The rockefeller university

Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61 alpha, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid(1-10). The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation(11). How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (similar to 10 angstrom) or are of insufficient quality(6-8). Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.

We introduce a novel assay for membrane fusion of solid supported membranes on silica beads and on coverslips. Fusion of the lipid bilayers is induced by bringing an optically trapped bead in contact with the coverslip surface while observing the bead's thermal motion with microsecond temporal and nanometer spatial resolution using a three-dimensional position detector. The probability of fusion is controlled by the membrane tension on the particle. We show that the progression of fusion can be monitored by changes in the three-dimensional position histograms of the bead and in its rate of diffusion. We were able to observe all fusion intermediates including transient fusion, formation of a stalk, hemifusion and the completion of a fusion pore. Fusion intermediates are characterized by axial but not lateral confinement of the motion of the bead and independently by the change of its rate of diffusion due to the additional drag from the stalk-like connection between the two membranes. The detailed information provided by this assay makes it ideally suited for studies of early events in pure lipid bilayer fusion or fusion assisted by fusogenic molecules.

The CMG helicase is composed of Cdc45, Mcm2-7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7-4.8 angstrom. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2-7, braced by Cdc45-GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2-7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.

A measurement of vector boson (V) production in conjunction with a D*(2010)(+) meson is presented. Using a data sample corresponding to 9.7 fb(-1) of proton-antiproton collisions at center-of-mass energy root s = 1.96 TeV produced by the Fermilab Tevatron, we reconstruct V + D*+ samples with the CDF II detector. The D*+ is fully reconstructed in the D*(2010)(+) -> D-0 (-> K-pi(+))pi(+) decay mode. This technique is sensitive to the associated production of vector boson plus charm or bottom mesons. We measure the ratio of production cross sections sigma(W + D*) = sigma(W) = [1.75 +/- 0.13(stat) +/- 0.09(stat)]% and sigma(Z + D*)/sigma(Z) = [1.5 +/- 0.4(stat) +/- 0.2(stat)]% and perform a differential measurement of d sigma(W + D*)/dp(T)(D*). Event properties are utilized to determine the fraction of V + D*(2010)(+) events originating from different production processes. The results are in agreement with the predictions obtained with the PYTHIA program, limiting possible contribution from non-standard-model physics processes.

Adult tissue stem cells (SCs) reside in niches, which orchestrate SC behavior. SCs are typically used sparingly and exist in quiescence unless activated for tissue growth. Whether parsimonious SC use is essential to conserve long-term tissue-regenerating potential during normal homeostasis remains poorly understood. Here, we examine this issue by conditionally ablating a key transcription factor Forkhead box C1 (FOXC1) expressed in hair follicle SCs (HFSCs). FOXC1-deficient HFSCs spend less time in quiescence, leading to markedly shortened resting periods between hair cycles. The enhanced hair cycling accelerates HFSC expenditure, and impacts hair regeneration in aging mice. Interestingly, although FOXC1-deficient HFs can still form a new bulge that houses HFSCs for the next hair cycle, the older bulge is left unanchored. As the new hair emerges, the entire old bulge, including its reserve HFSCs and SC-inhibitory inner cell layer, is lost. We trace this mechanism first, to a marked increase in cell cycle-associated transcripts upon Foxc1 ablation, and second, to a downstream reduction in E-cadherin-mediated inter-SC adhesion. Finally, we show that when the old bulge is lost with each hair cycle, overall levels of SC-inhibitory factors are reduced, further lowering the threshold for HFSC activity. Taken together, our findings suggest that HFSCs have restricted potential in vivo, which they conserve by coupling quiescence to adhesion-mediated niche maintenance, thereby achieving long-term tissue homeostasis.

Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture -independent, or "metagenomic," methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth's microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture -independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence -based, high -throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.

Clustered regularly interspaced short palindromic repeat (CRISPR) loci encode an adaptive immune system of prokaryotes. Within these loci, sequences intercalated between repeats known as "spacers" specify the targets of CRISPR immunity. The majority of spacers match sequences present in phages and plasmids; however, it is not known whether there are differences in the immunity provided against these diverse invaders. We studied this issue using the Staphylococcus epidermidis CRISPR system, which harbors spacers matching both phages and plasmids. We determined that this CRISPR system provides similar levels of defense against the conjugative plasmid pG0400 and the bacteriophage CNPX. However, whereas antiplasmid immunity was very sensitive to the introduction of mismatches in the target sequence, mutations in the phage target were largely tolerated. Placing the phage and plasmid targets into a vector that can be both conjugated and transduced, we demonstrated that the route of entry of the target has no impact on the effect of the mismatches on immunity. Instead, we established that the specific sequences of each spacer/target determine the susceptibility of the S. epidermidis CRISPR system to mutations. Therefore, spacers that are more resistant to mismatches would provide long-term immunity against phages and plasmids that otherwise would escape CRISPR targeting through the accumulation of mutations in the target sequence. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements. IMPORTANCE CRISPR-Cas loci protect bacteria and archaea from both phage infection and plasmid invasion. These loci harbor short sequences of phage and plasmid origin known as "spacers" that specify the targets of CRISPR-Cas immunity. The presence of a spacer sequence matching a phage or plasmid ensures host immunity against infection by these genetic elements. In turn, phages and plasmids constantly mutate their targets to avoid recognition by the spacers of the CRISPR-Cas immune system. In this study, we demonstrated that different spacer sequences vary in their ability to tolerate target mutations that allow phages and plasmids to escape from CRISPR-Cas immunity. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements.

Estrogen surge following progesterone withdrawal at parturition plays an important role in initiating maternal behavior in various rodent species. Systemic estrogen treatment shortens the latency to onset of maternal behavior in nulliparous female rats that have not experienced parturition. In contrast, nulliparous laboratory mice show rapid onset of maternal behavior without estrogen treatment, and the role of estrogen still remains unclear. Here the effect of systemic estrogen treatment (for 2 h, 1 day, 3 days, and 7 days) after progesterone withdrawal was examined on maternal behavior of C57BL/6 mice. This estrogen regimen led to different effects on nursing, pup retrieval, and nest building behaviors. Latency to nursing was shortened by estrogen treatment within 2 h. Moreover, pup retrieval and nest building were decreased. mRNA expression was also investigated for estrogen receptor a (ER alpha) and for genes involved in regulating maternal behavior, specifically, the oxytocin receptor (OTR) and vasopressin receptor in the medial amygdala (MeA) and medial preoptic area (MPOA). Estrogen treatment led to decreased ER alpha mRNA in both regions. Although OTR mRNA was increased in the MeA, OTR and vasopressin receptor mRNA were reduced in the MPOA, showing region-dependent transcription regulation. To determine the mechanisms for the actions of estrogen treatment, the contribution of estrogen synthesis in the brain was examined. Blockade of estrogen synthesis in the brain by systemic letrozole treatment in ovariectomized mice interfered with pup retrieval and nest building but not nursing behavior, indicating different contributions of estrogen synthesis to maternal behavior. Furthermore, letrozole treatment led to an increase in ER alpha mRNA in the MeA but not in the MPOA, suggesting that involvement of estrogen synthesis is brain region dependent. Altogether, these results suggest that region-dependent estrogen synthesis leads to differential transcriptional activation due to exogenous estrogen treatment, and thereby results in different effects on maternal behavior.

Intracellular accumulation of hyperphosphorylated tau protein is linked to neuronal degeneration in Alzheimer's disease (AD). Mounting evidence suggests that tau phosphorylation and O-N-acetylglucosamine glycosylation (O-GlcNAcylation) are mutually exclusive post-translational modifications. O-GlcNAcylation depends on 3-5% of intracellular glucose that enters the hexosamine biosynthetic pathway. To our knowledge, the existence of an imbalance between tau phosphorylation and O-GlcNAcylation has not been reported in animal models of AD, as yet. Here, we used triple trans genic (3xTg-AD) mice at 12 months, an age at which hyperphosphorylated tau is already detected and associated with cognitive decline. In these mice, we showed that tau was hyperphosphorylated on both Ser396 and Thr205 in the hippocampus, and to a lower extent and exclusively on Thr205 in the frontal cortex. Tau O-GlcNAcylation, assessed in tau immunoprecipitates, was substantially reduced in the hippocampus of 3xTg-AD mice, with no changes in the frontal cortex or in the cerebellum. No changes in the expression of the three major enzymes involved in O-GlcNAcylation, i.e., glutamine fructose-6-phosphate amidotransferase, O-linked beta-N-acetylglucosamine transferase, and O-GlcNAc hydrolase were found in the hippocampus of 3xTg-AD mice. These data demonstrate that an imbalance between tau phosphorylation and O-GlcNAcylation exists in AD mice, and strengthens the hypothesis that O-GlcNAcylation might be targeted by disease modifying drugs in AD. (C) 2016 Elsevier Ltd. All rights reserved.

Although epidemiological evidence suggests a human genetic basis of pulmonary tuberculosis (PTB) susceptibility, the identification of specific genes and alleles influencing PTB risk has proven to be difficult. Previous genome-wide association (GWA) studies have identified only three novel loci with modest effect sizes in sub-Saharan African and Russian populations. We performed a GWA study of 550,352 autosomal SNPs in a family-based discovery Moroccan sample (on the full population and on the subset with PTB diagnosis at <25 years), which identified 143 SNPs with p < 1 x 10(-4). The replication study in an independent case/control sample identified four SNPs displaying a p < 0.01 implicating the same risk allele. In the combined sample including 556 PTB subjects and 650 controls these four SNPs showed suggestive association (2 x 10(-6) < p < 4 x 10(-5)): rs358793 and rs17590261 were intergenic, while rs6786408 and rs916943 were located in introns of FOXP1 and AGMO, respectively. Both genes are involved in the function of macrophages, which are the site of latency and reactivation of Mycobacterium tuberculosis. The most significant finding (p = 2 x 10(-6)) was obtained for the AGMO SNP in an early (<25 years) age-at- onset subset, confirming the importance of considering age-at-onset to decipher the genetic basis of PTB. Although only suggestive, these findings highlight several avenues for future research in the human genetics of PTB.