The rockefeller university

Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.

Piwi-interacting RNAs (piRNAs), long thought to be restricted to germline, have recently been discovered in neurons of Aplysia, with a role in the epigenetic regulation of gene expression underlying long-term memory. We here ask whether piwi/piRNAs are also expressed and have functional roles in the mammalian brain. Large-scale RNA sequencing and subsequent analysis of protein expression revealed the presence in brain of several piRNA biogenesis factors including a mouse piwi (Mili), as well as small RNAs, albeit at low levels, resembling conserved piRNAs in mouse testes [primarily LINE1 (long interspersed nuclear element1) retrotransposon- derived]. Despite the seeming low expression of these putative piRNAs, single-base pair CpG methylation analyses across the genome of Mili/piRNA-deficient (Mili(-/-)) mice demonstrate that brain genomic DNA is preferentially hypomethylated within intergenic areas and LINE1 promoter areas of the genome. Furthermore, Mili mutant mice exhibit behavioral deficits such as hyperactivity and reduced anxiety. These results suggest that putative piRNAs exist in mammalian brain, and similar to the role of piRNAs in testes, they may be involved in the silencing of retrotransposons, which in brain have critical roles in contributing to genomic heterogeneity underlying adaptation, stress response, and brain pathology. We also describe the presence of another class of small RNAs in the brain, with features of endogenous siRNAs, which may have taken over the role of invertebrate piRNAs in their capacity to target both transposons, as well as protein-coding genes. Thus, RNA interference through gene and retrotransposon silencing previously encountered in Aplysia may also have potential roles in the mammalian brain.

Two papers in this issue of Molecular Cell provide insights into how the multisubunit Mediator coactivator complex dynamically links enhancer-bound activators to the RNA polymerase II machinery at the core promoter.

Cobblestone lissencephaly (COB) is a severe brain malformation in which overmigration of neurons and glial cells into the arachnoid space results in the formation of cortical dysplasia. COB occurs in a wide range of genetic disorders known as dystroglycanopathies, which are congenital muscular dystrophies associated with brain and eye anomalies and range from Walker-Warburg syndrome to Fukuyama congenital muscular dystrophy. Each of these conditions has been associated with alpha-dystroglycan defects or with mutations in genes encoding basement membrane components, which are known to interact with alpha-dystroglycan. Our screening of a cohort of 25 families with recessive forms of COB identified six families affected by biallelic mutations in TMTC3 (encoding transmembrane and tetratricopeptide repeat containing 3), a gene without obvious functional connections to alpha-dystroglycan. Most affected individuals showed brainstem and cerebellum hypoplasia, as well as ventriculomegaly. However, the minority of the affected individuals had eye defects or elevated muscle creatine phosphokinase, separating the TMTC3 COB phenotype from typical congenital muscular dystrophies. Our data suggest that loss of TMTC3 causes COB with minimal eye or muscle involvement.

Pathogenic amino acid substitutions of the common E3 component (hE3) of the human alpha-ketoglutarate dehydrogenase and the pyruvate dehydrogenase complexes lead to severe metabolic diseases (E3 deficiency), which usually manifest themselves in cardiological and/or neurological symptoms and often cause premature death. To date, 14 disease-causing amino acid substitutions of the hE3 component have been reported in the clinical literature. None of the pathogenic protein variants has lent itself to high-resolution structure elucidation by X-ray or NMR. Hence, the structural alterations of the hE3 protein caused by the disease-causing mutations and leading to dysfunction, including the enhanced generation of reactive oxygen species by selected disease-causing variants, could only be speculated. Here we report results of an examination of the effects on the protein structure of ten pathogenic mutations of hE3 using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), a new and state-of-the-art approach of solution structure elucidation. On the basis of the results, putative structural and mechanistic conclusions were drawn regarding the molecular pathogenesis of each disease-causing hE3 mutation addressed in this study. (C) 2016 Elsevier B.V. All rights reserved.

Mild versus severe psoriasis is often distinguished by clinical measures such as the extent of skin involvement or Psoriasis Area and Severity Index score, both of which use arbitrary boundaries. It is widely assumed that severe psoriasis involves higher levels of skin inflammation, but comparative molecular profiles of mild versus severe disease have not been performed. In this study, we used immunohistochemistry, reverse transcription PCR, and gene arrays to determine the phenotype of North American patients with mild psoriasis (n = 34, mean PASI score = 5.5) versus severe psoriasis (n = 23, mean PASI score = 23.2). Overall, skin inflammation, defined as the sum of T-cell infiltration/activation and IL-17-mediated epidermal responses, was not higher in severe psoriasis lesions. Surprisingly, mild psoriasis was characterized by higher numbers of T cells in skin lesions, higher IL-17A expression, and stronger expression of the core psoriasis transcriptome. In contrast, severe psoriasis was characterized by stronger expression of some epidermal response genes (TGFA, CALM1, SMPD3, and IL1RL2). However, a key molecular distinction was higher expression of negative immune regulatory genes (CTLA4, CD69 and PD-L1) in mild lesions compared with severe psoriasis lesions. These data have important implications for treating psoriasis across the spectrum of disease, as well as for potential mechanisms that allow psoriasis to progress to more extensive cutaneous disease.

The earliest aspects of human embryogenesis remain mysterious. To model patterning events in the human embryo, we used colonies of human embryonic stem cells (hESCs) grown on micropatterned substrate and differentiated with BMP4. These gastruloids recapitulate the embryonic arrangement of the mammalian germ layers and provide an assay to assess the structural and signaling mechanisms patterning the human gastrula. Structurally, high density hESCs localize their receptors to transforming growth factor 13 at their lateral side in the center of the colony while maintaining apical localization of receptors at the edge. This relocalization insulates cells at the center from apically applied ligands while maintaining response to basally presented ones. In addition, BMP4 directly induces the expression of its own inhibitor, NOGGIN, generating a reaction-diffusion mechanism that underlies patterning. We develop a quantitative model that integrates edge sensing and inhibitors to predict human fate positioning in gastruloids and, potentially, the human embryo.

There is growing evidence that human genetic variants contribute to liver fibrosis in subjects with hepatitis C virus (HCV) monoinfection, but this aspect has been little investigated in patients coinfected with HCV and human immunodeficiency virus (HIV). We performed the first genome-wide association study of liver fibrosis progression in patients coinfected with HCV and HIV, using the well-characterized French National Agency for Research on AIDS and Viral Hepatitis CO13 HEPAVIH cohort. Liver fibrosis was assessed by elastography (FibroScan), providing a quantitative fibrosis score. After quality control, a genome-wide association study was conducted on 289 Caucasian patients, for a total of 8,426,597 genotyped (Illumina Omni2.5 BeadChip) or reliably imputed single-nucleotide polymorphisms. Single-nucleotide polymorphisms with P values <10(-6) were investigated in two independent replication cohorts of European patients infected with HCV alone. Two signals of genome-wide significance (P < 5 x 10(-8)) were obtained. The first, on chromosome 3p25 and corresponding to rs61183828 (P = 3.8 x 10(-9)), was replicated in the two independent cohorts of patients with HCV monoinfection. The cluster of single-nucleotide polymorphisms in linkage disequilibrium with rs61183828 was located close to two genes involved in mechanisms affecting both cell signaling and cell structure (CAV3) or HCV replication (RAD18). The second signal, obtained with rs11790131 (P = 9.3 x 10(-9)) on chromosome region 9p22, was not replicated. Conclusion: This genome-wide association study identified a new locus associated with liver fibrosis severity in patients with HIV/HCV coinfection, on chromosome 3p25, a finding that was replicated in patients with HCV monoinfection; these results provide new relevant hypotheses for the pathogenesis of liver fibrosis in patients with HIV/HCV coinfection that may help define new targets for drug development or new prognostic tests, to improve patient care. (Hepatology 2016;64:1462-1472)