Targeted rescue of cancer-associated IDH1 mutant activity using an engineered synthetic antibody

We have utilized a high-diversity phage display library to engineer antibody fragments (Fabs) that can modulate the activity of the enzyme isocitrate dehydrogenase 1 (IDH1). We show that a conformation-specific Fab can reactivate an IDH1 mutant associated with brain tumors. The results show that this strategy is a first step towards developing "activator drugs" for a large number of genetic disorders where mutations have disrupted protein function

Self-rated health disparities among disadvantaged older adults in ethnically diverse urban neighborhoods in a Middle Eastern country.

OBJECTIVES: This paper examines differentials in self-rated health (SRH) among older adults (aged 60+ years) across three impoverished and ethnically diverse neighborhoods in post-conflict Lebanon and assesses whether variations are explained by social and economic factors. DESIGN: Data were drawn from the Older Adult Component (n = 740) of the Urban Health Survey, a population-based cross-sectional study conducted in 2003 in a formal community (Nabaa), an informal settlement (Hey El-Sellom), and a refugee camp for Palestinians (Burj El-Barajneh) in Beirut, Lebanon. The role of the social capital and economic security constructs in offsetting poor SRH was assessed using multivariate ordinal logistic regression analyses. RESULTS: Older adults in Nabaa fared better in SRH compared to those in Hey El-Sellom and Burj El-Barajneh, with a prevalence of good, average, and poor SRH being respectively, 41.5%, 37.0%, and 21.5% in Nabaa, 33.3%, 23.9%, and 42.7% in Hey El-Sellom, and 25.2%, 31.3%, and 43.5% in Burj El-Barajneh. The economic security construct attenuated the odds of poorer SRH in Burj El-Barajneh as compared to Nabaa from 2.57 (95% confidence interval, CI: 1.89-3.79) to 1.42 (95% CI: 0.96-2.08), but had no impact on this association in Hey El-Sellom (odds ratio, OR: 2.12, 95% CI: 1.39-3.24). The incorporation of the social capital construct in the fully adjusted model rendered this association insignificant in Hey El-Sellom (OR: 1.49, 95% CI: 0.96-2.32), and led to further reductions in the magnitude of the association in Burj El-Barajneh camp (OR: 1.18, 95% CI: 0.80-1.76). CONCLUSIONS: The social context in which older adults live and their financial security are key in explaining disparities in SRH in marginalized communities. Social capital and economic security, often overlooked in policy and public health interventions, need to be integrated in dimensions of well-being of older adults, especially in post-conflict settings

Towards population inversion of electrically pumped Er ions sensitized by Si nanoclusters

This study reports the estimation of the inverted Er fraction in a system of Er doped silicon oxide sensitized by Si nanoclusters, made by magnetron sputtering. Electroluminescence was obtained from the sensitized erbium, with a power efficiency of 10¿2 %. By estimating the density of Er ions that are in the first excited state, we find that up to 20% of the total Er concentration is inverted in the best device, which is one order of magnitude higher than that achieved by optical pumping of similar materials

A Novel Fluorescent Clinical Method to Rapidly Quantify Plasma Volume

Objectives To determine the performance of a rapid fluorescent indicator technique for measuring plasma volume (PV). Methods This was an open-label, observational evaluation of a two-component intravenous visible fluorescent dye technique to rapidly measure PV in 16 healthy subjects and 16 subjects with chronic kidney disease (8 stage 3 and 8 stage 4 CKD), at 2 clinical research sites. The method consisted of a single intravenous injection of 12 mg of a large 150-kDa carboxy-methyl dextran conjugated to a fluorescent rhodamine-derived dye as the PV marker (PVM), and 35 mg of a small 5-kDa carboxy-methyl dextran conjugated to fluorescein, the renal clearance marker. Dye concentrations were quantified 15 min after the injections for initial PV measurements using the indicator-dilution principle. Additional samples were taken over 8 h to evaluate the stability of the PVM as a determinant of PV. Blood volumes (BV) were calculated based on PV and the subject’s hematocrit. Pharmacokinetic parameters were calculated from the plasma concentration data taken over several days using noncompartmental methods (Phoenix WinNonlin®). Linear correlation and Bland-Altman plots were used to compare visible fluorescent injectate-measured PV compared to Nadler’s formula for estimating PV. Finally, 8 healthy subjects received 350 mL infusion of a 5% albumin solution in normal saline over 30 min and a repeat PV determination was then carried out. Results PV and BV varied according to weight and body surface area, with PV ranging from 2,115 to 6,234 mL and 28.6 to 41.9 mL/kg when weight adjusted. Both parameters were stable for > 6 h with repeated plasma measurements of the PVM. There was no difference between healthy subjects and CKD subjects. Overall, there was general agreement with Nadler’s estimation formula for the mean PV in subjects. A 24-h repeat dose measurement in 8 healthy subjects showed PV variability of 98 ± 121 mL (mean = 3.8%). Additionally, following an intravenous bolus of 350 mL of a 5% albumin solution in normal saline in 8 healthy subjects, the mean (SD) measured increase in PV was 356 (±50.0) mL post-infusion. There were no serious adverse events reported during the study. Conclusions This minimally invasive fluorescent dye approach safely allowed for rapid, accurate, and reproducible determination of PV, BV, and dynamic monitoring of changes following fluid administration

RNA buffers the phase separation behavior of prion-like RNA binding proteins

Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNAcritically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.1125sciescopu

Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling

Background: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response. Findings: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway. Conclusions: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity. Keywords: Prolactin signaling, Synthetic antibody, Phage display, Alloster

Context-led capacity building in time of crisis: fostering non-communicable diseases (NCD) research skills in the Mediterranean Middle East and North Africa.

BACKGROUND: This paper examines one EC-funded multinational project (RESCAP-MED), with a focus on research capacity building (RCB) concerning non-communicable diseases (NCDs) in the Mediterranean Middle East and North Africa. By the project's end (2015), the entire region was engulfed in crisis. OBJECTIVE: Designed before this crisis developed in 2011, the primary purpose of RESCAP-MED was to foster methodological skills needed to conduct multi-disciplinary research on NCDs and their social determinants. RESCAP-MED also sought to consolidate regional networks for future collaboration, and to boost existing regional policy engagement in the region on the NCD challenge. This analysis examines the scope and sustainability of RCB conducted in a context of intensifying political turmoil. METHODS: RESCAP-MED linked two sets of activities. The first was a framework for training early- and mid-career researchers through discipline-based and writing workshops, plus short fellowships for sustained mentoring. The second integrated public-facing activities designed to raise the profile of the NCD burden in the region, and its implications for policymakers at national level. Key to this were two conferences to showcase regional research on NCDs, and the development of an e-learning resource (NETPH). RESULTS: Seven discipline-based workshops (with 113 participants) and 6 workshops to develop writing skills (84 participants) were held, with 18 fellowship visits. The 2 symposia in Istanbul and Beirut attracted 280 participants. Yet the developing political crisis tagged each activity with a series of logistical challenges, none of which was initially envisaged. The immediacy of the crisis inevitably deflected from policy attention to the challenges of NCDs. CONCLUSIONS: This programme to strengthen research capacity for one priority area of global public health took place as a narrow window of political opportunity was closing. The key lessons concern issues of sustainability and the paramount importance of responsively shaping a context-driven RCB

Characterization of Engineered Actin Binding Proteins That Control Filament Assembly and Structure

Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate.We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs), are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel "reduced genetic code" phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively.From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020