The rockefeller university

Objective: To determine live-birth rates (LBRs) at various ages in very poor prognosis patients, who are defined as poor responders under the Bologna criteria. Design: Retrospective cohort study. Setting: Academically affiliated private fertility center. Patient(s): Among 483 patients, who under the Bologna criteria (three or fewer oocytes, >40 years of age, and/or antimullerian hormone [AMH] <1.1 ng/mL [2/3 criteria minimum]) were poor responders, 278 (381 fresh IVF cycles) qualified for the study because they had at least one embryo on day 3 for transfer. Intervention(s): IVF cycles in women with low functional ovarian reserve, involving androgen and CoQ10 supplementation and ovarian stimulation with daily gonadotropin dosages of 300-450 IU of FSH and 150 IU of hMG in microdose agonist cycles. Main Outcome Measure(s): Age-specific LBRs per ET. Result(s): Ages did not differ between nonelective (ne) single ET (SET), ne2-ET, and neR3-ET cycles (41.3 +/- 3.9, 41.7 +/- 3.1, and 42.4 +/- 2.1 years, respectively). Patients with neSETs demonstrated significantly lower AMH and higher FSH levels and required higher gonadotropin dosages than ne2-ET and neR3-ET patients. LBRs declined with age. Above age 42, three or more embryos are required to achieve reasonable LBRs and two or more to avoid futility under American Society for Reproductive Medicine (ASRM) guidelines. Conclusion(s): Very poor prognosis patients can still achieve acceptable pregnancy rates at least till their mid-40s if they reach ET. The degree to which egg donation is emphasized as the only treatment option in such patients, therefore, requires reconsideration. Above age 42, at least two, and preferably three embryos, are however required to exceed futility, as defined by ASRM. (C) 2015 by American Society for Reproductive Medicine.

Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132-713) and STAT3 (127-722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

While most animals thermotax only to regulate their temperature, female mosquitoes are attracted to human body heat during pursuit of a blood meal. Here we elucidate the basic rules of Aedes aegypti thermotaxis and test the function of candidate thermoreceptors in this important behavior. We show that host-seeking mosquitoes are maximally attracted to thermal stimuli approximating host body temperatures, seeking relative warmth while avoiding both relative cool and stimuli exceeding host body temperature. We found that the cation channel TRPA1, in addition to playing a conserved role in thermoregulation and chemosensation, is required for this specialized host-selective thermotaxis in mosquitoes. During host-seeking, AaegTRPA1(-/-) mutants failed to avoid stimuli exceeding host temperature, and were unable to discriminate between host-temperature and high-temperature stimuli. TRPA1-dependent tuning of thermotaxis is likely critical for mosquitoes host-seeking in a complex thermal environment in which humans are warmer than ambient air, but cooler than surrounding sun-warmed surfaces.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85 alpha, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85 alpha dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85 alpha that is functionally equivalent to native p85 alpha. Analytical ultracentrifugation studies showed that p85 alpha undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85 alpha using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85 alpha dimer. Our study provides new insight into the structure and assembly of the p85 alpha homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

We study the behaviour of double-stranded RNA eunder twist and tension using oxRNA, a recently developed coarse-grained model of RNA. Introducing explicit salt-dependence into the model allows us to directly compare our results to data from recent single-molecule experiments. The model reproduces extension curves as a function of twist and stretching force, including the buckling transition and the behaviour of plectoneme structures. For negative supercoiling, we predict denaturation bubble formation in plectoneme end-loops, suggesting preferential plectoneme localisation in weak base sequences. OxRNA exhibits a positive twist-stretch coupling constant, in agreement with recent experimental observations. (C) 2015 AIP Publishing LLC.

BackgroundAchieving timely accrual into clinical research studies remains a challenge for clinical translational research. We developed an evaluation measure, the Accrual Index (AI), normalized for sample size and study duration, using data from the protocol and study management databases. We applied the AI retrospectively and prospectively to assess its utility. MethodsAccrual Target, Projected Time to Accrual Completion (PTAC), Evaluable Subjects, Dates of Recruitment Initiation, Analysis, and Completion were defined. AI is (% Accrual Target accrued/% PTAC elapsed). Changes to recruitment practices were described, and data extracted from study management databases. ResultsDecember 2014 (or final) AI was analyzed for 101 studies initiating recruitment from 2007 to 2014. Median AI was 1 for protocols initiating recruitment in 2011, 2013, and 2014. The AI varied widely for studies pre-2013. Studies with AI > 4 utilized convenience samples for recruitment. Data-justified PTAC was refined in 2013-2014 after which the AI range narrowed. Protocol characteristics were not associated with study AI. ConclusionProtocol AI reflects the relative agreement between accrual feasibility assessment (PTAC), and accrual performance, and is affected by recruitment practices. The AI may be useful in managing accountability, modeling accrual, allocating recruitment resources, and testing innovations in recruitment practices.

Background: Neutrophil activation induces citrullination of intracellular targets of anticitrullinated peptide antibodies (ACPA), which are specific for rheumatoid arthritis (RA). Citrullinated fibrinogen is bound by ACPA but it is less well understood how extracellular proteins are citrullinated. The cells that produce fibrinogen, hepatocytes, do not express peptidyl arginine deiminase (PAD) enzymes nor do PAD enzymes include N-terminal signal peptides to direct them into the secretory pathway. We hypothesized that dying neutrophils release PAD in the extracellular space, and that this could cause citrullination of target extracellular antigens relevant to RA such as fibrinogen. Methods: HL60 cells were differentiated into neutrophil-like cells by treatment with all-trans retinoic acid (ATRA). Differentiation was confirmed by CD11b staining, PAD4, PAD2 and myeloperoxidase expression, cell division, and nuclear morphology. Death was induced with various stimuli, including freeze-thaw to induce necrosis, Ionomycin and PMA to induce NETosis, and UV-B to induce apoptosis. Death markers were assessed by immunohistochemistry and flow cytometry. To quantify extracellular citrullination, dying ATRA-differentiated HL60 cells were cultured with fibrinogen for 24 hours and supernatants were probed for fibrinogen citrullination, PAD2 and PAD4 by western blot. Results: While both NETotic and necrotic ATRA differentiated HL60 cells citrullinated fibrinogen, apoptotic cells did not citrullinate fibrinogen, even when allowed to undergo secondary necrosis. Incubation of necrotic neutrophil lysates with fibrinogen also causes fibrinogen citrullination. PAD2 and PAD4 were detected by western blot of supernatants of ATRA-differentiated HL60 cells undergoing necrotic and NETotic death, but not apoptotic or secondarily necrotic cell death. Conclusion: We implicate granulocytes undergoing inflammatory cell death as a mechanism for altering extracellular self-proteins that may be targets of autoimmunity linked to inflammatory diseases such as rheumatoid arthritis.

The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation.

Background. Using a multicenter, active surveillance network from 2 rotavirus seasons (2012 and 2013), we assessed the vaccine effectiveness of RV5 (RotaTeq) and RV1 (Rotarix) rotavirus vaccines in preventing rotavirus gastroenteritis hospitalizations and emergency department (ED) visits for numerous demographic and secular strata. Methods. We enrolled children hospitalized or visiting the ED with acute gastroenteritis (AGE) for the 2012 and 2013 seasons at 7 medical institutions. Stool specimens were tested for rotavirus by enzyme immunoassay and genotyped, and rotavirus vaccination histories were compared for rotavirus-positive cases and rotavirus-negative AGE controls. We calculated the vaccine effectiveness (VE) for preventing rotavirus associated hospitalizations and ED visits for each vaccine, stratified by vaccine dose, season, clinical setting, age, predominant genotype, and ethnicity. Results. RV5-specific VE analyses included 2961 subjects, 402 rotavirus cases (14%) and 2559 rotavirus-negative AGE controls. RV1-specific VE analyses included 904 subjects, 100 rotavirus cases (11%), and 804 rotavirus-negative AGE controls. Over the 2 rotavirus seasons, the VE for a complete 3-dose vaccination with RV5 was 80% (confidence interval [CI], 74%-84%), and VE for a complete 2-dose vaccination with RV1 was 80% (CI, 68%-88%). Statistically significant VE was observed for each year of life for which sufficient data allowed analysis (7 years for RV5 and 3 years for RV1). Both vaccines provided statistically significant genotype-specific protection against predominant circulating rotavirus strains. Conclusions. In this large, geographically and demographically diverse sample of US children, we observed that RV5 and RV1 rotavirus vaccines each provided a lasting and broadly heterologous protection against rotavirus gastroenteritis.