The rockefeller university

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

IMPORTANCE Methotrexate is a first-line systemic agent for treating of psoriasis, although its onset of effects is slower and overall it is less effective than tumor necrosis factor blockers. OBJECTIVE To differentiate the response of psoriatic disease to adalimumab and methotrexate sodium. DESIGN, SETTING, AND PARTICIPANTS Single-center, randomized, assessor-blind, 2-arm clinical trial of 30 patients from the outpatient dermatology center of Tufts Medical Center, enrolled from August 18, 2009, to October 11, 2011. Patients aged 18 to 85 years with chronic plaque-type psoriasis, a minimum Physician Global Assessment score of 3 (higher scores indicate more severe disease), and a psoriatic plaque of at least 2 cm were randomized in a 1: 1 fashion to receive subcutaneous adalimumab or oral methotrexate. Skin biopsy specimens obtained at baseline and weeks 1, 2, 4, and 16 were given a histologic grade by blinded assessors to evaluate treatment response. Analyses were conducted from April 16, 2013, to January 5, 2015. INTERVENTIONS A 16-week course of subcutaneous adalimumab (40 mg every 2 weeks after a loading dose) or low-dosage oral methotrexate sodium (7.5-25 mg/wk). MAIN OUTCOMES AND MEASURES Changes in genomic, immunohistochemical, and messenger RNA (mRNA) profiles. RESULTS Methotrexate responders experienced significant downregulation of helper T-cell-related (T(H)1, T(H)17, and T(H)22) mRNA expression compared with methotrexate nonresponders. Comparisons among adalimumab-treated patients were limited by the number of nonresponders (n = 1). Between adalimumab and methotrexate responders, we found no significant differences in gene expression at any study point or in the expression of T-cell-related-mRNA at week 16. Adalimumab responders demonstrated early downregulation of chemokine (C-C motif) ligand 20 (CCL20) mRNA(mean [SE] at week 2, -1.83 [0.52], P < .001; week 16, -3.55 [0.54], P < .001) compared with late downregulation for methotrexate responders (week 2, 0.02 [0.51], P = .96; week 16, -2.96 [0.51], P < .001). Similar differences were observed with interleukin 22 (IL22) mRNA showing early downregulation for adalimumab responders (week 2, -3.17 [1.00], P < .001; week 16, -3.58 [1.00], P < .001) compared with late downregulation for methotrexate responders (week 2, -0.44 [0.68], P = .64; week 16, -5.14 [0.68], P < .001). Analysis of variance findings for key mRNA and immunohistochemical marker expression over the study course were significant only for CCL20 (P = .03) and IL22 (P = .006) mRNA comparing adalimumab and methotrexate responders. CONCLUSIONS AND RELEVANCE Methotrexate is an immunomodulator with effects on helper T-cell signaling in psoriasis. Similar genomic and immunohistochemical response signatures and levels of mRNA downregulation at study completion among adalimumab and methotrexate responders suggest a disease-driven instead of therapeutic-driven pathway regulation. Adalimumab and methotrexate responses are differentiated by patterns of normalization of CCL20 and IL22 mRNA expression and may explain the varied onset and degree of clinical responses by each treatment.

The TGF beta signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGF beta ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGF beta 1 signaling. Coco synergizes with TGF beta 1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGF beta 1 and enhances TGF beta 1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGF beta signaling.

Intravenous immunoglobulins (IVIGs) are beneficial and safe for various diseases other than primary immunodeficiencies. Over the years, IVIG has been given for autoimmune diseases as an off-label adjunct therapy. While other biologic agents are indicated for rheumatoid arthritis (RA), IVIG may have a role for specific subgroups of RA patients where anti-cytokine blockers or rituximab may be unwarranted. Such subgroups may include patients with vasculitis, overlap rhupus syndrome, severe infections with active disease, and pregnancy. In addition, IVIG may be considered for juvenile chronic arthritis (JCA) and adult Still's disease. We review the literature for IVIG treatment in RA patients and for these subgroups. (C) 2015 Elsevier B.V. All rights reserved.

Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (K-d = similar to 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure-activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub-induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone post-translational modification.

RNA polymerase inhibitors like the CBR class that target the enzyme's complex catalytic center are attractive leads for new antimicrobials. Catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg2+ ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. We report crystal structures of CBR inhibitor/Escherichia coli RNA polymerase complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3'-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.

The discovery of steroid hormone receptors in brain regions that mediate every aspect of brain function has broadened the definition of 'neuroendocrinology' to include the reciprocal communication between the brain and the body via hormonal and neural pathways. The brain is the central organ of stress and adaptation to stress because it perceives and determines what is threatening, as well as the behavioral and physiological responses to the stressor. The adult and developing brain possess remarkable structural and functional plasticity in response to stress, including neuronal replacement, dendritic remodeling, and synapse turnover. Stress causes an imbalance of neural circuitry subserving cognition, decision-making, anxiety and mood that can alter expression of those behaviors and behavioral states. This imbalance, in turn, affects systemic physiology via neuroendocrine, autonomic, immune and metabolic mediators. In the short term, as for increased fearful vigilance and anxiety in a threatening environment, these changes may be adaptive. But, if the danger passes and the behavioral state persists along with the changes in neural circuitry, such maladaptation may need intervention with a combination of pharmacological and behavioral therapies, as is the case for chronic anxiety and depression. There are important sex differences in the brain responses to stressors that are in urgent need of further exploration. Moreover, adverse early-life experience, interacting with alleles of certain genes, produce lasting effects on brain and body over the life-course via epigenetic mechanisms. While prevention is most important, the plasticity of the brain gives hope for therapies that take into consideration brain-body interactions.

Background A substantial fraction of all American healthcare expenditures are potentially wasted, and practices that are not evidence-based could contribute to such waste. We sought to characterize whether Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT) tests of preoperative patients are used in a way unsupported by evidence and potentially wasteful. Methods and Findings We evaluated prospectively-collected patient data from 19 major teaching hospitals and 8 hospital-affiliated surgical centers in 7 states (Delaware, Florida, Maryland, Massachusetts, New Jersey, New York, Pennsylvania) and the District of Columbia. A total of 1,053,472 consecutive patients represented every patient admitted for elective surgery from 2009 to 2012 at all 27 settings. A subset of 682,049 patients (64.7%) had one or both tests done and history and physical (H&P) records available for analysis. Unnecessary tests for bleeding risk were defined as: PT tests done on patients with no history of abnormal bleeding, warfarin therapy, vitamin K-dependent clotting factor deficiency, or liver disease; or aPTT tests done on patients with no history of heparin treatment, hemophilia, lupus anticoagulant antibodies, or von Willebrand disease. We assessed the proportion of patients who received PT or aPTT tests who lacked evidence-based reasons for testing. Conclusions This study sought to bring the availability of big data together with applied comparative effectiveness research. Among preoperative patients, 26.2% received PT tests, and 94.3% of tests were unnecessary, given the absence of findings on H&P. Similarly, 23.3% of preoperative patients received aPTT tests, of which 99.9% were unnecessary. Among patients with no H&P findings suggestive of bleeding risk, 6.6% of PT tests and 7.1% of aPTT tests were either a false positive or a true positive (i.e. indicative of a previously-undiagnosed potential bleeding risk). Both PT and aPTT, designed as diagnostic tests, are apparently used as screening tests. Use of unnecessary screening tests raises concerns for the costs of such testing and the consequences of false positive results.