The rockefeller university

We have isolated Clostridium perfringens type B, an epsilon toxin-secreting bacillus, from a young woman at clinical presentation of Multiple Sclerosis (MS) with actively enhancing lesions on brain MRI. This finding represents the first time that C. perfringens type B has been detected in a human. Epsilon toxin's tropism for the blood-brain barrier (BBB) and binding to oligodendrocytes/myelin makes it a provocative candidate for nascent lesion formation in MS. We examined a well-characterized population of MS patients and healthy controls for carriage of C. perfringens toxinotypes in the gastrointestinal tract. The human commensal Clostridium perfringens type A was present in approximately 50% of healthy human controls compared to only 23% in MS patients. We examined sera and CSF obtained from two tissue banks and found that immunoreactivity to ETX is 10 times more prevalent in people with MS than in healthy controls, indicating prior exposure to ETX in the MS population. C. perfringens epsilon toxin fits mechanistically with nascent MS lesion formation since these lesions are characterized by BBB permeability and oligodendrocyte cell death in the absence of an adaptive immune infiltrate.

A complete understanding of nervous system function cannot be achieved without the identification of its component cell types. In this Perspective, we explore a series of related issues surrounding cell identity and how revolutionary methods for labeling and probing specific neuronal types have clarified this question. Specifically, we ask the following questions: what is the purpose of such diversity, how is it generated, how is it maintained, and, ultimately, how can one unambiguously identity one cell type from another? We suggest that each cell type can be defined by a unique and conserved molecular ground state that determines its capabilities. We believe that gaining an understanding of these molecular barcodes will advance our ability to explore brain function, enhance our understanding of the biochemical basis of CNS disorders, and aid in the development of novel therapeutic strategies.

Holliday junctions (HJs), the DNA intermediates of homologous recombination, need to be faithfully processed in order to preserve genome integrity. In human cells, the BLM helicase complex promotes nonnucleolytic dissolution of double HJs. In vitro, HJs may be nucleolytically processed by MUS81-EME1, GEN1, and SLX4-SLX1. Here, we exploit human SLX4-null cells to examine the requirements for HJ resolution in vivo. Lack of BLM and SLX4 or GEN1 and SLX4 is synthetically lethal in the absence of exogenous DNA damage, and lethality is a consequence of dysfunctional mitosis proceeding in the presence of unprocessed HJs. Thus, GEN1 activity cannot be substituted for the SLX4-associated nucleases, and one of the HJ resolvase activities, either of those associated with SLX4 or with GEN1, is required for cell viability, even in the presence of BLM. In vivo HJ resolution depends on both SLX4-associated MUS81-EME1 and SLX1, suggesting that they are acting in concert in the context of SLX4.

A measurement is presented of the ratio of the inclusive 3-jet cross section to the inclusive 2-jet cross section as a function of the average transverse momentum, < p(T1,2)>, of the two leading jets in the event. The data sample was collected during 2011 at a proton-proton centre-of-mass energy of 7 TeV with the CMS detector at the LHC, corresponding to an integrated luminosity of 5.0 fb(-1). The strong coupling constant at the scale of the Z boson mass is determined to be alpha(S)(M-Z) = 0.1148 +/- 0.0014 (exp.) +/- 0.0018 (PDF) +/- 0.0050 (theory), by comparing the ratio in the range 0.42 < < p(T1,2)> < 1.39 TeV to the predictions of perturbative QCD at next-to-leading order. This is the first determination of alpha(S)(M-Z) from measurements at momentum scales beyond 0.6 TeV. The predicted ratio depends only indirectly on the evolution of the parton distribution functions of the proton such that this measurement also serves as a test of the evolution of the strong coupling constant. No deviation from the expected behaviour is observed.

We established a human tissue explant model to facilitate study of cutaneous squamous cell carcinoma. We accomplished this by implanting debulked SCC, from surgical discard, into nude rats. Human SCC remained viable and continued to proliferate for at least 4 weeks and showed evidence of neovascularization. At 4 weeks, SCC implants showed a trend toward increased PCNA positive cells compared to fresh SCC cells/mm(2) tissue) supporting continued proliferation throughout engraftment. Von Willebrand's Factor (VWF) positive cells were found within implants and likely represented rat vessel neovascularization. Human Langerhans' (Langerin+) cells, but no T cells (CD3+, CD8+, FoxP3+), macrophages (CD163), or NK cells (NKp46), were present in SCC implants at 4 weeks. These findings support the possibility that LCs fail to migrate from cutaneous SCC and thus contribute to lack of effective antitumor response. Our findings also provide a novel model system for further study of primary cutaneous SCC.

Background. Tumor necrosis factor (TNF) is a key immune regulator of tuberculosis resistance, as exemplified by the highly increased risk of tuberculosis disease among individuals receiving TNF-blocker therapy. Methods. We determined the extent of TNF production after stimulation with BCG or BCG plus interferon gamma (IFN-gamma) using a whole blood assay in 392 children belonging to 135 nuclear families from an area hyperendemic for tuberculosis in South Africa. We conducted classical univariate and bivariate genome-wide linkage analysis of TNF production using the data from both stimulation protocols by means of an extension of the maximum-likelihood-binomial method for quantitative trait loci to multivariate analysis. Results. Stimulation of whole blood by either BCG or BCG plus IFN-gamma resulted in a range of TNF release across subjects. Extent of TNF production following both stimulation protocols was highly correlated (r = 0.81). We failed to identify genetic linkage of TNF release when considering each stimulus separately. However, using a multivariate approach, we detected a major pleiotropic locus (P < 10(-5)) on chromosome region 11p15, termed TNF locus 1 (TNF1), that controlled TNF production after stimulation by both BCG alone and BCG plus IFN-gamma. Conclusions. The TNF1 locus was mapped in the vicinity of the TST1 locus, previously identified in the same family sample, that controls tuberculin skin test (TST) negativity per se, that is, T-cell-independent resistance to Mycobacterium tuberculosis infection. This suggested that there is a connection between TST negativity per se and TNF production.

Regulation of gene expression is a vital part of the cellular stress response, yet the full set of proteins that orchestrate this regulation remains unknown. Snt2 is a Saccharomyces cerevisiae protein whose function has not been well characterized that was recently shown to associate with Ecm5 and the Rpd3 deacetylase. Here, we confirm that Snt2, Ecm5, and Rpd3 physically associate. We then demonstrate that cells lacking Rpd3 or Snt2 are resistant to hydrogen peroxide (H2O2)-mediated oxidative stress and use chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to show that Snt2 and Ecm5 recruit Rpd3 to a small number of promoters and in response to H2O2, colocalize independently of Rpd3 to the promoters of stress response genes. By integrating ChIP-seq and expression analyses, we identify target genes that require Snt2 for proper expression after H2O2. Finally, we show that cells lacking Snt2 are also resistant to nutrient stress imparted by the TOR (target of rapamycin) pathway inhibitor rapamycin and identify a common set of genes targeted by Snt2 and Ecm5 in response to both H2O2 and rapamycin. Our results establish a function for Snt2 in regulating transcription in response to oxidative stress and suggest Snt2 may also function in multiple stress pathways.

IL-12R1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL-12R1 is a receptor chain of both the IL-12 and the IL-23 receptor and deficiency of IL-12R1 thus abolishes both IL-12 and IL-23 signaling. IL-12R1 deficiency is caused by bi-allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL-12R1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL-12R1 protein. In addition to disease-causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database (www.LOVD.nl/IL12RB1). In this article, we review the function of IL-12R1 and molecular genetics of human IL12RB1. (C) 2013 Wiley Periodicals, Inc.

Nuclear shape and size vary between species, during development, and in many tissue pathologies, but the causes and effects of these differences remain poorly understood. During fertilization, sperm nuclei undergo a dramatic conversion from a heavily compacted form into decondensed, spherical pronuclei, accompanied by rapid nucleation of microtubules from centrosomes. Here we report that the assembly of the spherical nucleus depends on a critical balance of microtubule dynamics, which is regulated by the chromatin-binding protein Developmental pluripotency-associated 2 (Dppa2). Whereas microtubules normally promote sperm pronuclear expansion, in Dppa2-depleted Xenopus egg extracts excess microtubules cause pronuclear assembly defects, leading to abnormal morphology and disorganized DNA replication. Dppa2 inhibits microtubule polymerization in vitro, and Dppa2 activity is needed at a precise time and location during nascent pronuclear formation. This demonstrates a strict spatiotemporal requirement for local suppression of microtubules during nuclear formation, fulfilled by chromatin-bound microtubule regulators.

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.