A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation.

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose

Derivation of 30 human embryonic stem cell lines—improving the quality

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process, we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal, but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked, and they are available for researchers

A systematic review on health resilience to economic crises

Background The health effects of recent economic crises differ markedly by population group. The objective of this systematic review is to examine evidence from longitudinal studies on factors influencing resilience for any health outcome or health behaviour among the general population living in countries exposed to financial crises. Methods We systematically reviewed studies from six electronic databases (EMBASE, Global Health, MEDLINE, PsycINFO, Scopus, Web of Science) which used quantitative longitudinal study designs and included: (i) exposure to an economic crisis; (ii) changes in health outcomes/behaviours over time; (iii) statistical tests of associations of health risk and/or protective factors with health outcomes/behaviours. The quality of the selected studies was appraised using the Quality Assessment Tool for Quantitative Studies. PRISMA reporting guidelines were followed. Results From 14,584 retrieved records, 22 studies met the eligibility criteria. These studies were conducted across 10 countries in Asia, Europe and North America over the past two decades. Ten socio-demographic factors that increased or protected against health risk were identified: gender, age, education, marital status, household size, employment/occupation, income/ financial constraints, personal beliefs, health status, area of residence, and social relations. These studies addressed physical health, mortality, suicide and suicide attempts, mental health, and health behaviours. Women’s mental health appeared more susceptible to crises than men’s. Lower income levels were associated with greater increases in cardiovascular disease, mortality and worse mental health. Employment status was associated with changes in mental health. Associations with age, marital status, and education were less consistent, although higher education was associated with healthier behaviours. Conclusions Despite widespread rhetoric about the importance of resilience, there was a dearth of studies which operationalised resilience factors. Future conceptual and empirical research is needed to develop the epidemiology of resilience

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

INTRODUCTION Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years

Use of machine learning to shorten observation-based screening and diagnosis of autism

The Autism Diagnostic Observation Schedule-Generic (ADOS) is one of the most widely used instruments for behavioral evaluation of autism spectrum disorders. It is composed of four modules, each tailored for a specific group of individuals based on their language and developmental level. On average, a module takes between 30 and 60 min to deliver. We used a series of machine-learning algorithms to study the complete set of scores from Module 1 of the ADOS available at the Autism Genetic Resource Exchange (AGRE) for 612 individuals with a classification of autism and 15 non-spectrum individuals from both AGRE and the Boston Autism Consortium (AC). Our analysis indicated that 8 of the 29 items contained in Module 1 of the ADOS were sufficient to classify autism with 100% accuracy. We further validated the accuracy of this eight-item classifier against complete sets of scores from two independent sources, a collection of 110 individuals with autism from AC and a collection of 336 individuals with autism from the Simons Foundation. In both cases, our classifier performed with nearly 100% sensitivity, correctly classifying all but two of the individuals from these two resources with a diagnosis of autism, and with 94% specificity on a collection of observed and simulated non-spectrum controls. The classifier contained several elements found in the ADOS algorithm, demonstrating high test validity, and also resulted in a quantitative score that measures classification confidence and extremeness of the phenotype. With incidence rates rising, the ability to classify autism effectively and quickly requires careful design of assessment and diagnostic tools. Given the brevity, accuracy and quantitative nature of the classifier, results from this study may prove valuable in the development of mobile tools for preliminary evaluation and clinical prioritization—in particular those focused on assessment of short home videos of children—that speed the pace of initial evaluation and broaden the reach to a significantly larger percentage of the population at risk

Ergocalciferol and Microcirculatory Function in Chronic Kidney Disease and Concomitant Vitamin D Deficiency: An Exploratory, Double Blind, Randomised Controlled Trial

Vitamin D deficiency and endothelial dysfunction are non-traditional risk factors for cardiovascular events in chronic kidney disease. Previous studies in chronic kidney disease have failed to demonstrate a beneficial effect of vitamin D on arterial stiffness, left ventricular mass and inflammation but none have assessed the effect of vitamin D on microcirculatory endothelial function.We conducted a randomised controlled trial of 38 patients with non diabetic chronic kidney disease stage 3-4 and concomitant vitamin D deficiency (<16 ng/dl) who received oral ergocalciferol (50,000 IU weekly for one month followed by 50,000 IU monthly) or placebo over 6 months. The primary outcome was change in microcirculatory function measured by laser Doppler flowmetry after iontophoresis of acetylcholine. Secondary endpoints were tissue advanced glycation end products, sublingual functional capillary density and flow index as well as macrovascular parameters. Parallel in vitro experiments were conducted to determine the effect of ergocalciferol on cultured human endothelial cells.Twenty patients received ergocalciferol and 18 patients received placebo. After 6 months, there was a significant improvement in the ergocalciferol group in both endothelium dependent microcirculatory vasodilatation after iontophoresis of acetylcholine (p = 0.03) and a reduction in tissue advanced glycation end products (p = 0.03). There were no changes in sublingual microcirculatory parameters. Pulse pressure (p = 0.01) but not aortic pulse wave velocity was reduced. There were no significant changes in bone mineral parameters, blood pressure or left ventricular mass index suggesting that ergocalciferol improved endothelial function independently of these parameters. In parallel experiments, expression of endothelial nitric oxide synthase and activity were increased in human endothelial cells in a dose dependent manner.Ergocalciferol improved microcirculatory endothelial function in patients with chronic kidney disease and concomitant vitamin D deficiency. This process may be mediated through enhanced expression and activity of endothelial nitric oxide synthase.Clinical trials.gov NCT00882401

Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations

Leaf-cutting ant fungi produce cell wall degrading pectinase complexes reminiscent of phytopathogenic fungi

<p>Abstract</p> <p>Background</p> <p>Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus <it>Leucocoprinus gongylophorus </it>that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.</p> <p>Results</p> <p>We tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in <it>Acromyrmex echinatior </it>leaf-cutting ants. Seven (21%) of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade.</p> <p>Conclusions</p> <p>Pectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by <it>Acromyrmex </it>leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to those normally found in phytopathogens.</p

Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: Interim results of a prospective cohort study

<p>Abstract</p> <p>Background</p> <p>We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.</p> <p>Methods</p> <p>Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.</p> <p>Results</p> <p>At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (<it>mam</it>) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).</p> <p>Conclusion</p> <p>This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the <it>mam </it>gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.</p