The rockefeller university

Janus kinase (JAK) inhibitors have achieved positive responses in myeloproliferative neoplasms, but at the expense of decreased natural killer (NK) cell numbers and compromised function. Selective JAK2 inhibition may also have a role in preventing and treating graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Although JAK inhibitors can impair monocyte-derived dendritic cell (moDC) activation and function and suppress effector T-cell responses, the effects on NK cells and the relevant mechanisms remain undefined. Using common gamma(c) cytokines and distinct human dendritic cell (DC) subtypes, we compared the effects of a JAK2-specific (TG101348) with a less selective JAK1/ 2 (ruxolitinib) inhibitor on NK-cell activation and function. Ruxolitinib treatment completely blocked IL2, IL15, and DC-mediated STAT5 phosphorylation, along with the capacity of NK cells to secrete IFN gamma or lyse NK cell-sensitive targets. Only NK-cell proliferation stimulated by moDCs resisted ruxolitinib treatment. In contrast, TG101348 treatment of stimulated NK cells resulted in far less functional compromise. TG101348 completely inhibited only soluble IL15-mediated STAT5 phosphorylation, which Langerhans-type DCs (LCs), presenting membrane-bound IL15 in trans, could salvage. These results demonstrate that ruxolitinib's nonselective inhibition of JAK1/2 results in profound NK-cell dysfunction by blocking downstream pSTAT5, hence providing a persuasive rationale for the development of selective JAK2 inhibitors for immunotherapeutic applications. (C) 2016 AACR.

Dengue virus (DENV) infection in the presence of reactive, non-neutralizing immunoglobulin G (IgG) (RNNIg) is the greatest risk factor for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Progression to DHF/DSS is attributed to antibody-dependent enhancement (ADE); however, because only a fraction of infections occurring in the presence of RNNIg advance to DHF/DSS, the presence of RNNIg alone cannot account for disease severity. We discovered that DHF/DSS patients respond to infection by producing IgGs with enhanced affinity for the activating Fc receptor Fc gamma RIIIA due to afucosylated Fc glycans and IgG1 subclass. RNNIg enriched for afucosylated IgG1 triggered platelet reduction in vivo and was a significant risk factor for thrombocytopenia. Thus, therapeutics and vaccines restricting production of afucosylated, IgG1 RNNIg during infection may prevent ADE of DENV disease.

We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a beta-barrel membrane protein, and the G-protein-coupled receptor NTR1, an alpha-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.

Based on the energy conservation law, a new formula for the photon momentum was proposed in 11 I. In this work, some defects of this new proposal are discussed. It is shown that this new proposal can lead to negative energy of photons. In addition, the group velocity of massive particles exceeds the light velocity which violates the causality principle. Finally, we show that the momentum conservation law combined with the conventional formula for the photon momentum can predict a rational velocity for the velocity of the relativistic scattered particles which revalidate the conventional formula for the photon momentum. (C) 2017 Elsevier GmbH. All rights reserved.

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo. Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels.

CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application.

Vision influences behavior, but ongoing behavior also modulates vision in animals ranging from insects to primates. The function and biophysical mechanisms of most such modulations remain unresolved. Here, we combine behavioral genetics, electrophysiology, and high-speed videography to advance a function for behavioral modulations of visual processing in Drosophila. We argue that a set of motion-sensitive visual neurons regulate gaze-stabilizing head movements. We describe how, during flight turns, Drosophila perform a set of head movements that require silencing their gaze-stability reflexes along the primary rotation axis of the turn. Consistent with this behavioral requirement, we find pervasive motor-related inputs to the visual neurons, which quantitatively silence their predicted visual responses to rotations around the relevant axis while preserving sensitivity around other axes. This work proposes a function for a behavioral modulation of visual processing and illustrates how the brain can remove one sensory signal from a circuit carrying multiple related signals.

Ff filamentous phage (fd, M13 and f1) of Escherichia coli have been the workhorse of phage display technology for the past 30 years. Dominance of Ff over other bacteriophage in display technology stems from the titres that are about 100-fold higher than any other known phage, efficacious transformation ensuring large library size and superior stability of the virion at high temperatures, detergents and pH extremes, allowing broad range of biopanning conditions in screening phage display libraries. Due to the excellent understanding of infection and assembly requirements, Ff phage have also been at the core of phage-assisted continual protein evolution strategies (PACE). This chapter will give an overview of the Ff filamentous phage structure and biology, emphasizing those properties of the Ff phage life cycle and virion that are pertinent to phage display applications.

Hypertension in male and aging female rodents is associated with glutamate-dependent plasticity in the hypothalamus, but existing models have failed to capture distinct transitional menopausal phases that could have a significant impact on the synaptic plasticity and emergent hypertension. In rodents, accelerated ovarian failure (AOF) induced by systemic injection of 4-vinylcyclohexane diepoxide mimics the estrogen fluctuations seen in human menopause including the perimenopause transition (peri-AOF) and postmenopause (post-AOF). Thus, we used the mouse AOF model to determine the impact of slow-pressor angiotensin II (Ang ll) administration on blood pressure and on the subcellular distribution of obligatory N-methyl-D-aspartate (NMDA) receptor GIuN1 subunits in the paraventricular hypothalamic nu-cleus (PVN), a key estrogen-responsive cardiovascular regulatory area. Estrogen-sensitive neuronal profiles were identified in mice expressing enhanced green fluorescent protein under the promoter for estrogen receptor (ER) beta, a major ER in the PVN. Slow-pressor Angll increased arterial blood pressure in mice at peri-and post-AOF time points. In control oil-injected (nonhypertensive) mice, Angll decreased the total number of GIuN1 in ERR-containing PVN dendrites. In contrast, Angll resulted in a reapportionment of GIuN1 from the cytoplasm to the plasma membrane of ER beta-containing PVN dendrites in peri-AOF mice. Moreover, in post-AOF mice, Angll increased total GIuN1, dendritic size and radical production in ERR-containing neurons. These results indicate that unique patterns of hypothalamic glutamate receptor plasticity and dendritic structure accompany the elevated blood pressure in peri-and post-AOF time points. Our findings suggest the possibility that distinct neurobiological processes are associated with the increased blood pressure during perimenopausal and postmenopausal periods.

The paraventricular nucleus of the thalamus (PVT) appears to participate in drug addiction. Recent evidence in rats shows that ethanol drinking is increased by orexin/hypocretin (OX) afferents from the hypothalamus, acting specifically in the anterior (aPVT) rather than posterior (pPVT) PVT subregion. The present study sought to identify neuropeptides transcribed within the PVT, which themselves might contribute to ethanol drinking and possibly mediate the actions of OX. We discovered that substance P (SP) in the aPVT can stimulate intermittent-access ethanol drinking, similar to OX, and that SP receptor [neurokinin 1 receptor/tachykinin receptor 1 (NK1R)] antagonists in this subregion reduce ethanol drinking. As with OX, this effect is site specific, with SP in the pPVT or dorsal third ventricle having no effect on ethanol drinking, and it is behaviorally specific, with SP in the aPVT reducing the drinking of sucrose and stimulating it in the pPVT. A close relationship between SP and OX was demonstrated by a stimulatory effect of local OX injection on SP mRNA and peptide levels, specifically in the aPVT but not pPVT, and a stimulatory effect of OX on SP expression in isolated thalamic neurons, reflecting postsynaptic actions. A functional relationship between OX and SP in the aPVT is suggested by our additional finding that ethanol drinking induced by OX is blocked by a local NK1R antagonist administered at a sub-threshold dose. These results, suggesting that SP in the aPVT mediates the stimulatory effect of OX on ethanol drinking, identify a new role for SP in the control of this behavior.