The rockefeller university

Background: ZFP36, also known as tristetraprolin or TTP, and ELAVL1, also known as HuR, are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embryonic kidney cells, and examined the combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. Results: Targets bound and negatively regulated by ZFP36 include transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance upon ZFP36 overexpression independent of effects from confounding features. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences ELAVL1 interacts with to stabilize transcripts. Conclusions: ZFP36-RNA target specificities in vivo are quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to unravel in vivo combinatorial regulation by RNA-binding proteins.

The prevalence of autism spectrum disorders (ASDs) has increased 20-fold over the past 50 years to >1% of US children. Although twin studies attest to a high degree of heritability, the genetic risk factors are still poorly understood. We analyzed data from two independent populations using u-statistics for genetically structured wide-locus data and added data from unrelated controls to explore epistasis. To account for systematic, but disease-unrelated differences in (non-randomized) genome-wide association studies (GWAS), a correlation between P-values and minor allele frequency with low granularity data and for conducting multiple tests in overlapping genetic regions, we present a novel study-specific criterion for 'genome-wide significance'. From recent results in a comorbid disease, childhood absence epilepsy, we had hypothesized that axonal guidance and calcium signaling are involved in autism as well. Enrichment of the results in both studies with related genes confirms this hypothesis. Additional ASD-specific variations identified in this study suggest protracted growth factor signaling as causing more severe forms of ASD. Another cluster of related genes suggests chloride and potassium ion channels as additional ASD-specific drug targets. The involvement of growth factors suggests the time of accelerated neuronal growth and pruning at 9-24 months of age as the period during which treatment with ion channel modulators would be most effective in preventing progression to more severe forms of autism. By extension, the same computational biostatistics approach could yield profound insights into the etiology of many common diseases from the genetic data collected over the last decade.

Many prokaryotes possess an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR loci produce small guide RNAs (crRNAs) that, in conjunction with flanking CRISPR-associated (cas) genes, combat viruses and block plasmid transfer by an antisense targeting mechanism. CRISPR-Cas systems have been classified into three types (I to III) that employ distinct mechanisms of crRNA biogenesis and targeting. The type III-A system in Staphylococcus epidermidis RP62a blocks the transfer of staphylococcal conjugative plasmids and harbors nine cas-csm genes. Previous biochemical analysis indicated that Cas10, Csm2, Csm3, Csm4, and Csm5 form a crRNA-containing ribonucleoprotein complex; however, the roles of these genes toward antiplasmid targeting remain unknown. Here, we determined the cas-csm genes that are required for antiplasmid immunity and used genetic and biochemical analyses to investigate the functions of predicted motifs and domains within these genes. We found that many mutations affected immunity by impacting the formation of the Cas10-Csm complex or crRNA biogenesis. Surprisingly, mutations in the predicted nuclease domains of the members of the Cas10-Csm complex had no detectable effect on antiplasmid immunity or crRNA biogenesis. In contrast, the deletion of csm6 and mutations in the cas10 Palm polymerase domain prevented CRISPR immunity without affecting either complex formation or crRNA production, suggesting their involvement in target destruction. By delineating the genetic requirements of this system, our findings further contribute to the mechanistic understanding of type III CRISPR-Cas systems.

Chronic inflammation may contribute to neuropsychological impairments in individuals with HIV, and modulation of this inflammatory response by opiate receptor ligands is important in light of the prevalence of drug use in HIV populations. Exogenous MOR and KOR agonists have differential effects on central nervous system (CNS) immunity and, while some data suggest KOR agonists are immunosuppressive, the KOR agonist dynorphin has been shown to stimulate human monocyte chemotaxis. In this study, we examined mRNA levels of endogenous opioid receptors OPRK1 and OPRM1, prodynorphin (PDYN), macrophage scavenger receptor CD163, and microglia/macrophage marker CD68 in the caudate and anterior cingulate of postmortem brains from HIV-positive and HIV-negative subjects. Brain tissues of HIV-infected (n = 24) and control subjects (n = 15) were obtained from the Manhattan HIV Brain Bank. Quantification of the gene mRNA was performed using SYBR Green RT-PCR. CD68 and CD163 were increased in HIV-positive (HIV+) compared to HIV-negative (HIV-) individuals in both brain regions. There were higher OPRK1 (P <0.005), and lower PDYN mRNA (P <0.005) levels in the anterior cingulate of HIV+ compared to HIV- subjects. This difference between the clinical groups was not found in the caudate. There was no difference in the levels of OPRM1 mRNA between HIV+ and HIV- subjects. Using linear regression analysis, we examined the relationship of OPRK1 and PDYN mRNA levels in the HIV+ subjects with seven cognitive domain T scores of a neuropsychological test battery. Within the HIV+ subjects, there was a positive correlation between anterior cingulate PDYN mRNA levels and better T-scores in the motor domain. Within the HIV+ subjects there were also positive correlations of both OPRK1 and PDYN mRNA levels with the anti-inflammatory marker CD163, but not with proinflammatory CD68 levels. In this setting, decreased PDYN mRNA may reflect a homeostatic mechanism to reduce monocyte migration, accompanied by compensatory increases in the cognate receptor (KOR) to dampen pro-inflammatory responses. It is possible that enhanced neuroprotection and better motor performance are associated with higher levels of dynorphin and the recruitment of neuroprotective CD163-positive macrophages. Further studies are needed to test this hypothesis.

Transit-amplifying cells (TACs) are an early intermediate in tissue regeneration. Here, using hair follicles (HFs) as a paradigm, we show that emerging TACs constitute a signaling center that orchestrates tissue growth. Whereas primed stem cells (SCs) generate TACs, quiescent SCs only proliferate afterTACs form and begin expressing Sonic Hedgehog (SHH). TAC generation is independent of autocrine SHH, but the TAC pool wanes if they can't produce SHH. We trace this paradox to two direct actions of SHH: promoting quiescent-SC proliferation and regulating dermal factors that stoke TAC expansion. Ingrained within quiescent SCs' special sensitivity to SHH signaling is their high expression of GAS1. Without sufficient input from quiescent SCs, replenishment of primed SCs for the next hair cycle is compromised, delaying regeneration and eventually leading to regeneration failure. Our findings unveil TACs as transient but indispensable integrators of SC niche components and reveal an intriguing interdependency of primed and quiescent SC populations on tissue regeneration.

Optical fiber spectroscopy is a versatile tool for measuring diffuse reflectance and extracting absorption information that can noninvasively quantify the presence of chromophores such as oxyhemoglobin and deoxy-hemoglobin in tissues. Cerebrovascular abnormalities were widely recognized in Alzheimer's disease (AD) patients. We analyzed blood volume fraction and level of oxygenated hemoglobin in Tg6799 mice, which are transgenic mice expressing five different familial Alzheimer disease-associated mutations in the human amyloid precursor protein and presenilin-1 genes. Diffuse reflectance spectra were iteratively fit as weighted sums of oxy- and deoxy-hemoglobin. Our observations showed slightly hypoxic conditions and significantly increased blood volume in the Alzheimer's mice versus wild type. These results suggest that hyperperfusion of our AD mice may be a compensating mechanism for impaired cerebral vascular function and somehow relevant with early stage of AD patients. Ongoing work focuses on developing a cannula fixture that allows measurement in awake, behaving animals.

In vitro mimics of cellular machines have been recently engineered and utilized to investigate processes within cells. These devices can provide novel insights into biological mechanisms and have the potential to improve biotechnological processes such as separation. In particular, several devices have been designed to mimic translocation through nuclear pore complexes (NPCs). We describe here the fabrication of a biomimetic NPC using nanoporous filters lined with FG-repeats of proteins that create a selectivity barrier. We show the utility of this nanoselective filter as a testbed for the investigation of nucleocytoplasmic transport and demonstrate that this device closely reproduces key features of trafficking through the NPC.

Measurements of the differential and double-differential Drell-Yan cross sections are presented using an integrated luminosity of 4.5 (4.8) fb(-1) in the dimuon (dielectron) channel of proton-proton collision data recorded with the CMS detector at the LHC at = 7 TeV. The measured inclusive cross section in the Z-peak region (60-120 GeV) is sigma(a""a"") = 986.4 +/- 0.6 (stat.) +/- 5.9 (exp. syst.) +/- 21.7 (th. syst.) +/- 21.7 (lum.) pb for the combination of the dimuon and dielectron channels. Differential cross sections d sigma/dm for the dimuon, dielectron, and combined channels are measured in the mass range 15 to 1500 GeV and corrected to the full phase space. Results are also presented for the measurement of the double-differential cross section d(2)sigma/dm d|y| in the dimuon channel over the mass range 20 to 1500 GeV and absolute dimuon rapidity from 0 to 2.4. These measurements are compared to the predictions of perturbative QCD calculations at next-to-leading and next-to-next-to-leading orders using various sets of parton distribution functions.