The rockefeller university

Nicotine, acting through nicotinic acetylcholine receptors (nAChRs), increases the firing rate of both orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC), yet nicotine and other nAChR agonists decrease food intake in mice. Viral-mediated knockdown of the 4 nAChR subunit in all neuronal cell types in the ARC prevents the nicotinic agonist cytisine from decreasing food intake, but it is not known whether the 4 subunit is selectively expressed in anorexigenic neurons or how other nAChR subtypes are distributed in this nucleus. Using translating ribosome affinity purification (TRAP) on ARC tissue from mice with ribosomes tagged in either AgRP or POMC cells, we examined nAChR subunit mRNA levels using real-time PCR. Both AgRP and POMC cells express a comparable panel of nAChR subunits with differences in 7 mRNA levels and a trend for difference in 4 levels, but no differences in 4 expression. Immunoprecipitation of assembled nAChRs revealed that the 4 subunit forms assembled channels with 3, 2 and 4, but not other subunits found in the ARC. Finally, using cell type-selective, virally delivered small hairpin RNAs targeting either the 4 or 7 subunit, we examined the contribution of each subunit in either AgRP or POMC cells to the behavioural response to nicotine, refining the understanding of nicotinic regulation of this feeding circuit. These experiments identify a more complex set of nAChRs expressed in ARC than in other hypothalamic regions. Thus, the ARC appears to be a particular target of nicotinic modulation.

Background: Propofol produces memory impairment at concentrations well below those abolishing consciousness. Episodic memory, mediated by the hippocampus, is most sensitive. Two potentially overlapping scenarios may explain how gamma-aminobutyric acid receptor type A (GABA(A)) potentiation by propofol disrupts episodic memory-the first mediated by shifting the balance from excitation to inhibition while the second involves disruption of rhythmic oscillations. We use a hippocampal network model to explore these scenarios. The basis for these experiments is the proposal that the brain represents memories as groups of anatomically dispersed strongly connected neurons. Methods: A neuronal network with connections modified by synaptic plasticity was exposed to patterned stimuli, after which spiking output demonstrated evidence of stimulus-related neuronal group development analogous to memory formation. The effect of GABA(A) potentiation on this memory model was studied in 100 unique networks. Results: GABA(A) potentiation consistent with moderate propofol effects reduced neuronal group size formed in response to a patterned stimulus by around 70%. Concurrently, accuracy of a Bayesian classifier in identifying learned patterns in the network output was reduced. Greater potentiation led to near total failure of group formation. Theta rhythm variations had no effect on group size or classifier accuracy. Conclusions: Memory formation is widely thought to depend on changes in neuronal connection strengths during learning that enable neuronal groups to respond with greater facility to familiar stimuli. This experiment suggests the ability to form such groups is sensitive to alteration in the balance between excitation and inhibition such as that resulting from administration of a gamma-aminobutyric acid-mediated anesthetic agent.

Cortical deep projection neurons (DPNs) are implicated in neurodevelopmental disorders. Although recent findings emphasize post-mitotic programs in projection neuron fate selection, the establishment of primate DPN identity during layer formation is not well understood. The subplate lies underneath the developing cortex and is a post-mitotic compartment that is transiently and disproportionately enlarged in primates in the second trimester. The evolutionary significance of subplate expansion, the molecular identity of its neurons, and its contribution to primate corticogenesis remain open questions. By modeling subplate formation with human pluripotent stem cells (hPSCs), we show that all classes of cortical DPNs can be specified from subplate neurons (SPNs). Post-mitotic WNT signaling regulates DPN class selection, and DPNs in the caudal fetal cortex appear to exclusively derive from SPNs. Our findings indicate that SPNs have evolved in primates as an important source of DPNs that contribute to cortical lamination prior to their known role in circuit formation.

Inborn errors of IFN-gamma immunity underlie Mendelian susceptibility to mycobacterial disease (MSMD). Autosomal recessive complete IL-12R beta 1 deficiency is the most frequent genetic etiology of MSMD. Only two of the 84 known mutations are copy number variations (CNVs), identified in two of the 213 IL-12R beta 1-deficient patients and two of the 164 kindreds reported. These two CNVs are large deletions found in the heterozygous or homozygous state. We searched for novel families with IL-12R beta 1 deficiency due to CNVs. We studied six MSMD patients from five unrelated kindreds displaying adverse reactions to BCG vaccination. Three of the patients also presented systemic salmonellosis, two had mucocutaneous candidiasis, and one had disseminated histoplasmosis. We searched for CNVs and other variations by IL12RB1-targeted next-generation sequencing (NGS). We identified six new IL-12R beta 1-deficient patients with a complete loss of IL-12R beta 1 expression on phytohemagglutinin-activated T cells and/or EBV-transformed B cells. The cells of these patients did not respond to IL-12 and IL-23. Five different CNVs encompassing IL12RB1 (four deletions and one duplication) were identified in these patients by NGS coverage analysis, either in the homozygous state (n = 1) or in trans (n = 4) with a single-nucleotide variation (n = 3) or a small indel (n = 1). Seven of the nine mutations are novel. Interestingly, four of the five CNVs were predicted to be driven by nearby Alu elements, as well as the two previously reported large deletions. The IL12RB1 locus is actually enriched in Alu elements (44.7%), when compared with the rest of the genome (10.5%). The IL12RB1 locus is Alu-enriched and therefore prone to rearrangements at various positions. CNVs should be considered in the genetic diagnosis of IL-12R beta 1 deficiency.

Background: Nitrogen (N) is a key macronutrient essential for plant growth, and its availability has a strong influence on crop development. The application of synthetic N feitilizers on crops has increased substantially in recent decades; however, the applied N is not fully utilized due to the low N use efficiency of crops. To overcome this limitation, it is importamt to understand the genome wide responses and functions of key genes and potential regulatory factors in N metabolism. Results: Here, we characterized changes in the rice (Oryza sativa) transcriptome, including genes, newly identified putative long non-coding RNAs (IncRNAs), and microRNAs (miRNAs) and their target mRNAs in response to N starvation using four different transcriptome approaches. Analysis of rice genes involved in N metabolism and/or transport using strand-specific RNA-Seq identified 2588 novel putative IncRNA encoding loci. Analysis of previously published RNA-Seq datasets levealed a group of N starvation-responsive IncRNAs showing differential expression under other abiotic stress conditions Poly A-primed sequencing (2P-Seq) revealed alternatively polyadenylated isoforms of N starvation-responsive incRNAs and provided precise 3' end information on the transcript models of these IncRNAs. Analysis of small RNA-Seq data identified N starvation-responsive miRNAs and down regulation of miR169 family members, causing de repression of NF-YA, as confirmed by strand specific RNA-Seq and qRT-PCR. Moreover, we profiled the N starvation-responsive down-regulation of root-specific miRNA, osa-miR444a.4-3p, and Degradome sequencing confirmed MADS25 as a novel target gene. Conclusions: in this study, we used a combination of multiple RNA Seq analyses to extensively profile the expression of genes, newly identified IncRNAs, and microRNAs in N-starved i rice roots and shoots. Data generated in this study provide an in-depth understanding of the regulatory pathways modulated by N starvation-responsive miRNAs. The results of comprehensive, large-scale data analysis provide valuable information on multiple aspects of the rice transcriptome, which may be useful in understanding the responses of rice plants to changes in the N supply status of soil.

We observed earlier that mineralo-organic nanoparticles form in human body fluids when the concentrations of calcium, carbonate and phosphate exceed saturation. The particles have been shown to represent mineral precursors in developing bones and teeth as well as in ectopic calcification and kidney stones. Recent studies suggest that the mineral particles may also be involved in other physiological processes, including immune tolerance against the gut microbiota and food antigens. We review here the involvement of mineralo-organic nanoparticles in physiological and pathological processes and discuss recent findings that reveal novel and unexpected roles for these particles in the human body.

Background: In Australia, community-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage sequence type (ST) 93 has rapidly risen to dominance since being described in the early 1990s. We examined 459 ST93 genome sequences from Australia, New Zealand, Samoa, and Europe to investigate the evolutionary history of ST93, its emergence in Australia and subsequent spread overseas. Results: Comparisons with other S. aureus genomes indicate that ST93 is an early diverging and recombinant lineage, comprising of segments from the ST59/ST121 lineage and from a divergent but currently unsampled Staphylococcal population. However, within extant ST93 strains limited genetic diversity was apparent with the most recent common ancestor dated to 1977 (95% highest posterior density 1973-1981). An epidemic ST93 population arose from a methicillin-susceptible progenitor in remote Northern Australia, which has a proportionally large Indigenous population, with documented overcrowded housing and a high burden of skin infection. Methicillin-resistance was acquired three times in these regions, with a Glade harboring a staphylococcal cassette chromosome mec (SCCmec) IVa expanding and spreading to Australia's east coast by 2000. We observed sporadic and non-sustained introductions of ST93-MRSA-IVa to the United Kingdom. In contrast, in New Zealand, ST93-MRSAIVa was sustainably transmitted with clonal expansion within the Pacific Islander population, who experience similar disadvantages as Australian Indigenous populations. Conclusion: ST93 has a highly recombinant genome including portions derived from an early diverging S. aureus population. Our findings highlight the need to understand host population factors in the emergence and spread of antimicrobial resistant community pathogens.

Elastin-like polypeptides (ELP) exhibit an inverse temperature transition or lower critical solution temperature (LCST) transition phase behavior in aqueous solutions. In this paper, the thermal responsive properties of the canonical ELP, poly(VPGVG), and its reverse sequence poly(VGPVG) were investigated by turbidity measurements of the cloud point behavior, circular dichroism (CD) measurements, and all-atom molecular dynamics (MD) simulations to gain a molecular understanding of mechanism that controls hysteretic phase behavior. It was shown experimentally that both poly(VPGVG) and poly(VGPVG) undergo a transition from soluble to insoluble in aqueous solution upon heating above the transition temperature (TO. However, poly(VPGVG) resolubilizes upon cooling below its T-v, whereas the reverse sequence, poly(VGPVG), remains aggregated despite significant undercooling below the T-t. The results from MD simulations indicated that a change in sequence order results in significant differences in the dynamics of the specific residues, especially valines, which lead to extensive changes in the conformations of VPGVG and VGPVG pentamers and, consequently, dissimilar propensities for secondary structure formation and overall structure of polypeptides. These changes affected the relative hydrophilicities of polypeptides above T-v where poly(VGPVG) is more hydrophilic than poly(VPGVG) with more extended conformation and larger surface area, which led to formation of strong interchain hydrogen bonds responsible for stabilization of the aggregated phase and the observed thermal hysteresis for poly(VGPVG).

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

How the brain preserves information about multiple simultaneous items is poorly understood. We report that single neurons can represent multiple stimuli by interleaving signals across time. We record single units in an auditory region, the inferior colliculus, while monkeys localize 1 or 2 simultaneous sounds. During dual-sound trials, we find that some neurons fluctuate between firing rates observed for each single sound, either on a whole-trial or on a sub-trial timescale. These fluctuations are correlated in pairs of neurons, can be predicted by the state of local field potentials prior to sound onset, and, in one monkey, can predict which sound will be reported first. We find corroborating evidence of fluctuating activity patterns in a separate dataset involving responses of inferotemporal cortex neurons to multiple visual stimuli. Alternation between activity patterns corresponding to each of multiple items may therefore be a general strategy to enhance the brain processing capacity, potentially linking such disparate phenomena as variable neural firing, neural oscillations, and limits in attentional/memory capacity.