for time to first CVD event). The cumulative incidence of the first occurrence of any CVD event during EDIC was significantly higher in participants with CAN at DCCT closeout compared with those without CAN. The association remained marginally significant after adjustment for multiple risk factors, including the EDIC updated mean HbA1c. When analyzed as a continuous variable, R-R variation was significantly lower at DCCT closeout in participants who experienced a CVD event compared with those who did not (P = 0.0012). In the DCCT/EDIC cohort, individuals diagnosed with CAN at DCCT closeout experienced a higher long-term risk of CVD events during follow-up in EDIC. This association was not independent of historic glycemic exposure and its metabolic memory effect, the principal determinant of both long-term CVD risk and CAN in type 1 diabetes.


Factors Associated With Short-term Relapse in Patients With Pemphigus Who Receive Rituximab as First-line Therapy: A Post Hoc Analysis of a Randomized Clinical Trial.
Rituximab and short-term corticosteroid therapy are the criterion standard treatments for patients with newly diagnosed moderate to severe pemphigus. To examine factors associated with short-term relapse in patients with pemphigus treated with rituximab. This post hoc analysis of a randomized clinical trial (Comparison Between Rituximab Treatment and Oral Corticosteroid Treatment in Patients With Pemphigus [RITUX 3]) conducted from January 1, 2010, to December 31, 2015, included patients from 20 dermatology departments of tertiary care centers in France from the RITUX 3 trial and 3 newly diagnosed patients treated according to the trial protocol. Data analysis was performed from February 1 to June 30, 2019. Patients randomly assigned to the rituximab group in the RITUX 3 trial and the 3 additional patients were treated with 1000 mg of intravenous rituximab on days 0 and 14 and 500 mg at months 12 and 18 combined with a short-term prednisone regimen. Baseline (pretreatment) clinical and biological characteristics (Pemphigus Disease Area Index [PDAI] score, ranging from 0-250 points, with higher values indicating more severe disease) and changes in anti-desmoglein (DSG) 1 and anti-DSG3 values as measured by enzyme-linked immunosorbent assay during the 3 months after rituximab treatment were compared between patients with disease relapse and those who maintained clinical remission during the first 12 months after treatment. The positive and negative predictive values of these factors were calculated. Among 47 patients (mean [SD] age, 54.3 [17.0] years; 17 [36%] male and 30 [64%] female) included in the study, the mean (SD) baseline PDAI score for patients with relapsing disease was higher than that of the patients with nonrelapsing disease (54 [33] vs 28 [24]; P = .03). At month 3, 7 of 11 patients with relapsing disease (64%) vs 7 of 36 patients with nonrelapsing disease (19%) had persistent anti-DSG1 antibody values of 20 IU/mL or higher and/or anti-DSG3 antibody values of 130 IU/mL or higher (P = .01). A PDAI score of 45 or higher defining severe pemphigus and/or persistent anti-DSG1 antibody values of 20 IU/mL or higher and/or anti-DSG3 antibody values of 130 IU/mL or higher at month 3 provided a positive predictive value of 50% (95% CI, 27%-73%) and a negative predictive value of 94% (95% CI, 73%-100%) for the occurrence of relapse after rituximab. The findings suggest that initial PDAI score and changes in anti-DSG antibody values after the initial cycle of rituximab might help differentiate a subgroup of patients with high risk of relapse who might benefit from maintenance rituximab infusion at month 6 from a subgroup of patients with low risk of relapse who do not need early maintenance therapy. NCT00784589.


General practitioners' perceptions, attitudes and experiences of frailty and frailty screening.
General practitioners (GPs) are uniquely positioned to support frailty identification and management. However, awareness of frailty and its treatment remains an emergent concept for many. Consequently, our aim was to explore GPs' perceptions, attitudes and experiences of frailty and frailty screening. A qualitative focus group study was conducted with 22 South Australian GPs. GPs were recruited through a combination of purposive, convenience and snowball sampling. Data were analysed using a thematic analysis approach. GPs saw frailty as a cycle of worsening decline punctuated by experience of negative outcomes. Participants largely felt that they already knew who their frail patients were without the need for formal screening. Consequently, there was varied support for formal screening, largely dependent on its intended purpose. Few GPs had actively intervened to prevent the onset or progression of frailty, with most strategies aimed at stabilisation and management. This study suggests that Australian GPs may be open to a proactive approach to frailty assessment and treatment, given appropriate training and resources.


TAMkin: a versatile package for vibrational analysis and chemical kinetics.
TAMkin is a program for the calculation and analysis of normal modes, thermochemical properties and chemical reaction rates. At present, the output from the frequently applied software programs ADF, CHARMM, CPMD, CP2K, Gaussian, Q-Chem, and VASP can be analyzed. The normal-mode analysis can be performed using a broad variety of advanced models, including the standard full Hessian, the Mobile Block Hessian, the Partial Hessian Vibrational approach, the Vibrational Subsystem Analysis with or without mass matrix correction, the Elastic Network Model, and other combinations. TAMkin is readily extensible because of its modular structure. Chemical kinetics of unimolecular and bimolecular reactions can be analyzed in a straightforward way using conventional transition state theory, including tunneling corrections and internal rotor refinements. A sensitivity analysis can also be performed, providing important insight into the theoretical error margins on the kinetic parameters. Two extensive examples demonstrate the capabilities of TAMkin: the conformational change of the biological system adenylate kinase is studied, as well as the reaction kinetics of the addition of ethene to the ethyl radical. The important feature of batch processing large amounts of data is highlighted by performing an extended level of theory study, which TAMkin can automate significantly.


Phosphorylation of LSD1 by PKCα is crucial for circadian rhythmicity and phase resetting.
The circadian clock is a self-sustaining oscillator that controls daily rhythms. For the proper circadian gene expression, dynamic changes in chromatin structure are important. Although chromatin modifiers have been shown to play a role in circadian gene expression, the in vivo role of circadian signal-modulated chromatin modifiers at an organism level remains to be elucidated. Here, we provide evidence that the lysine-specific demethylase 1 (LSD1) is phosphorylated by protein kinase Cα (PKCα) in a circadian manner and the phosphorylated LSD1 forms a complex with CLOCK:BMAL1 to facilitate E-box-mediated transcriptional activation. Knockin mice bearing phosphorylation-defective Lsd1(SA/SA) alleles exhibited altered circadian rhythms in locomotor behavior with attenuation of rhythmic expression of core clock genes and impaired phase resetting of circadian clock. These data demonstrate that LSD1 is a key component of the molecular circadian oscillator, which plays a pivotal role in rhythmicity and phase resetting of the circadian clock.


Is there enough evidence to support hip capsular reconstruction? A systematic review of biomechanical studies.
The aim of this study was to review and summarize the available biomechanical data on hip capsular reconstruction to guide clinical decision-making. A literature search was completed in December 2020 using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to identify biomechanical cadaver studies on hip capsular reconstruction, hip capsulectomy or hip capsular defect. The investigated parameters included maximum distraction force, capsular state affecting range of motion (ROM), rotation and translation. Four studies met al. the inclusion-exclusion criteria. The median effective force for resisting maximum distraction for the reconstruction state, capsular defect state and the intact state was 171, 111 and 206 N, respectively. The defect capsule force was significantly lower (P = 0.00438) than the intact capsule force. The reconstruction state had a higher distraction force than that of the capsular defect, but due to heterogeneity, the overall effect size was not statistically significant. The capsular reconstruction state reduced excess motion and the degree of instability compared to the capsular defect state but restored the hip close to its native capsular state in the cadaveric model. When compared to capsulectomy/defect state, hip capsular reconstruction significantly improved the rotational stability and effective force at maximum distraction and minimized translation. However, no conclusions can be made regarding the most effective protocol due to the high heterogeneity between the four studies. Further biomechanical studies are needed to test various types of grafts under the same protocol.


Polymorphisms in the 5' flanking region of IL12RB2 are associated with susceptibility to periodontal diseases in the Japanese population.
The expression of interleukin (IL)-12Rbeta2 molecule is a crucial regulatory factor in the T-helper type (Th) 1 differentiation of T cells. To elucidate the role of the cell-mediated immune (CMI) response in the pathogenesis of periodontitis, Japanese periodontal patients were subjected to single nucleotide polymorphism (SNP) analyses of the 5' flanking region of IL12RB2, whose variants are frequently detected in lepromatous leprosy patients, in which the very weak cellular immune response is caused by low expression of IL-12Rbeta2. The gene polymorphisms of the 5' flanking region of IL12RB2 were examined in subjects with several types of periodontal disease and in healthy controls. Serum immunoglobulin (Ig) G antibody titres against periodontopathic bacteria were measured and compared in periodontal patients with and without variant alleles of IL12RB2. The frequencies of variant alleles of IL12RB2 were significantly higher in aggressive periodontitis patients as compared with healthy controls or chronic periodontitis patients. Serum IgG titres against all periodontal bacteria examined in subjects carrying variant alleles were higher than those in subjects without variant alleles. IL-12Rbeta2 SNPs could be useful as genetic markers to access the susceptibility of the general population to periodontal disease. Low CMI responses or high humoral responses are associated with the pathogenesis of inflammatory periodontal diseases.


Infected Nonhealing Wound in a Kidney Transplant Recipient: Successful Treatment With Topical Homologous Platelet-Rich Gel.
Impaired would healing is a known adverse effect of chronic immunosuppression. Solid-organ transplant recipients undergoing major abdominal surgery have an increased risk of wound-related complications compared with the general population. In this subset of patients, surgical site infections and wound dehiscence must be aggressively treated to avoid sepsis, graft loss, and death. Recently, topical application of platelet-rich plasma has been proposed as an alternative therapeutic option to enhance wound healing in difficult cases. Unfortunately, randomized controlled trials evaluating the efficacy of platelet-rich plasma compared with standard or advanced wound management are lacking, and the literature mostly refers to anecdotal reports in patients with no evidence of wound infection. This report documents a kidney transplant recipient who experienced spontaneous bladder rupture because of gangrenous cystitis. After an exploratory laparotomy and bladder repair, the patient developed a deep surgical site infection by multidrug resistant Acinetobacter baumannii and extensive wound dehiscence. Advanced wound management and vacuum-assisted closure therapy were ineffective. Topical homologous platelet-rich gel was used resulting in significant wound healing, without infections or immunologic complications.


Prognostic implications of left ventricular global longitudinal strain in heart failure patients with narrow QRS complex treated with cardiac resynchronization therapy: a subanalysis of the randomized EchoCRT trial.
Left ventricular (LV) global longitudinal strain (GLS) reflects LV systolic function and correlates inversely with the extent of LV myocardial scar and fibrosis. The present subanalysis of the Echocardiography Guided CRT trial investigated the prognostic value of LV GLS in patients with narrow QRS complex. Left ventricular (LV) global longitudinal strain (GLS) was measured on the apical 2-, 4- and 3-chamber views using speckle tracking analysis. Measurement of baseline LV GLS was feasible in 755 patients (374 with cardiac resynchronization therapy (CRT)-ON and 381 with CRT-OFF). The median value of LV GLS in the overall population was 7.9%, interquartile range 6.2-10.1%. After a mean follow-up period of 19.4 months, 95 patients in the CRT-OFF group and 111 in the CRT-ON group reached the combined primary endpoint of all-cause mortality and heart failure hospitalization. Each 1% absolute unit decrease in LV GLS was independently associated with 11% increase in the risk to reach the primary endpoint (Hazard ratio 1.11; 95% confidence interval 95% 1.04-1.17, P < 0.001), after adjusting for ischaemic cardiomyopathy and randomization treatment among other clinically relevant variables. When categorizing patients according to quartiles of LV GLS, the primary endpoint occurred more frequently in patients in the lowest quartile (<6.2%) treated with CRT-ON vs. CRT-OFF (45.6% vs. 28.7%, P = 0.009) whereas, no differences were observed in patients with LV GLS ≥6.2% treated with CRT-OFF vs. CRT-ON (23.7% vs. 24.5%, respectively; P = 0.62). Low LV GLS is associated with poor outcome in heart failure patients with QRS width <130 ms, independent of randomization to CRT or not. Importantly, in the group of patients with the lowest LV GLS quartile, CRT may have a detrimental effect on clinical outcomes.


Adaptive cognitive testing in cerebrovascular disease and vascular dementia.
To examine whether brevity can be combined with precision in measuring global cognitive ability in patients with cerebrovascular disease (CVD) or vascular dementia (VaD). Longer tests (e.g. the CAMCOG) are precise but inefficient, whereas brief tests (e.g. the MMSE) are efficient but imprecise. A simulated computerized adaptive testing (CAT) algorithm using existing CAMCOG data from 284 patients with CVD of whom 55 were diagnosed with VaD. CAT was used to estimate each individual patient's total score on a large precise test (the CAMCOG). CAT repeatedly selected only items of appropriate difficulty, depending on whether the previous item was (in)correctly responded to. CAT estimates were compared with total scores on the whole CAMCOG. Even though there was an average test reduction of more than 40%, CAT estimates were in very high agreement with the whole test results (intraclass correlation >0.97) and had similar accuracy for the diagnosis of dementia (area under the curve = 0.94). CAT combines efficiency with precision in the measurement of global cognitive ability in CVD patients.


Hormone replacement therapy and ischemic stroke severity in women: a case-control study.
To investigate whether ischemic stroke severity differed among women who were receiving hormone replacement therapy (HRT) as compared with those who were not receiving these drugs. Estrogen has a neuroprotective effect in animal models of ischemic stroke, but data reflecting the impact of HRT on ischemic stroke severity in humans are lacking. All women receiving HRT at the time of admission for acute ischemic stroke to an academic medical center over 3 years were identified by medical record review (n = 58). HRT users were matched with 116 HRT nonusers by age and number of stroke risk factors. Stroke severity was assessed retrospectively with the Canadian Neurological SCALE: Data were analyzed with nonparametric univariate tests (Spearman rank and chi(2) tests) and linear regression modeling using nonparametric matched-pair analysis. History of congestive heart failure or coronary artery disease (p = 0.01), atrial fibrillation (p = 0.02), and African American race (p = 0.04), were significantly associated with greater stroke severity in the univariate analysis. There was a nonsignificant trend toward lesser stroke severity in HRT users (median Canadian Neurological Scale score, 10, vs 9.5 in non-HRT users, p = 0.08). Multivariate analysis showed no independent effect of HRT use on stroke severity (F = 1.24, p = 0.17). There was no significant effect of HRT status on stroke severity. Because this was a retrospective analysis, prospective studies are also needed to further elucidate any potential neuroprotective effect of hormone replacement.


Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.
We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry. Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation. A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells. These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segregation, metabolic processes, and stress responses. Additionally, the known enzymes, including E1, E2, and E3 of the sumoylation cascade were found to be auto-sumoylated. Taken together, these results show that protein sumoylation is broadly involved in many cellular functions and this mass spectrometry-based proteomic approach is useful in studying the regulation of protein sumoylation in the cells.


Performance of Pugs, German Shepherds, and Greyhounds (Canis lupus familiaris) on an odor-discrimination task.
Public opinion and the scientific literature alike reflect a widespread assumption that there are differences in behavior between dog breeds. Direct empirical behavioral assessments of such differences, however, are rare and have produced mixed results. One area where breed differences are often assumed is olfaction, where German Shepherds, hounds, and Labradors are commonly used for odor-detection work, whereas toy breeds and brachycephalic dogs, such as Pugs, are not. Choice of breed for scent detection work, however, may be driven more by historical choices than data. In this article we directly assessed the ability of German Shepherds, Pugs, and Greyhounds to acquire a simple olfactory discrimination, and their ability to maintain performance when the target odorant was diluted. Our results show that contrary to expectations, Pugs significantly outperformed the German Shepherds in acquiring the odor discrimination and maintaining performance when the odorant concentration was decreased. Nine of 10 Greyhounds did not complete acquisition training because they failed a motivation criterion. These results indicate that Pugs outperformed German Shepherds in the dimensions of olfaction assessed. Greyhounds showed a general failure to participate. Overall, our results highlight the importance of direct behavioral measurement of assumed behavioral breed differences.


Interaction between cytokines and ammonia in the mitochondrial permeability transition in cultured astrocytes.
Hepatic encephalopathy (HE) is the major neurological complication occurring in patients with acute and chronic liver failure. Elevated levels of blood and brain ammonia are characteristic of HE, and astrocytes are the primary target of ammonia toxicity. In addition to ammonia, recent studies suggest that inflammation and associated cytokines (CKs) also contribute to the pathogenesis of HE. It was previously established that ammonia induces the mitochondrial permeability transition (mPT) in cultured astrocytes. As CKs have been shown to cause mitochondrial dysfunction in other conditions, we examined whether CKs induce the mPT in cultured astrocytes. Cultures treated with tumor necrosis factor-α, interleukin-1β, interleukin-6, and interferon-γ, individually or in a mixture, resulted in the induction of the mPT in a time-dependent manner. Simultaneous treatment of cultures with a mixture of CKs and ammonia showed a marked additive effect on the mPT. As oxidative stress (OS) is known to induce the mPT, so we examined the effect of CKs and ammonia on hemeoxygenase-1 (HO-1) protein expression, a marker of OS. Such treatment displayed a synergistic effect in the upregulation of HO-1. Antioxidants significantly blocked the additive effects on the mPT by CKs and ammonia, suggesting that OS represents a major mechanism in the induction of the mPT. Treatment of cultures with minocycline, an antiinflammatory agent, which is known to inhibit OS, also diminished the additive effects on the mPT caused by CKs and ammonia. Induction of the mPT in astrocytes appears to represent a major pathogenetic factor in HE, in which CKs and ammonia are critically involved.


A chromosomal Agrobacterium tumefaciens gene required for effective plant signal transduction.
The vir gene products of Agrobacterium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.


Integration of genetic and physical maps of the Primula vulgaris S locus and localization by chromosome in situ hybridization.
Heteromorphic flower development in Primula is controlled by the S locus. The S locus genes, which control anther position, pistil length and pollen size in pin and thrum flowers, have not yet been characterized. We have integrated S-linked genes, marker sequences and mutant phenotypes to create a map of the P. vulgaris S locus region that will facilitate the identification of key S locus genes. We have generated, sequenced and annotated BAC sequences spanning the S locus, and identified its chromosomal location. We have employed a combination of classical genetics and three-point crosses with molecular genetic analysis of recombinants to generate the map. We have characterized this region by Illumina sequencing and bioinformatic analysis, together with chromosome in situ hybridization. We present an integrated genetic and physical map across the P. vulgaris S locus flanked by phenotypic and DNA sequence markers. BAC contigs encompass a 1.5-Mb genomic region with 1 Mb of sequence containing 82 S-linked genes anchored to overlapping BACs. The S locus is located close to the centromere of the largest metacentric chromosome pair. These data will facilitate the identification of the genes that orchestrate heterostyly in Primula and enable evolutionary analyses of the S locus.


Characterizing the subharmonic response of phospholipid-coated microbubbles for carotid imaging.
The subharmonic vibration of BR14 (Bracco Research S.A., Geneva, Switzerland) contrast agent microbubbles is investigated within the preferable frequency range for carotid ultrasound imaging (8-12 MHz). The response of the bubbles was recorded optically with an ultra-fast recording camera (Brandaris 128) at three acoustic pressures (50, 100 and 120 kPa). The vibration of the microbubbles was measured as a function of the excitation frequency and its frequency content was determined. Among 390 recordings, 40% showed subharmonic oscillations. It was observed that for smaller microbubbles (diameter < 3 μm) the frequency of the maximum subharmonic response increases for increasing pressures (shell hardening) opposite to what has been reported for larger microbubbles (3 μm < diameter < 15 μm). These findings are well predicted by the model proposed by Marmottant et al. (2005) after including the dilatational shell viscosity of the microbubbles measured by Van der Meer et al. (2007), which indicates a marked shear-thinning behavior of the phospholipid shell.


Head and neck pedicled flap autonomization using a new high-resolution indocyanine green fluorescence video-angiography device.
In head and neck oncologic surgery a reconstructive phase is often required and pedicled flaps are still a viable option, though they may need a pedicle division performed at a later stage. Several techniques are commonly used for perfusion assessment of the flaps, with indocyanine green (ICG) fluorescence video-angiography representing a promising tool. We used ICG video-angiography to evaluate the perfusion of two of the most commonly adopted pedicled flaps in the head and neck field (the supraclavicular and the paramedian forehead flap) before and after second-stage pedicle division, allowing a safer in-setting. Moreover, the new high-resolution device that we have employed added further accuracy to the traditional video-angiography, providing a real-time flap-to-normal skin ICG ratio. Indeed, ICG video-angiography proved to be a useful tool in head and neck reconstructive surgery and it may allow an earlier second-stage pedicle division.


Hispolon from Phellinus linteus has antiproliferative effects via MDM2-recruited ERK1/2 activity in breast and bladder cancer cells.
The MDM2 proto-oncogene is overexpressed in many human tumors. Although MDM2 inhibits tumor-suppressor function of p53, there exists a p53-independent role for MDM2 in tumorigenesis. Therefore, downregulation of MDM2 has been considered an attractive therapeutic strategy. Hispolon extracted from Phellinus species was found to induce epidermoid and gastric cancer cell apoptosis. However, the mechanisms are not fully understood. Herein, we report our findings that hispolon inhibited breast and bladder cancer cell growth, regardless of p53 status. Furthermore, p21(WAF1), a cyclin-dependent kinase inhibitor, was elevated in hispolon-treated cells. MDM2, a negative regulator of p21(WAF1), was ubiquitinated and degraded after hispolon treatment. We also found that activated ERK1/2 (extracellular signal-regulated kinase1/2) was recruited to MDM2 and involved in mediating MDM2 ubiquitination. Based on this finding, we investigated whether the sensitivity of cells to hispolon was related to ERK1/2 activity. The results indicated that cells with higher ERK1/2 activity were more sensitive to hispolon. In addition, hispolon-induced caspase-7 cleavage was inhibited by the ERK1/2 inhibitor, U0126. In conclusion, hispolon ubiquitinates and downregulates MDM2 via MDM2-recruited activated ERK1/2. Therefore, hispolon may be a potential anti-tumor agent in breast and bladder cancers.


Antipyrine and lidocaine are cleared faster in horses than in humans: acetaminophen may be handled similarly.
The following studies were designed to evaluate plasma elimination kinetics of intravenously administered antipyrine, acetaminophen and lidocaine among 9 healthy adult horses and 9 healthy drug-free humans (3 each per drug group), in order to compare potential species differences in drug-metabolizing ability. Acetaminophen is largely biotransformed in humans by hepatic glucuronide and sulfate conjugation, whereas both antipyrine and lidocaine are oxidized by hepatic microsomal mixed-function oxidases. Thus, plasma clearances of these drugs are thought to reflect differences in hepatic oxidative and conjugative activity, and possibly hepatic blood flow in the case of lidocaine. Results showed that mean (+/- SD, n = 3) acetaminophen clearance was similar in both horses (4.84 +/- 0.637 ml/min/kg) and humans (4.68 +/- 0.691 ml/min/kg). However, antipyrine clearance was 10 times greater in horses (5.83 +/- 2.21 ml/min/kg) than in humans (0.536 +/- 0.110 ml/min/kg), which may reflect enhanced hepatic microsomal activity in horses. Although lidocaine clearance in humans was similar to estimated hepatic blood flow (20.6 +/- 5.81 ml/min/kg), clearance in horses was more than 2 times greater (52.0 +/- 11.7 ml/min/kg). The cause of the higher clearance of lidocaine in horses (like dogs) remains unexplained, and may involve significant metabolism of lidocaine at extrahepatic, extravascular sites, for intravascular degradation and renal excretion of intact lidocaine in horses was negligible. Although precise biochemical mechanisms underlying pharmacokinetic parameters for these drugs in horses were not determined, it is nonetheless concluded from antipyrine results that horses may have an enhanced ability (compared with humans) to clear drugs from the circulation that are primarily metabolized in the liver by phase I oxidative reactions.(ABSTRACT TRUNCATED AT 250 WORDS)


Illicit Cigarette Trade in Five South American Countries: A Gap Analysis for Argentina, Brazil, Chile, Colombia, and Peru.
Because of its nature, it is very hard to measure illicit tobacco trade in any product. In the case of Latin American countries, there is scant information on the magnitude and characteristics of this cigarette trade. The goal of this article is to provide estimates on the evolution of the illicit cigarette trade in five South American countries: Argentina, Brazil, Chile, Colombia, and Peru. Gap analysis estimates for cigarette tax evasion/avoidance (a comparison on the evolution of the difference between registered cigarette sales and measured population consumption) are developed for Argentina, Brazil, Chile, Colombia, and Peru. Nationally representative surveys, conducted regularly, are used to measure population consumption. Confidence intervals constructed by bootstrapping sample estimates are generated to statistically evaluate the evolution of the gap. Illicit cigarette trade has increased as a percentage of total sales in Brazil in recent years. In the case of Argentina, after a relative decrease between 2005 and 2009, it seems to have stabilized. There is no statistical evidence to argue that there has been an increase of illicit cigarette trade in Chile, Colombia, and Peru, despite substantial price increases in Chile and tax increase in both Colombia and Peru. Using simple statistical methods, it is possible to assess the trend in illicit tobacco trade over time to better inform policy makers. Getting reliable and regular population consumption surveys can also help to track illicit tobacco trade. Claims by tobacco industry of a positive association between price/tax changes and illicit trade are unsubstantiated. Evolution of illicit cigarette trade in five Latin American countries shows different trajectories, not in line with tobacco industry estimates, which highlight the importance of producing solid, independent estimates. There are inexpensive methodologies that can provide estimates of the evolution of the relative importance of illicit trade and can be used to inform policy makers.


SRC-induced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1.
The Rac activator Tiam1 is required for adherens junction (AJ) maintenance, and its depletion results in AJ disassembly. Conversely, the oncoprotein Src potently induces AJ disassembly and epithelial-mesenchymal transition (EMT). Here, we show that Tiam1 is phosphorylated on Y384 by Src. This occurs predominantly at AJs, is required for Src-induced AJ disassembly and cell migration, and creates a docking site on Tiam1 for Grb2. We find that Tiam1 is associated with ERK. Following recruitment of the Grb2-Sos1 complex, ERK becomes activated and triggers the localized degradation of Tiam1 at AJs, likely involving calpain proteases. Furthermore, we demonstrate that, in human tumors, Y384 phosphorylation positively correlates with Src activity, and total Tiam1 levels are inversely correlated. Thus, our data implicate Tiam1 phosphorylation and consequent degradation in Src-mediated EMT and resultant cell motility and establish a paradigm for regulating local concentrations of Rho-GEFs.


Functional significance of mutations in the Snf2 domain of ATRX.
ATRX is a member of the Snf2 family of chromatin-remodelling proteins and is mutated in an X-linked mental retardation syndrome associated with alpha-thalassaemia (ATR-X syndrome). We have carried out an analysis of 21 disease-causing mutations within the Snf2 domain of ATRX by quantifying the expression of the ATRX protein and placing all missense mutations in their structural context by homology modelling. While demonstrating the importance of protein dosage to the development of ATR-X syndrome, we also identified three mutations which primarily affect function rather than protein structure. We show that all three of these mutant proteins are defective in translocating along DNA while one mutant, uniquely for a human disease-causing mutation, partially uncouples adenosine triphosphate (ATP) hydrolysis from DNA binding. Our results highlight important mechanistic aspects in the development of ATR-X syndrome and identify crucial functional residues within the Snf2 domain of ATRX. These findings are important for furthering our understanding of how ATP hydrolysis is harnessed as useful work in chromatin remodelling proteins and the wider family of nucleic acid translocating motors.


Clonotypic analysis of the fetal B cell repertoire: evidence for an early and predominant expression of idiotypes associated with the VH 36-60 family.
Determining the mechanisms by which B cells develop that culminates in the generation of a diverse repertoire is critical to our understanding of how the immune response is regulated. The B cell specificity repertoire appears to be developmentally acquired in a predetermined, temporally ordered fashion. Thus, in the Balb/c mouse, the ontogenetic appearance of functional B cells specific for various hapten probes occurs in the order of dinitrophenol (DNP), fluorescein (Fl), and phosphorylcholine (PC). In addition, when the influenza virus hemagglutinin molecule was used as an antigen probe, similar conclusions were drawn regarding a patterned acquisition of the specificity repertoire. More recently, we have used a fetal organ culture system to show that hapten-responsive B cells appear in the same predictable, temporal order in vitro. The importance of these studies was that the effects of environmental influences were minimized in the absence of circulation and cell migration, and therefore the results indicated that the patterned emergence of the specificity repertoire observed was largely the result of genetic regulatory processes. Recent molecular findings may relate to such genetic regulatory processes. The VH genes in Balb/c mice have been grouped into eight families based on sequence homology, and have been mapped relative to the constant region genes. Yancopoulos et al. and Perlmutter et al. have shown that the VH gene segments closest to the JH cluster, the VH1B 7183 family, are preferentially utilized in transformed fetal pre-B cell lines and in fetal B cell hybridomas. This led to the hypothesis that the developmental control of the expression of VH gene segments is related to chromosomal organization. A logical extension of these findings is that the programmed appearance of particular clonotypes in ontogeny may be explained, in part, by the preferential use of particular VH gene segments. However, to what extent the transformed B cell lines represent members of the functional expressed repertoire could not be evaluated. In the studies described herein, the fetal organ culture system was used to assess the early expressed repertoire at the clonotypic level using idiotypic analysis. Anti-DNP secreting clones were derived from fetal organ cultures and tested for the presence of two idiotypes, 36 and MOPC 460 (460). The 36 idiotype is a predominant DNP clonotype of the neonatal repertoire, while the 460 idiotype is a major cross-reactive idiotype of the adult DNP response.(ABSTRACT TRUNCATED AT 400 WORDS)


Hierarchical and automated cell-type annotation and inference of cancer cell of origin with Census.
Cell-type annotation is a time-consuming yet critical first step in the analysis of single-cell RNA-seq data, especially when multiple similar cell subtypes with overlapping marker genes are present. Existing automated annotation methods have a number of limitations, including requiring large reference datasets, high computation time, shallow annotation resolution, and difficulty in identifying cancer cells or their most likely cell of origin. We developed Census, a biologically intuitive and fully automated cell-type identification method for single-cell RNA-seq data that can deeply annotate normal cells in mammalian tissues and identify malignant cells and their likely cell of origin. Motivated by the inherently stratified developmental programs of cellular differentiation, Census infers hierarchical cell-type relationships and uses gradient-boosted \decision trees that capitalize on nodal cell-type relationships to achieve high prediction speed and accuracy. When benchmarked on 44 atlas-scale normal and cancer, human and mouse tissues, Census significantly outperforms state-of-the-art methods across multiple metrics and naturally predicts the cell-of-origin of different cancers. Census is pretrained on the Tabula Sapiens to classify 175 cell-types from 24 organs; however, users can seamlessly train their own models for customized applications. Census is available at Zenodo https://zenodo.org/records/7017103 and on our Github https://github.com/sjdlabgroup/Census.


Further assessment of forward pressure level for in situ calibration.
Quantifying ear-canal sound level in forward pressure has been suggested as a more accurate and practical alternative to sound pressure level (SPL) calibrations used in clinical settings. The mathematical isolation of forward (and reverse) pressure requires defining the Thévenin-equivalent impedance and pressure of the sound source and characteristic impedance of the load; however, the extent to which inaccuracies in characterizing the source and/or load impact forward pressure level (FPL) calibrations has not been specifically evaluated. This study examined how commercially available probe tips and estimates of characteristic impedance impact the calculation of forward and reverse pressure in a number of test cavities with dimensions chosen to reflect human ear-canal dimensions. Results demonstrate that FPL calibration, which has already been shown to be more accurate than in situ SPL calibration, can be improved particularly around standing-wave null frequencies by refining estimates of characteristic impedance. Better estimates allow FPL to be accurately calculated at least through 10 kHz using a variety of probe tips in test cavities of different sizes, suggesting that FPL calibration can be performed in ear canals of all sizes. Additionally, FPL calibration appears a reasonable option when quantifying the levels of extended high-frequency (10-18 kHz) stimuli.


Improving risk indexes for Alzheimer's disease and related dementias for use in midlife.
Knowledge of a person's risk for Alzheimer's disease and related dementias (ADRDs) is required to triage candidates for preventive interventions, surveillance, and treatment trials. ADRD risk indexes exist for this purpose, but each includes only a subset of known risk factors. Information missing from published indexes could improve risk prediction. In the Dunedin Study of a population-representative New Zealand-based birth cohort followed to midlife (N = 938, 49.5% female), we compared associations of four leading risk indexes with midlife antecedents of ADRD against a novel benchmark index comprised of nearly all known ADRD risk factors, the Dunedin ADRD Risk Benchmark (DunedinARB). Existing indexes included the Cardiovascular Risk Factors, Aging, and Dementia index (CAIDE), LIfestyle for BRAin health index (LIBRA), Australian National University Alzheimer's Disease Risk Index (ANU-ADRI), and risks selected by the Lancet Commission on Dementia. The Dunedin benchmark was comprised of 48 separate indicators of risk organized into 10 conceptually distinct risk domains. Midlife antecedents of ADRD treated as outcome measures included age-45 measures of brain structural integrity [magnetic resonance imaging-assessed: (i) machine-learning-algorithm-estimated brain age, (ii) log-transformed volume of white matter hyperintensities, and (iii) mean grey matter volume of the hippocampus] and measures of brain functional integrity [(i) objective cognitive function assessed via the Wechsler Adult Intelligence Scale-IV, (ii) subjective problems in everyday cognitive function, and (iii) objective cognitive decline measured as residualized change in cognitive scores from childhood to midlife on matched Weschler Intelligence scales]. All indexes were quantitatively distributed and proved informative about midlife antecedents of ADRD, including algorithm-estimated brain age (β's from 0.16 to 0.22), white matter hyperintensities volume (β's from 0.16 to 0.19), hippocampal volume (β's from -0.08 to -0.11), tested cognitive deficits (β's from -0.36 to -0.49), everyday cognitive problems (β's from 0.14 to 0.38), and longitudinal cognitive decline (β's from -0.18 to -0.26). Existing indexes compared favourably to the comprehensive benchmark in their association with the brain structural integrity measures but were outperformed in their association with the functional integrity measures, particularly subjective cognitive problems and tested cognitive decline. Results indicated that existing indexes could be improved with targeted additions, particularly of measures assessing socioeconomic status, physical and sensory function, epigenetic aging, and subjective overall health. Existing premorbid ADRD risk indexes perform well in identifying linear gradients of risk among members of the general population at midlife, even when they include only a small subset of potential risk factors. They could be improved, however, with targeted additions to more holistically capture the different facets of risk for this multiply determined, age-related disease.


Self-assembling amphipathic alpha-helical peptides induce the formation of active protein aggregates in vivo.
We recently found that several self-assembling alpha, beta, or surfactant-like peptides, when terminally attached to proteins, can promote the in vivo assembly of active protein aggregates (or active inclusion bodies, AIBs) in Escherichia coil. In this work, we systematically examined the AIBs induced by an amphipathic alpha-helical peptide 18Awt (EWLKAFYEKVLEKLKELF) and its variants with altered ion pairs. Transmission electron microscopic and Fourier transform infrared spectroscopic analyses suggested that the AIBs appeared to adopt an amorphous mesh-like structure, and were likely induced by helical structures formed by the assembly of the 18A peptides. Confocal fluorescent micrographic analysis revealed that the AIBs resided around the periphery of the cell membrane or in the cytoplasm, depending on the distribution of ion pairs on the 18A peptides, which suggested that the association between the aggregates and the cell membrane was mediated by the lipid-18A interaction. Two of these 18A peptide variants were further used in constructing cleavable self-aggregating tags (cSAT) in conjunction with an intein molecule for protein purification, and verified using two model proteins. This extends the cSAT approach for laboratory and potentially industrial uses. Our study might also provide new insights into aggregation-related diseases.


Tracking climate impacts on the migratory monarch butterfly.
Understanding the impacts of climate on migratory species is complicated by the fact that these species travel through several climates that may be changing in diverse ways throughout their complete migratory cycle. Most studies are not designed to tease out the direct and indirect effects of climate at various stages along the migration route. We assess the impacts of spring and summer climate conditions on breeding monarch butterflies, a species that completes its annual migration cycle over several generations. No single, broad-scale climate metric can explain summer breeding phenology or the substantial year-to-year fluctuations observed in population abundances. As such, we built a Poisson regression model to help explain annual arrival times and abundances in the Midwestern United States. We incorporated the climate conditions experienced both during a spring migration/breeding phase in Texas as well as during subsequent arrival and breeding during the main recruitment period in Ohio. Using data from a state-wide butterfly monitoring network in Ohio, our results suggest that climate acts in conflicting ways during the spring and summer seasons. High spring precipitation in Texas is associated with the largest annual population growth in Ohio and the earliest arrival to the summer breeding ground, as are intermediate spring temperatures in Texas. On the other hand, the timing of monarch arrivals to the summer breeding grounds is not affected by climate conditions within Ohio. Once in Ohio for summer breeding, precipitation has minimal impacts on overall abundances, whereas warmer summer temperatures are generally associated with the highest expected abundances, yet this effect is mitigated by the average seasonal temperature of each location in that the warmest sites receive no benefit of above average summer temperatures. Our results highlight the complex relationship between climate and performance for a migrating species and suggest that attempts to understand how monarchs will be affected by future climate conditions will be challenging.