The rockefeller university

Atopic dermatitis (AD), the most common chronic inflammatory skin disease, is driven by both terminal keratinocyte differentiation defects and strong type 2 immune responses. In contrast to chronic plaque-type psoriasis, AD is now understood to be a much more heterogeneous disease, with additional activation of TH22, TH17/IL-23, and TH1 cytokine pathways depending on the subtype of the disease. In this review we discuss our current understanding of the AD immune map in both patients with early-onset and those with chronic disease. Clinical studies with broad and targeted therapeutics have helped to elucidate the contribution of various immune axes to the disease phenotype. Importantly, immune activation extends well beyond lesional AD because nonlesional skin and the blood component harbor AD-specific inflammatory changes. For this reason, future therapeutics will need to focus on a systemic treatment approach, especially in patients with moderate-to-severe disease.

Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate-aspartate shuttle, which is important for cytoplasmic NAD(+) regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate-aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD(+)/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene.

Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes, we previously demonstrated that CME is independent of the AP-2 adaptor protein complex, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosomerestricted protein cohort interacts with clathrin and drives CME. Here, we used a novel cryomilling affinity isolation strategy to preserve transient low-affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, Trypanosoma brucei (Tb) EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites.

Patterns of daily human activity are controlled by an intrinsic circadian clock that promotes 24 hr rhythms in many behavioral and physiological processes. This system is altered in delayed sleep phase disorder (DSPD), a common form of insomnia in which sleep episodes are shifted to later times misaligned with the societal norm. Here, we report a hereditary form of DSPD associated with a dominant coding variation in the core circadian clock gene CRY1, which creates a transcriptional inhibitor with enhanced affinity for circadian activator proteins Clock and Bmal1. This gain-of-function CRY1 variant causes reduced expression of key transcriptional targets and lengthens the period of circadian molecular rhythms, providing a mechanistic link to DSPD symptoms. The allele has a frequency of up to 0.6%, and reverse phenotyping of unrelated families corroborates late and/or fragmented sleep patterns in carriers, suggesting that it affects sleep behavior in a sizeable portion of the human population.

BACKGROUND Myocarditis is inflammation of the heart muscle that can follow various viral infections. Why children only rarely develop life-threatening acute viral myocarditis (AVM), given that the causal viral infections are common, is unknown. Genetic lesions might underlie such susceptibilities. Mouse genetic studies demonstrated that interferon (IFN)-alpha/beta immunity defects increased susceptibility to virus-induced myocarditis. Moreover, variations in human TLR3, a potent inducer of IFNs, were proposed to underlie AVM. OBJECTIVES This study sought to evaluate the hypothesis that human genetic factors may underlie AVM in previously healthy children. METHODS We tested the role of TLR3-IFN immunity using human induced pluripotent stem cell-derived cardiomyocytes. We then performed whole-exome sequencing of 42 unrelated children with acute myocarditis (AM), some with proven viral causes. RESULTS We found that TLR3-and STAT1-deficient cardiomyocytes were not more susceptible to Coxsackie virus B3 (CVB3) infection than control cells. Moreover, CVB3 did not induce IFN-alpha/beta and IFN-alpha/beta-stimulated genes in control cardiomyocytes. Finally, exogenous IFN-alpha did not substantially protect cardiomyocytes against CVB3. We did not observe a significant enrichment of rare variations in TLR3-or IFN-alpha/beta-related genes. Surprisingly, we found that homozygous but not heterozygous rare variants in genes associated with inherited cardiomyopathies were significantly enriched in AM-AVM patients compared with healthy individuals (p = 2.22E-03) or patients with other diseases (p = 1.08E-04). Seven of 42 patients (16.7%) carried rare biallelic (homozygous or compound heterozygous) nonsynonymous or splice-site variations in 6 cardiomyopathy-associated genes (BAG3, DSP, PKP2, RYR2, SCN5A, or TNNI3). CONCLUSIONS Previously silent recessive defects of the myocardium may predispose to acute heart failure presenting as AM, notably after common viral infections in children. (C) 2017 by the American College of Cardiology Foundation.

Individuals with Parkinson's disease (PD) often suffer from comorbid depression. P11 (S100A10), a member of the S100 family of proteins, is expressed widely throughout the body and is involved in major depressive disorder and antidepressant response. Central p11 levels are reduced in postmortem tissue from depressed individuals; however, p11 has not yet been investigated in PD patients with depression or those without depression. We investigated p11 levels in postmortem PD brains and assessed whether peripheral p11 levels correlate with disease severity. Substantia nigra, putamen, and cortical p11 protein levels were assessed in postmortem brain samples from PD patients and matched controls. In a different set of postmortem brains, p11 mRNA expression was measured in dopaminergic cells from the substantia nigra. Both p11 protein and mRNA levels were decreased in PD patients. Peripheral p11 protein levels were investigated in distinct leukocyte populations from PD patients with depression and those without depression. Monocyte, natural killer (NK) cell, and cytotoxic T-cell p11 levels were positively associated with the severity of PD, and NK cell p11 levels were positively associated with depression scores. Given that inflammation plays a role in both PD and depression, it is intriguing that peripheral p11 levels are altered in immune cells in both conditions. Our data provide insight into the pathological alterations occurring centrally and peripherally in PD. Moreover, if replicated in other cohorts, p11 could be an easily accessible biomarker for monitoring the severity of PD, especially in the context of comorbid depression.

Channelrhodopsins (ChRs) are light-activated ion channels widely employed for photostimulation of excitable cells. This study focuses on ReaChR, a chimeric ChR variant with optimal properties for optogenetic applications. We combined electrophysiological recordings with infrared and UV-visible spectroscopic measurements to investigate photocurrents and photochemical properties of ReaChR. Our data imply that ReaChR is green-light activated (lambda(max) = 532 nm) with a nonrhodopsin- like action spectrum peaking at 610 nm for stationary photocurrents. This unusual spectral feature is associated with photoconversion of a previously unknown light-sensitive, blue-shifted photocycle intermediate L (lambda(max) = 495 nm), which is accumulated under continuous illumination. To explain the complex photochemical reactions, we propose a symmetrical two-cycle-model based on the two C-15 = N isomers of the retinal cofactor with either syn-or anti-configuration, each comprising six consecutive states D, K, L, M, N, and O. Ion conduction involves two states per cycle, the late M-(M-2) with a deprotonated retinal Schiff base and the consecutive green-absorbing N-state that both equilibrate via reversible reprotonation. In our model, a fraction of the deprotonated M-intermediate of the anti-cycle may be photoconverted-as the L-state-back to its inherent dark state, or to its M-state pendant (M') of the syn-cycle. The latter reaction pathway requires a C-13 - C-14, C-15 - N doubleisomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C-13 = C-14 double-bond isomerization.

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell's transduction channels, calcium's role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog's sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle's instantaneous force-displacement relation and measurement of a bundle's spontaneous oscillation.

The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice(1-5). Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFa superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.

The shortening of human telomeres has two opposing effects during cancer development. On the one hand, telomere shortening can exert a tumour-suppressive effect through the proliferation arrest induced by activating the kinases ATM and ATR at unprotected chromosome ends. On the other hand, loss of telomere protection can lead to telomere crisis, which is a state of extensive genome instability that can promote cancer progression. Recent data, reviewed here, provide new evidence for the telomere tumour suppressor pathway and has revealed that telomere crisis can induce numerous cancer-relevant changes, including chromothripsis, kataegis and tetraploidization.