The rockefeller university

While addition of the first-approved protease inhibitors (PIs), telaprevir and boceprevir, to pegylated interferon (PEG-IFN) and ribavirin (RBV) combination therapy significantly increased sustained virologic response (SVR) rates, PI-based triple therapy for the treatment of chronic hepatitis C virus (HCV) infection was prone to the emergence of resistant viral variants. Meanwhile, multiple direct acting antiviral agents (DAAs) targeting either the HCV NS3/4A protease, NS5A or NS5B polymerase have been approved and these have varying potencies and distinct propensities to provoke resistance. The pre-clinical in vivo assessment of drug efficacy and resistant variant emergence underwent a great evolution over the last decade. This field had long been hampered by the lack of suitable small animal models that robustly support the entire HCV life cycle. In particular, chimeric mice with humanized livers (humanized mice) and chimpanzees have been instrumental for studying HCV inhibitors and the evolution of drug resistance. In this review, we present the different in vivo HCV infection models and discuss their applicability to assess HCV therapy response and emergence of resistant variants.

Maternal consumption of a high-fat diet (HFD) during pregnancy is found to stimulate the genesis of hypothalamic orexigenic peptide neurons in the offspring, while HFD intake in adult animals produces a systemic low-grade inflammation which increases neuroimmune factors that may affect neurogenesis and neuronal migration. Building on this evidence and our recent study showing that the inflammatory chemokine, CCL2, stimulates the migration of hypothalamic neurons and expression of orexigenic neuropeptides, we tested here the possibility that prenatal exposure to a HFD in rats affects this chemokine system, both CCL2 and its receptors, CCR2 and CCR4, and alters its actions on hypothalamic neurons, specifically those expressing the neuropeptides, enkephalin (ENK) and galanin (GAL). Using primary dissociated hypothalamic neurons extracted from embryos on embryonic day 19, we found that prenatal HFD exposure compared to chow control actually reduces the expression of CCL2 in these hypothalamic neurons, while increasing CCR2 and CCR4 expression, and also reduces the sensitivity of hypothalamic neurons to CCL2. The HFD abolished the dose-dependent, stimulatory effect of CCL2 on the number of migrated neurons and even shifted its normal stimulatory effect on migrational velocity and distance traveled by control neurons to an inhibition of migration. Further, it abolished the dose-dependent, stimulatory effect of CCL2 on neuronal expression of ENK and GAL. These results demonstrate that prenatal HFD exposure greatly disturbs the functioning of the CCL2 chemokine system in embryonic hypothalamic neurons, reducing its endogenous levels and ability to promote the migration of neurons and their expression of orexigenic peptides. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Since their discovery in patients with autosomal dominant (AD) chronic mucocutaneous candidiasis (CMC) in 2011, heterozygous STAT1 gain-of-function (GOF) mutations have increasingly been identified worldwide. The clinical spectrum associated with them needed to be delineated. We enrolled 274 patients from 167 kindreds originating from 40 countries from 5 continents. Demographic data, clinical features, immunological parameters, treatment, and outcome were recorded. The median age of the 274 patients was 22 years (range, 1-71 years); 98% of them had CMC, with a median age at onset of 1 year (range, 0-24 years). Patients often displayed bacterial (74%) infections, mostly because of Staphylococcus aureus (36%), including the respiratory tract and the skin in 47% and 28% of patients, respectively, and viral (38%) infections, mostly because of Herpesviridae (83%) and affecting the skin in 32% of patients. Invasive fungal infections (10%), mostly caused by Candida spp. (29%), and mycobacterial disease (6%) caused by Mycobacterium tuberculosis, environmental mycobacteria, or Bacille Calmette-Guerin vaccines were less common. Many patients had autoimmune manifestations (37%), including hypothyroidism(22%), type 1 diabetes (4%), blood cytopenia (4%), and systemic lupus erythematosus (2%). Invasive infections (25%), cerebral aneurysms(6%), and cancers (6%) were the strongest predictors of poor outcome. CMC persisted in 39% of the 202 patients receiving prolonged antifungal treatment. Circulating interleukin-17A-producing T-cell count was low for most (82%) but not all of the patients tested. STAT1 GOF mutations underlie AD CMC, as well as an unexpectedly wide range of other clinical features, including not only a variety of infectious and autoimmune diseases, but also cerebral aneurysms and carcinomas that confer a poor prognosis.

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs)(1,2), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2-4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell-and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2(-/-) mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

We report a measurement of the forward-backward asymmetry, A(FB), in b (b) over bar pairs produced in proton-antiproton collisions and identified by muons from semileptonic b-hadron decays. The event sample is collected at a center-of-mass energy of root s = 1.96 TeV with the CDF II detector and corresponds to 6.9 fb(-1) of integrated luminosity. We obtain an integrated asymmetry of A(FB)(b (b) over bar) = (1.2 +/- 0.7)% at the particle level for b-quark pairs with invariant mass, m(b (b) over bar), down to 40 GeV/c(2) and measure the dependence of A(FB)(b (b) over bar) on m(b (b) over bar). The results are compatible with expectations from the standard model.

Antimetabolites that affect nucleotide metabolism are frontline chemotherapy agents in several cancers and often successfully target one carbon metabolism. However, the precise mechanisms and resulting determinants of their therapeutic value are unknown. We show that 5-fluorouracil (5-FU), a commonly used antimetabolite therapeutic with varying efficacy, induces specific alterations to nucleotide metabolism by disrupting pyrimidine homeostasis. An integrative metabolomics analysis of the cellular response to 5-FU reveals intracellular uracil accumulation, whereas deoxyuridine levels exhibited increased flux into the extracellular space, resulting in an induction of overflow metabolism. Subsequent analysis from mice bearing colorectal tumors treated with 5-FU show specific secretion of metabolites in tumor-bearing mice into serum that results from alterations in nucleotide flux and reduction in overflowmetabolism. Together, these findings identify a determinant of an antimetabolite response that may be exploited to more precisely define the tumors that could respond to targeting cancer metabolism.

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1-Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2-7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2-7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2-7 had to be phosphorylated for binding to phospho-Tof1-Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2-7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1-Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1-Ter complex.

While engagement of the inhibitory Fc gamma-receptor (Fc gamma R) IIB is an absolute requirement for in vivo antitumor activity of agonistic mouse anti-CD40 monoclonal antibodies (mAbs), a similar requirement for human mAbs has been disputed. By using a mouse model humanized for its Fc gamma Rs and CD40, we revealed that Fc gamma RIIB engagement is essential for the activity of human CD40 mAbs, while engagement of the activating Fc gamma RIIA inhibits this activity. By engineering Fc variants with selective enhanced binding to Fc gamma RIIB, but not to Fc gamma RIIA, significantly improved antitumor immunity was observed. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using appropriate preclinical models that accurately reflect the unique affinities and cellular expression of human Fc gamma R.