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Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5 alpha protein (rhTRIM5 alpha). Initially, we attempted to derive rhTRIM5 alpha-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5 alpha. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5 alpha sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5 alpha sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5 alpha recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5 alpha were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an 'assisted evolution' approach in which individual CA mutations that reduced rhTRIM5 alpha sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5 alpha-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5 alpha. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5 alpha on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIVmac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5 alpha is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection.

Measurements are presented of the production of primary K-S(0) and Lambda particles in proton-proton collisions at root s = 7 TeV in the region transverse to the leading charged-particle jet in each event. The average multiplicity and average scalar transverse momentum sum of K-S(0) and Lambda particles measured at pseudorapidities vertical bar eta vertical bar < 2 rise with increasing charged-particle jet p(T) in the range 1-10 GeV/c and saturate in the region 10-50 GeV/c. The rise and saturation of the strange-particle yields and transverse momentum sums in the underlying event are similar to those observed for inclusive charged particles, which confirms the impact-parameter picture of multiple parton interactions. The results are compared to recent tunes of the PYTHIA Monte Carlo event generator. The PYTHIA simulations underestimate the data by 15%-30% for K-S(0) mesons and by about 50% for Lambda baryons, a deficit similar to that observed for the inclusive strange-particle production in non-single-diffractive proton-proton collisions. The constant strange-to charged-particle activity ratios with respect to the leading jet p(T) and similar trends for mesons and baryons indicate that the multiparton-interaction dynamics is decoupled from parton hadronization, which occurs at a later stage.

Objectives: Disruption of circadian function has been observed in several human disorders, including bipolar disorder (BD). Research into these disorders can be facilitated by human cellular models that evaluate external factors (zeitgebers) that impact circadian pacemaker activity. Incorporating a firefly luciferase reporter system into human fibroblasts provides a facile, bioluminescent readout that estimates circadian phase, while leaving the cells intact. We evaluated whether this system can be adapted to clinical BD research and whether it can incorporate zeitgeber challenge paradigms. Methods: Fibroblasts from patients with bipolar I disorder (BD-I) (n = 13) and controls (n = 12) were infected ex vivo with a lentiviral reporter incorporating the promoter sequences for Bmal1, a circadian gene to drive expression of the firefly luciferase gene. Following synchronization, the bioluminescence was used to estimate period length. Phase response curves (PRCs) were also generated following forskolin challenge and the phase response patterns were characterized. Results: Period length and PRCs could be estimated reliably from the constructs. There were no significant case-control differences in period length, with a nonsignificant trend for differences in PRCs following the phase-setting experiments. Conclusions: An ex vivo cellular fibroblast-based model can be used to investigate circadian function in BD-I. It can be generated from specific individuals and this could usefully complement ongoing circadian clinical research.

Mineralo-organic nanoparticles form spontaneously in human body fluids when the concentrations of calcium and phosphate ions exceed saturation. We have shown previously that these mineralo-organic nanoparticles possess biomimetic properties and can reproduce the whole phenomenology of the so-called nanobacteria-mineralized entities initially described as the smallest microorganisms on earth. Here, we examine the possibility that various charged elements and ions may form mineral nanoparticles with similar properties in biological fluids. Remarkably, all the elements tested, including sodium, magnesium, aluminum, calcium, manganese, iron, cobalt, nickel, copper, zinc, strontium, and barium form mineralo-organic particles with bacteria-like morphologies and other complex shapes following precipitation with phosphate in body fluids. Upon formation, these mineralo-organic particles, which we term bions, invariably accumulate carbonate apatite during incubation in biological fluids; yet, the particles also incorporate additional elements and thus reflect the ionic milieu in which they form. Bions initially harbor an amorphous mineral phase that gradually converts to crystals in culture. Our results show that serum produces a dual inhibition-seeding effect on bion formation. Using a comprehensive proteomic analysis, we identify a wide range of proteins that bind to these mineral particles during incubation in medium containing serum. The two main binding proteins identified, albumin and fetuin-A, act as both inhibitors and seeders of bions in culture. Notably, bions possess several biomimetic properties, including the possibility to increase in size and number and to be sub-cultured in fresh culture medium. Based on these results, we propose that bions represent biological, mineralo-organic particles that may form in the body under both physiological and pathological homeostasis conditions. These mineralo-organic particles may be part of a physiological cycle that regulates the function, transport and disposal of elements and minerals in the human body.

In the mouse, a mature olfactory sensory neuron (OSN) of the main olfactory epithelium (MOE) expresses one allele of one of the 1200 odorant receptor (OR) genes in the genome. The mechanisms that underlie the one receptor-one neuron rule remain poorly understood. A popular experimental paradigm for OR gene choice is to delete an OR coding region by gene targeting or in a transgene. Here we have applied this Delta OR paradigm to SR1, also known as MOR256-3 or Olfr124. This gene is expressed in OSNs of the MOE, and in similar to 50% of the OSNs of the septal organ. In heterozygous Delta SR1 mice, we observe an unprecedented biallelic expression rate of 30% at the SR1 locus. In homozygous Delta SR1 mice, we find a significant increase in the number of septal organ OSNs that undergo apoptosis. As a population, Delta SR1 OSNs project their axons to 81-85 glomeruli in each half of the OB, and coexpress at least 77 OR genes as evaluated by single-cell molecular analysis. There are no obvious or simple rules for the set of OR genes that are coexpressed with the Delta SR1 allele. The frequencies of coexpression are different for Delta SR1 OSNs in the septal organ compared to those in the MOE. We propose that there are as many as five scenarios for the fate of individual Delta SR1 OSNs. (C) 2013 Elsevier Inc. All rights reserved.

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10.Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10.Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4(+) and NKp46(+) innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b(+) cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103(+) cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b(+) cDCs in the response to pathogens in vivo.

All brain functions require the coordinated activity of many neurons, and therefore there is considerable interest in estimating the amount of information that the discharge of a neural population transmits to its targets. In the past, such estimates had presented a significant challenge for populations of more than a few neurons, but we have recently described a novel method for providing such estimates for populations of essentially arbitrary size. Here, we explore the influence of some important aspects of the neuronal population discharge on such estimates. In particular, we investigate the roles of mean firing rate and of the degree and nature of correlations among neurons. The results provide constraints on the applicability of our new method and should help neuroscientists determine whether such an application is appropriate for their data.

Taylor's law (TL), a widely verified empirical relationship in ecology, states that the variance of population density is approximately a power-law function of mean density. The growth-rate theorem (GR) states that, in a subdivided population, the rate of change of the overall growth rate is proportional to the variance of the subpopulations' growth rates. We show that continuous-time exponential change implies GR at every time and, asymptotically for large time, TL with power-law exponent 2. We also show why diverse population-dynamic models predict TL in the limit of large time by identifying simple features these models share: If the mean population density and the variance of population density are (exactly or asymptotically) non-constant exponential functions of a parameter (e.g., time), then the variance of density is (exactly or asymptotically) a power-law function of mean density. (C) 2013 Elsevier Inc. All rights reserved.