The rockefeller university

Objectives: To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300. Methods: We reconstructed the phylogeny of S. aureus CC8 by mutation discovery at 112 genetic housekeeping loci from each of 174 isolates, sampled on five continents between 1957 and 2008. The distribution of antimicrobial resistance traits and of diverse mobile genetic elements was investigated in relation to the isolates' phylogeny. Results: Our analyses revealed the existence of nine phylogenetic clades within CC8. We identified at least eight independent events of methicillin resistance acquisition in CC8 and dated the origin of a methicillin-resistant progenitor of the notorious USA300 clone to the mid-1970s. Of the S. aureus isolates in our collection, 88% carried plasmidic rep gene sequences, with up to five different rep genes in individual isolates and a total of eight rep families. Mapping the plasmid content onto the isolates' phylogeny illustrated the stable carriage over decades of some plasmids and the more volatile nature of others. Strikingly, we observed trends of increasing antibiotic resistance during the evolution of several lineages, including USA300. Conclusions: We propose a model for the evolution of S. aureus CC8, involving a split into at least nine phylogenetic lineages and a subsequent series of acquisitions and losses of mobile genetic elements that carry diverse virulence and antimicrobial resistance traits. The evolution of MRSA USA300 towards resistance to additional antibiotic classes is of major concern.

Staphylococcus aureus virulence is regulated when secreted autoinducing peptides (AIPs) are recognized by a membrane-bound receptor histidine kinase (RHK), AgrC. Some AIPs are agonists of virulence gene expression, while others are antagonists. It is unclear how AIP binding regulates AgrC activity. Here, we reconstitute an AgrC family member, AgrC-I, using nanometer-scale lipid bilayer discs. We show that AgrC-I requires membranes rich in anionic lipids to function. The agonist, AIP-I, binds AgrC-I noncooperatively in a 2: 2 stoichiometry, while an antagonist ligand, AIP-II, functions as an inverse agonist of the kinase activity. We also demonstrate the kinase and sensor domains in AgrC are connected by a helical linker whose conformational state exercises rheostat-like control over the kinase activity. Binding of agonist or inverse-agonist peptides results in twisting of the linker in different directions. These two observations provide a view of the molecular motions triggered by ligand binding in an intact membrane-bound RHK.

The possible signaling role of prokineticin 2 (PK2) and its receptor, prokineticin receptor 2 (PKR2), on female reproduction was investigated. First, the expression of PKR2 and its co-localization with estrogen receptor (ER alpha) in the hypothalamus was examined. Sexually dimorphic expression of PKR2 in the preoptic area of the hypothalamus was observed. Compared to the male mice, there was more widespread PKR2 expression in the preoptic area of the hypothalamus in the female mice. The likely co-expression of PKR2 and ERa in the preoptic area of the hypothalamus was observed. The estrous cycles in female PK2-null, and PKR2-null heterozygous mice, as well as in PK2-null and PKR2-null compound heterozygous mice were examined. Loss of one copy of PK2 or PKR2 gene caused elongated and irregular estrous cycle in the female mice. The alterations in the estrous cycle were more pronounced in PK2-null and PKR2-null compound heterozygous mice. Consistent with these observations, administration of a small molecule PK2 receptor antagonist led to temporary blocking of estrous cycle at the proestrous phase in female mice. The administration of PKR2 antagonist was found to blunt the circulating LH levels. Taken together, these studies indicate PK2 signaling is required for the maintenance of normal female estrous cycles.

In this study, we compare biosynthetic gene richness and diversity of 96 soil microbiomes from diverse environments found throughout the southwestern and northeastern regions of the United States. The 454-pyroseqencing of nonribosomal peptide adenylation (AD) and polyketide ketosynthase (KS) domain fragments amplified from these microbiomes provide a means to evaluate the variation of secondary metabolite biosynthetic diversity in different soil environments. Through soil composition and AD-and KS-amplicon richness analysis, we identify soil types with elevated biosynthetic potential. In general, arid soils show the richest observed biosynthetic diversity, whereas brackish sediments and pine forest soils show the least. By mapping individual environmental amplicon sequences to sequences derived from functionally characterized biosynthetic gene clusters, we identified conserved soil type-specific secondary metabolome enrichment patterns despite significant sample-to-sample sequence variation. These data are used to create chemical biogeographic distribution maps for biomedically valuable families of natural products in the environment that should prove useful for directing the discovery of bioactive natural products in the future.

Toe clipping is used to identify and genotype preweanling mice, but the procedure generates concerns relevant to pain and distress. The few pertinent studies available evaluated mice between postnatal days (PND) 3 and 7, advocate the use of toe clipping in mice PND 7 or younger, and identify handling as the most distressing aspect of the procedure. Because both toe and tail clipping may be necessary in older mice to obtain sufficient DNA for genotyping, we surmised that performing these procedures concurrently to minimize handling would be beneficial. We also examined reflex development until PND 21 and adult behavior at 8 to 10 wk of age in mice toe clipped at PND 7 or 17 and the benefits of using topical vapocoolant anesthesia. C57BL/6J pups at PND 7 and 17 were assigned to 1 of 4 groups: 1) clipping of digit 3 of contralateral fore- and hindpaws; 2) toe clipping after topical vapocoolant anesthesia; 3) unclipped, unsprayed controls; and, 4) unclipped and vapocoolant-sprayed. Compared with unanesthetized pups, those sprayed with vapocoolant vocalized and struggled more when handled and had more bleeding, erythema, and swelling, which persisted for as long as 12 h after toe clipping. Reflex development, anxiety, locomotion, and motor coordination were not different among groups or with regard to the age of toe clipping. No tissue reaction was noted microscopically in paws collected at 10 wk of age. We conclude that the use of vapocoolant cannot be recommended due to its harmful effects and that toe clipping at PND 7 or 17 does not significantly affect the long-term welfare of mice.

Background: Severe genetic bottleneck occurs during HIV-1 sexual transmission whereby most infections are initiated by a single transmitted/founder (T/F) virus. Similar observations had been made in nonhuman primates exposed mucosally to SIV/SHIV. We previously reported variable clinical outcome in rhesus macaques inoculated intravaginally (ivg) with a high dose of R5 SHIVSF162P3N. Given the potential contributions of viral diversity to HIV-1 persistence and AIDS pathogenesis and recombination between retroviral genomes increases the genetic diversity, we tested the hypothesis that transmission of multiple variants contributes to heightened levels of virus replication and faster disease progression in the SHIVSF162P3N ivg-infected monkeys. Results: We found that the differences in viral replication and disease progression between the transiently viremic (TV; n = 2), chronically-infected (CP; n = 8) and rapid progressor (RP; n = 4) ivg-infected macaques cannot be explained by which variant in the inoculum was infecting the animal. Rather, transmission of a single variant was observed in both TV rhesus, with 1-2 T/F viruses found in the CPs and 2-4 in all four RP macaques. Moreover, the genetic relatedness of the T/F viruses in the CP monkeys with multivariant transmission was greater than that seen in the RPs. Biological characterization of a subset of T/F envelopes from chronic and rapid progressors revealed differences in their ability to mediate entry into monocyte-derived macrophages, with enhanced macrophage tropism observed in the former as compared to the latter. Conclusion: Our study supports the tenet that sequence diversity of the infecting virus contributes to higher steady-state levels of HIV-1 virus replication and faster disease progression and highlights the role of macrophage tropism in HIV-1 transmission and persistence.

Background: Phenethyl isothiocyanate (PEITC), present naturally in cruciferous vegetables, is a chemopreventive agent. It blocks initiation and post-initiation progression of carcinogenesis. Mechanism study in human prostate cancer cells revealed that PEITC is a dual inhibitor of aberrant DNA hypermethylation and histone deacetylases, reactivating silenced genes and regulating the androgen-mediated growth of tumor cells. The identity of the cellular organelle that initially interacts with PEITC has not been fully described. Methods: Human prostate cancer LNCaP cells were exposed to PEITC and the effects on cellular fine structure examined by transmission electron microscopic studies. Alteration of mitochondrial membrane potential and cytochrome c release were evaluated as early events of apoptosis, and the TUNEL method for quantifying apoptotic cells. Mitochondria were isolated for determining their protein expression. Results: Ultrastructural analyses have revealed condensed mitochondria and a perturbed mitochondrial cristae structure, which assumed a rounded and dilated shape within 4-hours of PEITC contact, and became more pronounced with longer PEITC exposure. They presented as the most prominent intracellular alterations in the early hours. Mitochondria structure alterations were demonstrated, for the first time, with the isothiocyanates. An increase in the number of smooth endoplasmic reticulum and vacuoles were also noted that is consistent with the presence of autophagy. Early events of apoptosis were detected, with cytochrome c released along with the appearance of mitochondrial alteration. Mitochondrial membrane potential was disrupted within 18 hours of PEITC exposure, preceding the appearance of apoptotic cells with DNA strand breaks. In parallel, the expression of the mitochondrial class III beta-tubulin in the outer membrane, which associates with the permeability transition pore, was significantly reduced as examined with isolated mitochondria. Conclusion: Mitochondria may represent the organelle target of the isothiocyanates, indicating that the isothiocyanates may be mitochondria-interacting agents to inhibit carcinogenesis.

Correlation between gut microbiota and host phylogeny could reflect codiversification over shared evolutionary history or a selective environment that is more similar in related hosts. These alternatives imply substantial differences in the relationship between host and symbiont, but can they be distinguished based on patterns in the community data themselves? We explored patterns of phylogenetic correlation in the distribution of gut bacteria among species of turtle ants (genus Cephalotes), which host a dense gut microbial community. We used 16S rRNA pyrosequencing from 25 Cephalotes species to show that their gut community is remarkably stable, from the colony to the genus level. Despite this overall similarity, the existing differences among species' microbiota significantly correlated with host phylogeny. We introduced a novel analytical technique to test whether these phylogenetic correlations are derived from recent bacterial evolution, as would be expected in the case of codiversification, or from broader shifts more likely to reflect environmental filters imposed by factors such as diet or habitat. We also tested this technique on a published data set of ape microbiota, confirming earlier results while revealing previously undescribed patterns of phylogenetic correlation. Our results indicated a high degree of partner fidelity in the Cephalotes microbiota, suggesting that vertical transmission of the entire community could play an important role in the evolution and maintenance of the association. As additional comparative microbiota data become available, the techniques presented here can be used to explore trends in the evolution of host-associated microbial communities.

Background: Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown. Results: Here, we show that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence of Slit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance in vivo. Conclusions: These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain.

A search for forbidden and exotic Z boson decays in the diphoton mass spectrum is presented for the first time in hadron collisions, based on data corresponding to 10.0 fb(-1) of integrated luminosity from proton-antiproton collisions at root s = 1.96 TeV collected by the CDF experiment. No evidence of signal is observed, and 95% credibility level Bayesian upper limits are set on the branching ratios of decays of the Z boson to a photon and neutral pion (which is detected as a photon), a pair of photons, and a pair of neutral pions. The observed branching ratio limits are 2.01 x 10(-5) for Z -> pi(0)gamma, 1.46 x 10(-5) for Z -> gamma gamma, and 1.52 x 10(-5) for Z -> pi(0)pi(0). The Z -> pi(0)gamma and Z -> gamma gamma limits improve the most stringent results from other experiments by factors of 2.6 and 3.6, respectively. The Z -> pi(0)pi(0) branching ratio limit is the first experimental result on this decay.