The rockefeller university

ChIP-seq is increasingly being used for genome-wide profiling of histone modification marks. It is of particular importance to compare ChIP-seq data of two different conditions, such as disease vs. control, and identify regions that show differences in ChIP enrichment. We have developed a powerful and easy to use program, called diffReps, to detect those differential sites from ChIP-seq data, with or without biological replicates. In addition, we have developed two useful tools for ChIP-seq analysis in the diffReps package: one for the annotation of the differential sites and the other for finding chromatin modification "hotspots". diffReps is developed in PERL programming language and runs on all platforms as a command line script. We tested diffReps on two different datasets. One is the comparison of H3K4me3 between two human cell lines from the ENCODE project. The other is the comparison of H3K9me3 in a discrete region of mouse brain between cocaine-and saline-treated !

G-protein-gated inward rectifier K+ (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5 angstrom resolution crystal structure of the mammalian GIRK2 channel in complex with beta gamma G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K+ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four beta gamma G-protein subunits at the interfaces between four K+ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with 'membrane delimited' activation of GIRK channels by G!

Among signaling components downstream of phytochrome A (phyA), HY5, HFR1 and LAF1 are transcription factors that regulate expression of phyA-responsive genes. Previous work has shown that FHY1/FHL distribute phyA signals directly to HFR1 and LAF1, both of which regulate largely independent pathways, but the relationship of HY5 to these two factors was unclear. Here, we investigated the genetic relationship among the genes encoding these three transcription factors, HY5, HFR1 and LAF1. Analyses of double and triple mutants showed that HY5, a basic leucine zipper (bZIP) factor, HFR1, a basic helix-loop-helix (bHLH) factor, and LAF1, a Myb factor, independently transmit phyA signals downstream. We showed that HY5 but not its homolog, HYH, could interact with HFR1 and LAF1; on the other hand, FHY1 and its homolog, FHL did not interact with HY5 or HYH. Together, our results suggest that HY5 transmits phyA signals through an FHY1/FHL-independent pathway but it may also modulate!

Time-reversal symmetry breaking is a key feature of many classes of natural sounds, originating in the physics of sound production. While attention has been paid to the response of the auditory system to "natural stimuli," very few psychophysical tests have been performed. We conduct psychophysical measurements of time-frequency acuity for stylized representations of "natural"-like notes (sharp attack, long decay) and the time-reversed versions of these notes (long attack, sharp decay). Our results demonstrate significantly greater precision, arising from enhanced temporal acuity, for such sounds over their time-reversed versions, without a corresponding decrease in frequency acuity. These data inveigh against models of auditory processing that include tradeoffs between temporal and frequency acuity, at least in the range of notes tested and suggest the existence of statistical priors for notes with a sharp-attack and a long-decay. We are additionally able to calculate a minimal theoretical bound on the sophistication of the nonlinearities in auditory processing. We find that among the best studied classes of nonlinear time-frequency representations, only matching pursuit, spectral derivatives, and reassigned spectrograms are able to satisfy this criterion.

Biological systems provide us with a diverse source of peptide-based ligands for cellular adhesion. Controlling and assessing the ligand surface density as well as tailoring the surface chemistry to have specific cellular adhesion properties are important in biomaterials design. In the following work, we provide a means for displaying peptide-based ligands on magnetic liposomes in which the surface density and chemistry may be controlled. Simultaneously, the conjugated vesicles provide a fluorescent signal for examining steric hindrance among surface ligands. In addition, the inherent magnetic and fluorescence features of this system revealed potential for magnet-based cell isolation and fluorescent labeling of adhered cells, respectively. Adhered cells were found to remain viable and proliferative, thereby allowing them to be used for subsequent evaluation. In a specific demonstration, we control the density of fibronectin-mimetic ligands on the polydiacetylene liposome surfaces. We find that steric limitation occurring at over 20% surface density result in decreased cell adhesion, in accord with related techniques. The magnetic-liposome system offers the means for not only separating cells adhered to the biomaterial, but also providing the ability to control and assess the biomaterial surface. This may prove particularly useful for examining combinations of peptide-based ligands or for evaluating the molecular-level ligand accessibility and its effect on cell attachment to a biomaterial surface.

The nucleus accumbens (Acb) contains subpopulations of neurons defined by their receptor content and potential involvement in sensorimotor gating and other behaviors that are dysfunctional in schizophrenia. In Acb neurons, the NMDA NR1 (NR1) subunit is coexpressed not only with the dopamine D1 receptor (D1R), but also with the mu-opioid receptor (mu-OR), which mediates certain behaviors that are adversely impacted by schizophrenia. The NMDA-NR1 subunit has been suggested to play a role in the D1R trafficking and behavioral dysfunctions resulting from systemic administration of apomorphine, a D1R and dopamine D2 receptor agonist that impacts prepulse inhibition to auditory-evoked startle (AS). Together, this evidence suggests that the NMDA receptor may regulate D1R trafficking in Acb neurons, including those expressing mu-OR, in animals exposed to auditory startle and apomorphine. We tested this hypothesis by combining spatial-temporal gene deletion technology, dual labeli!

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.