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Summary: Despite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the ∼4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the ∼560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set. [Copyright &y& Elsevier]

Summary: GDE1 is a mammalian glycerophosphodiesterase (GDE) implicated by in vitro studies in the regulation of glycerophophoinositol (GroPIns) and possibly other glycerophospho (GroP) metabolites. Here, we show using untargeted metabolomics that GroPIns is profoundly (>20-fold) elevated in brain tissue from GDE1(−/−) mice. Furthermore, two additional GroP metabolites not previously identified in eukaryotic cells, glycerophosphoserine (GroPSer) and glycerophosphoglycerate (GroPGate), were also highly elevated in GDE1(−/−) brains. Enzyme assays with synthetic GroP metabolites confirmed that GroPSer and GroPGate are direct substrates of GDE1. Interestingly, our metabolomic profiles also revealed that serine (both L-and D-) levels were significantly reduced in brains of GDE1(−/−) mice. These findings designate GroPSer as a previously unappreciated reservoir for free serine in the nervous system and suggest that GDE1, through recycling serine from GroPSer, may impact D-serine-dependent neural signaling processes in vivo. [ABSTRACT FROM AUTHOR]

Summary: Apicomplexan parasites release factors via specialized secretory organelles (rhoptries, micronemes) that are thought to control host cell responses. In order to explore parasite-mediated modulation of host cell signaling pathways, we exploited a phylogenomic approach to characterize the Toxoplasma gondii kinome, defining a 44 member family of coccidian-specific secreted kinases, some of which have been previously implicated in virulence. Comparative genomic analysis suggests that “ROPK” genes are under positive selection, and expression profiling demonstrates that most are differentially expressed between strains and/or during differentiation. Integrating diverse genomic-scale analyses points to ROP38 as likely to be particularly important in parasite biology. Upregulating expression of this previously uncharacterized gene in transgenic parasites dramatically suppresses transcriptional responses in the infected cell. Specifically, parasite ROP38 downregulates host genes associated with MAPK signaling and the control of apoptosis and proliferation. These results highlight the value of integrative genomic approaches in prioritizing candidates for functional validation. [ABSTRACT FROM AUTHOR]

Summary: Vaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy. [ABSTRACT FROM AUTHOR]

Summary: Developing cortex generates endogenous activity that modulates the formation of functional units, but how this activity is altered to support mature function is poorly understood. Using recordings from the visual cortex of preterm human infants and neonatal rats, we report a “bursting” period of visual responsiveness during which the weak retinal output is amplified by endogenous network oscillations, enabling a primitive form of vision. This period ends shortly before delivery in humans and eye opening in rodents with an abrupt switch to the mature visual response. The switch is causally linked to the emergence of an activated state of continuous cortical activity dependent on the ascending neuromodulatory systems involved in arousal. This switch is sensory system specific but experience independent and also involves maturation of retinal processing. Thus, the early development of visual processing is governed by a conserved, intrinsic program that switches thalamocortical response properties in anticipation of patterned vision. [ABSTRACT FROM AUTHOR]

Summary: Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of ∼3, compared with conventional cryoEM. The reconstruction was carried out to 13 Å resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions. [ABSTRACT FROM AUTHOR]

Summary: The mammalian brain exhibits profuse interregional connectivity. How information flow is rapidly and flexibly switched among connected areas remains poorly understood. Task-dependent changes in the power and interregion coherence of network oscillations suggest that such oscillations play a role in signal routing. We show that switching one of several convergent pathways from an asynchronous to an oscillatory state allows accurate selective transmission of population-coded information, which can be extracted even when other convergent pathways fire asynchronously at comparable rates. We further show that the band-pass filtering required to perform this information extraction can be implemented in a simple spiking network model with a single feed-forward interneuron layer. This constitutes a mechanism for flexible signal routing in neural circuits, which exploits sparsely synchronized network oscillations and temporal filtering by feed-forward inhibition. Video Abstract: Display Omitted [ABSTRACT FROM AUTHOR]

Summary: Repetitive antigen stimulation by prime-boost vaccination or pathogen reencounter increases memory CD8+ T cell numbers, but the impact on memory CD8+ T cell differentiation is unknown. Here we showed that repetitive antigen stimulations induced accumulation of memory CD8+ T cells with uniform effector memory characteristics. However, genome-wide microarray analyses revealed that each additional antigen challenge resulted in the differential regulation of several hundred new genes in the ensuing memory CD8+ T cell populations and, therefore, in stepwise diversification of CD8+ T cell transcriptomes. Thus, primary and repeatedly stimulated (secondary, tertiary, and quaternary) memory CD8+ T cells differed substantially in their molecular signature while sharing expression of a small group of genes and biological pathways, which may constitute a core signature of memory differentiation. These results reveal the complex regulation of memory CD8+ T cell differentiation and identify potential new molecular targets to dissect the function of memory cells generated by repeated antigen stimulation. [Copyright &y& Elsevier]

Summary: Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors—NF-κB, STAT4, and T-bet—with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-κB therefore cooperate at multiple cis elements to enable NF-κB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8+ T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals. [Copyright &y& Elsevier]

Summary: The organization of bristles on the Drosophila notum has long served as a popular model of robust tissue patterning. During this process, membrane-tethered Delta activates intracellular Notch signaling in neighboring epithelial cells, which inhibits Delta expression. This induces lateral inhibition, yielding a pattern in which each Delta-expressing mechanosensory organ precursor cell in the epithelium is surrounded on all sides by cells with active Notch signaling. Here, we show that conventional models of Delta-Notch signaling cannot account for bristle spacing or the gradual refinement of this pattern. Instead, the pattern refinement we observe using live imaging is dependent upon dynamic, basal actin-based filopodia and can be quantitatively reproduced by simulations of lateral inhibition incorporating Delta-Notch signaling by transient filopodial contacts between nonneighboring cells. Significantly, the intermittent signaling induced by these filopodial dynamics generates a type of structured noise that is uniquely suited to the generation of well-ordered, tissue-wide epithelial patterns. [Copyright &y& Elsevier]

Summary: The parietal lobe has long been viewed as a collection of architectonic and functional subdivisions. Though much parietal research has focused on mechanisms of visuospatial attention and control-related processes, more recent functional neuroimaging studies of memory retrieval have reported greater activity in left lateral parietal cortex (LLPC) when items are correctly identified as previously studied (“old”) versus unstudied (“new”). These studies have suggested functional divisions within LLPC that may provide distinct contributions toward recognition memory judgments. Here, we define regions within LLPC by developing a parcellation scheme that integrates data from resting-state functional connectivity MRI and functional MRI. This combined approach results in a 6-fold parcellation of LLPC based on the presence (or absence) of memory-retrieval-related activity, dissociations in the profile of task-evoked time courses, and membership in large-scale brain networks. This parcellation should serve as a roadmap for future investigations aimed at understanding LLPC function. [Copyright &y& Elsevier]

Recent genome-scale analyses of genetic interactions in model microbes have revealed the inherent functional organization of the cell as a dense network of highly interconnected pathways. While classical one gene at a time paradigms offer limited insight into cellular systems, genome-scale approaches are making considerable headway. Indeed, where small organic compounds are ideal probes of biological complexity, systematic chemical genomic methods are emerging as requisite and powerful approaches to describing both the small molecule probe and network with which it interacts. Here, we highlight various chemical genomic approaches that are being pioneered in model microbes. [Copyright &y& Elsevier]

Although a number of genomic and biochemical technologies are now used to elucidate the mechanisms of action of bioactive small molecules, affinity-based isolation of molecular targets is a classic, but still powerful, approach. This review highlights recent cases where biochemical isolation of target proteins of bioactive small molecules highlighted general strategies for a successful isolation and identification of molecular targets. This review is intended to be both an update on the most recent findings for those already active in the field of forward chemical genetics and a guide for scientists entering this burgeoning field. [Copyright &y& Elsevier]

Summary: Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4+ T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses. [Copyright &y& Elsevier]

Summary: Signal transducer and activator of transcription 4 (STAT4) and STAT6 are key factors in the specification of helper T cells; however, their direct roles in driving differentiation are not well understood. Using chromatin immunoprecipitation and massive parallel sequencing, we quantitated the full complement of STAT-bound genes, concurrently assessing global STAT-dependent epigenetic modifications and gene transcription by using cells from cognate STAT-deficient mice. STAT4 and STAT6 each bound over 4000 genes with distinct binding motifs. Both played critical roles in maintaining chromatin configuration and transcription of a core subset of genes through the combination of different epigenetic patterns. Globally, STAT4 had a more dominant role in promoting active epigenetic marks, whereas STAT6 had a more prominent role in antagonizing repressive marks. Clusters of genes negatively regulated by STATs were also identified, highlighting previously unappreciated repressive roles of STATs. Therefore, STAT4 and STAT6 play wide regulatory roles in T helper cell specification. [Copyright &y& Elsevier]

Summary: Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunity''s microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis. [Copyright &y& Elsevier]

Summary: Sensory systems are known to adapt their coding strategies to the statistics of their environment, but little is still known about the perceptual implications of such adjustments. We investigated how auditory spatial processing adapts to stimulus statistics by presenting human listeners and anesthetized ferrets with noise sequences in which interaural level differences (ILD) rapidly fluctuated according to a Gaussian distribution. The mean of the distribution biased the perceived laterality of a subsequent stimulus, whereas the distribution''s variance changed the listeners'' spatial sensitivity. The responses of neurons in the inferior colliculus changed in line with these perceptual phenomena. Their ILD preference adjusted to match the stimulus distribution mean, resulting in large shifts in rate-ILD functions, while their gain adapted to the stimulus variance, producing pronounced changes in neural sensitivity. Our findings suggest that processing of auditory space is geared toward emphasizing relative spatial differences rather than the accurate representation of absolute position. [Copyright &y& Elsevier]

Summary: Analyses of biological processes would benefit from accurate definitions of protein complexes. High-throughput mass spectrometry data offer the possibility of systematically defining protein complexes; however, the predicted compositions vary substantially depending on the algorithm applied. We determine consensus compositions for 409 core protein complexes from Saccharomyces cerevisiae by merging previous predictions with a new approach. Various analyses indicate that the consensus is comprehensive and of high quality. For 85 out of 259 complexes not recorded in GO, literature search revealed strong support in the form of coprecipitation. New complexes were verified by an independent interaction assay and by gene expression profiling of strains with deleted subunits, often revealing which cellular processes are affected. The consensus complexes are available in various formats, including a merge with GO, resulting in 518 protein complex compositions. The utility is further demonstrated by comparison with binary interaction data to reveal interactions between core complexes. [Copyright &y& Elsevier]

Summary: When E. coli cells express unneeded protein, they grow more slowly. Such penalty to fitness associated with making proteins is called protein cost. Protein cost is an important component in the cost-benefit tradeoffs that underlie the evolution of protein circuits, but its origins are still poorly understood. Here, we ask how the protein cost varies during the exponential growth phase of E. coli. We find that cells growing exponentially following an upshift from overnight culture show a large cost when producing unneeded proteins. However, after several generations, while still in exponential growth, the cells enter a phase where cost is much reduced despite vigorous unneeded protein production. We find that this reduced-cost phase depends on the ppGpp system, which adjusts the amount of ribosomes in the cell and does not occur after a downshift from rich to poor medium. These findings suggest that protein cost is a transient phenomenon that happens upon an upshift in conditions and that cost is reduced when ribosomes and other cellular systems have increased to their appropriate steady-state level in the new condition. [Copyright &y& Elsevier]

Summary: The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional signaling components, we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly represented. Included in this group are members of the Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein α subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These insights to the G protein signaling network reveal the essential genome as an untapped resource for identifying new components and regulators of signal transduction pathways. [Copyright &y& Elsevier]

Summary: The development of new anticancer agents derived from natural resources requires a rapid identification of their molecular mechanism of action. To make this step short, we have initiated the proteomic profiling of HeLa cells treated with anticancer drugs representing a wide spectrum of mechanisms of action using two-dimensional difference gel electrophoresis (2D-DIGE). Unique proteome patterns were observed in HeLa cells treated with the HSP90 inhibitor geldanamycin, and were similar to the patterns induced by radicicol, a structurally different HSP90 inhibitor. On the other hand, etoposide and ICRF-193, compounds claimed to be topoisomerase II inhibitors, showed different proteomic profiles, which reflect their different biological activities as revealed by cell-cycle analysis. Thus far, combined data from 19 compounds have allowed their successful classification by cluster analysis according to the mechanism of action. [Copyright &y& Elsevier]

Summary: STAT3, an essential transcription factor with pleiotropic functions, plays critical roles in the pathogenesis of autoimmunity. Despite recent data linking STAT3 with inflammatory bowel disease, exactly how it contributes to chronic intestinal inflammation is not known. Using a T cell transfer model of colitis, we found that STAT3 expression in T cells was essential for the induction of both colitis and systemic inflammation. STAT3 was critical in modulating the balance of T helper 17 (Th17) and regulatory T (Treg) cells, as well as in promoting CD4+ T cell proliferation. We used chromatin immunoprecipitation and massive parallel sequencing (ChIP-Seq) to define the genome-wide targets of STAT3 in CD4+ T cells. We found that STAT3 bound to multiple genes involved in Th17 cell differentiation, cell activation, proliferation, and survival, regulating both expression and epigenetic modifications. Thus, STAT3 orchestrates multiple critical aspects of T cell function in inflammation and homeostasis. [Copyright &y& Elsevier]

Summary: The recoding of diverse sensory and motor signals by granule cells (GCs) is probably critical for the function of cerebellar circuits, yet the nature of these transformations and their significance for cerebellar information processing remain poorly understood. In cerebellum-like structures in fish, anti-Hebbian plasticity at parallel fiber synapses generates “negative images” that act to cancel predictable patterns of electrosensory input. Here I test the hypothesis that GCs enhance the capacity of Purkinje-like cells to generate specific negative images by selectively encoding combinations of sensory and motor signals. Using in vivo whole-cell recordings, I show (1) that a subset of GCs integrate sensory and motor signals conveyed by distinct mossy fiber classes and (2) that Purkinje-like cells exhibit plastic changes specific to the combinations of signals that individual GCs encode. Consistent with influential theories of cerebellar function, these findings suggest that selective GC output enhances the capacity of Purkinje-like cells to acquire selectivity through associative plasticity. [Copyright &y& Elsevier]

Summary: Scale-free dynamics, with a power spectrum following P ∝f −β, are an intrinsic feature of many complex processes in nature. In neural systems, scale-free activity is often neglected in electrophysiological research. Here, we investigate scale-free dynamics in human brain and show that it contains extensive nested frequencies, with the phase of lower frequencies modulating the amplitude of higher frequencies in an upward progression across the frequency spectrum. The functional significance of scale-free brain activity is indicated by task performance modulation and regional variation, with β being larger in default network and visual cortex and smaller in hippocampus and cerebellum. The precise patterns of nested frequencies in the brain differ from other scale-free dynamics in nature, such as earth seismic waves and stock market fluctuations, suggesting system-specific generative mechanisms. Our findings reveal robust temporal structures and behavioral significance of scale-free brain activity and should motivate future study on its physiological mechanisms and cognitive implications. [Copyright &y& Elsevier]

Summary: In many regions of the visual system, the activity of a neuron is normalized by the activity of other neurons in the same region. Here we show that a similar normalization occurs during olfactory processing in the Drosophila antennal lobe. We exploit the orderly anatomy of this circuit to independently manipulate feedforward and lateral input to second-order projection neurons (PNs). Lateral inhibition increases the level of feedforward input needed to drive PNs to saturation, and this normalization scales with the total activity of the olfactory receptor neuron (ORN) population. Increasing total ORN activity also makes PN responses more transient. Strikingly, a model with just two variables (feedforward and total ORN activity) accurately predicts PN odor responses. Finally, we show that discrimination by a linear decoder is facilitated by two complementary transformations: the saturating transformation intrinsic to each processing channel boosts weak signals, while normalization helps equalize responses to different stimuli. Video Abstract: Display Omitted [Copyright &y& Elsevier]

Summary: Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV. [Copyright &y& Elsevier]

Summary: Many promoters in eukaryotes have nucleosome-depleted regions (NDRs) containing transcription factor binding sites. However, the functional significance of NDRs is not well understood. Here, we examine NDR function in two cell cycle-regulated promoters, CLN2pr and HOpr, by varying nucleosomal coverage of the binding sites of their activator, Swi4/Swi6 cell-cycle box (SCB)-binding factor (SBF), and probing the corresponding transcriptional activity in individual cells with time-lapse microscopy. Nucleosome-embedded SCBs do not significantly alter peak expression levels. Instead, they induce bimodal, “on/off” activation in individual cell cycles, which displays short-term memory, or epigenetic inheritance, from the mother cycle. In striking contrast, the same SCBs localized in NDR lead to highly reliable activation, once in every cell cycle. We further demonstrate that the high variability in Cln2p expression induced by the nucleosomal SCBs reduces cell fitness. Therefore, we propose that the NDR function in limiting stochasticity in gene expression promotes the ubiquity and conservation of promoter NDR. PaperClip: Display Omitted [Copyright &y& Elsevier]

Summary: MicroRNAs are emerging as important regulators of diverse biological processes and pathologies in animals and plants. Though hundreds of human microRNAs are known, only a few have known functions. Here, we predict human microRNA functions by using a new method that systematically assesses the statistical enrichment of several microRNA-targeting signatures in annotated gene sets such as signaling networks and protein complexes. Some of our top predictions are supported by published experiments, yet many are entirely new or provide mechanistic insights to known phenotypes. Our results indicate that coordinated microRNA targeting of closely connected genes is prevalent across pathways. We use the same method to infer which microRNAs regulate similar targets and provide the first genome-wide evidence of pervasive cotargeting, in which a handful of “hub” microRNAs are involved in a majority of cotargeting relationships. Our method and analyses pave the way to systematic discovery of microRNA functions. [Copyright &y& Elsevier]

Summary: Enhancers determine tissue-specific gene expression programs. Enhancers are marked by high histone H3 lysine 4 mono-methylation (H3K4me1) and by the acetyl-transferase p300, which has allowed genome-wide enhancer identification. However, the regulatory principles by which subsets of enhancers become active in specific developmental and/or environmental contexts are unknown. We exploited inducible p300 binding to chromatin to identify, and then mechanistically dissect, enhancers controlling endotoxin-stimulated gene expression in macrophages. In these enhancers, binding sites for the lineage-restricted and constitutive Ets protein PU.1 coexisted with those for ubiquitous stress-inducible transcription factors such as NF-κB, IRF, and AP-1. PU.1 was required for maintaining H3K4me1 at macrophage-specific enhancers. Reciprocally, ectopic expression of PU.1 reactivated these enhancers in fibroblasts. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors determines the activity of a distinct group of enhancers. We suggest that this may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation. [Copyright &y& Elsevier]

Summary: The regulation of mitotic entry in somatic cells differs from embryonic cells, yet it is only for embryonic cells that we have a quantitative understanding of this process. To gain a similar insight into somatic cells, we developed a human cell extract system that recapitulates CDK1 activation and nuclear envelope breakdown in response to mitotic cyclins. As cyclin B concentrations increase, CDK1 activates in a three-stage nonlinear response, creating an ordering of substrate phosphorylations. This response is established by dual regulatory feedback loops involving WEE1/MYT1, which impose a cyclin B threshold, and CDC25, which allows CDK1 to escape the WEE1/MYT1 inhibition. This system also exhibits a complex response to cyclin A. Cyclin A promotes WEE1 phosphorylation to weaken the negative feedback loop and primes mitotic entry through cyclin B. This observation explains the requirement of both cyclin A and cyclin B to initiate mitosis in somatic cells. [Copyright &y& Elsevier]

Summary: To better understand yeast ribosome synthesis, we developed techniques for the rapid harvesting and analysis of metabolically labeled cultures. Modeling of the resulting kinetic data allowed predicted lifetimes and processing patterns to be compared with the experimental data. This supported a transcription time for the 35S primary transcripts of ∼170 s at 30°C (∼40 nt s−1), with a high fraction (∼70%) of nascent transcripts cleaved at the early processing sites that generate the 20S precursor to the 18S rRNA. This level of nascent transcript cleavage apparently conflicted with previous reports that modification of yeast pre-rRNA exclusively occurred on released transcripts. A second round of high-resolution kinetic labeling showed that 20S pre-rRNA predominately undergoes methylation as nascent transcripts, whereas the 27S precursor to the 25S/5.8S rRNAs was partially methylated on the nascent transcript. The results demonstrate that quantitative analyses of pre-rRNA processing can yield important biological insights. [Copyright &y& Elsevier]

Summary: A canonical feedforward circuit is proposed to underlie sensory cortical responses with balanced excitation and inhibition in layer 4 (L4). However, in another input layer, L6, sensory responses and the underlying synaptic circuits remain largely unclear. Here, cell-attached recordings in rat primary auditory cortex revealed that for the majority of L6 excitatory neurons, tonal stimuli did not drive spike responses, but suppressed spontaneous firings. Whole-cell recordings further revealed that the silencing resulted from tone-evoked strong inhibition arriving earlier than excitation. This pattern of inputs can be attributed to a parallel feedforward circuit with both excitatory and inhibitory inputs disynaptically relayed. In contrast, in the other neurons directly driven by thalamic input, stimuli evoked excitation preceding relatively weak inhibition, resulting in robust spike responses. Thus, the dichotomy of L6 response properties arises from two distinct patterns of excitatory-inhibitory interplay. The parallel circuit module generating preceding inhibition may provide a gating mechanism for conditional corticothalamic feedback. [Copyright &y& Elsevier]

Summary: The functional selectivity of human primary visual cortex (V1) for orientation and motion direction is established by around 3 months of age , but there have been few studies of the development of extrastriate visual areas that integrate outputs from V1 . We investigated sensitivity and topographical organization for global form and motion with high-density visual event-related potentials (VERPs) in 4- to 5-month-old infants and adults. Responses were measured to transitions between concentrically organized elements (short arc segments for form, dot trajectories for motion) and random arrangements. Adults showed topographically separate responses, with midline motion and more lateral form responses. Of 26 infants, 25 showed significant motion responses but only 13 showed form responses, suggesting more advanced development for extrastriate motion areas than form. Infants'' form and motion responses were topographically distinct but contrasted with the corresponding adult topographies, with infants'' motion responses more lateral than form responses. These results imply distinct neural sources at both ages and raise the possibility of substantial reorganization of extrastriate networks between infancy and adulthood. We speculate that global motion responses arise from area V5 in infants but are dominated by more medial areas such as V3/V3A and V6 in adults. [Copyright &y& Elsevier]

Summary: Dendritic cell (DC) populations consist of multiple subsets that are essential orchestrators of the immune system. Technological limitations have so far prevented systems-wide accurate proteome comparison of rare cell populations in vivo. Here, we used high-resolution mass spectrometry-based proteomics, combined with label-free quantitation algorithms, to determine the proteome of mouse splenic conventional and plasmacytoid DC subsets to a depth of 5,780 and 6,664 proteins, respectively. We found mutually exclusive expression of pattern recognition pathways not previously known to be different among conventional DC subsets. Our experiments assigned key viral recognition functions to be exclusively expressed in CD4+ and double-negative DCs. The CD8α+ DCs largely lack the receptors required to sense certain viruses in the cytoplasm. By avoiding activation via cytoplasmic receptors, including retinoic acid-inducible gene I, CD8α+ DCs likely gain a window of opportunity to process and present viral antigens before activation-induced shutdown of antigen presentation pathways occurs. [Copyright &y& Elsevier]

Summary: Sequential neural activity patterns are as ubiquitous as the outputs they drive, which include motor gestures and sequential cognitive processes. Neural sequences are long, compared to the activation durations of participating neurons, and sequence coding is sparse. Numerous studies demonstrate that spike-time-dependent plasticity (STDP), the primary known mechanism for temporal order learning in neurons, cannot organize networks to generate long sequences, raising the question of how such networks are formed. We show that heterosynaptic competition within single neurons, when combined with STDP, organizes networks to generate long unary activity sequences even without sequential training inputs. The network produces a diversity of sequences with a power law length distribution and exponent −1, independent of cellular time constants. We show evidence for a similar distribution of sequence lengths in the recorded premotor song activity of songbirds. These results suggest that neural sequences may be shaped by synaptic constraints and network circuitry rather than cellular time constants. [Copyright &y& Elsevier]

Summary: Understanding the neural code underlying perception requires the mapping of physical stimulus parameters to both psychophysical decisions and neuronal responses. Here, we employed a novel psychophysical task in head-fixed rats to measure discriminability of vibrotactile whisker deflections. Rats could discriminate 90 Hz from 60 Hz pulsatile stimuli if stimulus intensity covaried with frequency. To pin down the physical parameters used by the rats to discriminate these vibrations, we manipulated stimulus amplitude to arrive at pairs of nondiscriminable stimuli. We found that vibrations matched in intensity (measured as mean absolute velocity), but differing in frequency, were no longer discriminable. Recordings of trigeminal ganglion neurons revealed that the distribution of neurometric sensitivities based on spike counts, but not interspike intervals, matched the rats'' inability to discriminate intensity-matched stimuli. In conclusion, we suggest that stimulus mean absolute velocity, encoded in primary afferent spike counts, plays a prominent role for whisker-mediated perception. [Copyright &y& Elsevier]

Summary: Advances in image acquisition and informatics technology have led to organism-scale spatiotemporal atlases of gene expression and protein distributions. To maximize the utility of this information for the study of developmental processes, a new generation of mathematical models is needed for discovery and hypothesis testing. Here, we develop a data-driven, geometrically accurate model of early Drosophila embryonic bone morphogenetic protein (BMP)-mediated patterning. We tested nine different mechanisms for signal transduction with feedback, eight combinations of geometry and gene expression prepatterns, and two scale-invariance mechanisms for their ability to reproduce proper BMP signaling output in wild-type and mutant embryos. We found that a model based on positive feedback of a secreted BMP-binding protein, coupled with the experimentally measured embryo geometry, provides the best agreement with population mean image data. Our results demonstrate that using bioimages to build and optimize a three-dimensional model provides significant insights into mechanisms that guide tissue patterning. [Copyright &y& Elsevier]

Summary: A paradigm in transcriptional regulation is that graded increases in transcription factor (TF) concentration are translated into on/off transcriptional responses by cooperative TF binding to adjacent sites. Digital transcriptional responses underlie the definition of anatomical boundaries during development. Here we show that NF-κB, a TF controlling inflammation and immunity, is conversely an analog transcriptional regulator that uses clustered binding sites noncooperatively. We observed that increasing concentrations of NF-κB are translated into gradual increments in gene transcription. We provide a thermodynamic interpretation of the experimental observations by combining quantitative measurements and a minimal physical model of an NF-κB-dependent promoter. We demonstrate that NF-κB binds independently to adjacent sites to promote additive RNA Pol II recruitment and graded transcriptional outputs. These findings reveal an alternative mode of operation of clustered TF binding sites, which might function in biological conditions where the transcriptional output is proportional to the strength of an environmental input. [Copyright &y& Elsevier]

Summary: Neurons in visual, somatosensory, and auditory cortex can respond to the termination as well as the onset of a sensory stimulus. In auditory cortex, these off responses may underlie the ability of the auditory system to use sound offsets as cues for perceptual grouping. Off responses have been widely proposed to arise from postinhibitory rebound, but this hypothesis has never been directly tested. We used in vivo whole-cell recordings to measure the synaptic inhibition evoked by sound onset. We find that inhibition is invariably transient, indicating that off responses are not caused by postinhibitory rebound in auditory cortical neurons. Instead, on and off responses appear to be driven by distinct sets of synapses, because they have distinct frequency tuning and different excitatory-inhibitory balance. Furthermore, an on-on sequence causes complete forward suppression, whereas an off-on sequence causes no suppression at all. We conclude that on and off responses are driven by largely nonoverlapping sets of synaptic inputs. [Copyright &y& Elsevier]

High-resolution functional MRI (hr-fMRI) affords unique leverage on the functional properties of human medial temporal lobe (MTL) substructures. We review initial hr-fMRI efforts to delineate (1) encoding and retrieval processes within the hippocampal circuit, (2) hippocampal subfield contributions to pattern separation and pattern completion, and (3) the representational capabilities of distinct MTL subregions. Extant data reveal functional heterogeneity within human MTL and highlight the promise of hr-fMRI for bridging human, animal, and computational approaches to understanding MTL function. [Copyright &y& Elsevier]

Summary: Changes of ocular dominance in the visual cortex can be induced by visual manipulations during a critical period in early life. However, the role of critical period plasticity in normal development is unknown. Here we show that at the onset of this time window, the preferred orientations of individual cortical cells in the mouse are mismatched through the two eyes and the mismatch decreases and reaches adult levels by the end of the period. Deprivation of visual experience during this period irreversibly blocks the binocular matching of orientation preference, but has no effect in adulthood. The critical period of binocular matching can be delayed by long-term visual deprivation from birth, like that of ocular dominance plasticity. These results demonstrate that activity-dependent changes induced by normal visual experience during the well-studied critical period serve to match eye-specific inputs in the cortex, thus revealing a physiological role for critical period plasticity during normal development. [Copyright &y& Elsevier]

Summary: During natural vision, the entire visual field is stimulated by images rich in spatiotemporal structure. Although many visual system studies restrict stimuli to the classical receptive field (CRF), it is known that costimulation of the CRF and the surrounding nonclassical receptive field (nCRF) increases neuronal response sparseness. The cellular and network mechanisms underlying increased response sparseness remain largely unexplored. Here we show that combined CRF + nCRF stimulation increases the sparseness, reliability, and precision of spiking and membrane potential responses in classical regular spiking (RSC) pyramidal neurons of cat primary visual cortex. Conversely, fast-spiking interneurons exhibit increased activity and decreased selectivity during CRF + nCRF stimulation. The increased sparseness and reliability of RSC neuron spiking is associated with increased inhibitory barrages and narrower visually evoked synaptic potentials. Our experimental observations were replicated with a simple computational model, suggesting that network interactions among neuronal subtypes ultimately sharpen recurrent excitation, producing specific and reliable visual responses. [Copyright &y& Elsevier]

Summary: Cytokines produced during infection/inflammation activate adaptive central nervous system (CNS) responses, including acute stress responses mediated by the hypothalamo-pituitary-adrenal (HPA) axis. The mechanisms by which cytokines engage HPA control circuitry remain unclear, though stimulated release of prostanoids from neighboring vascular cells has been implicated in this regard. How specific vascular cell types, endothelial cells (ECs) versus perivascular cells (PVCs; a subset of brain-resident macrophages), participate in this response remains unsettled. We exploited the phagocytic activity of PVCs to deplete them in rats by central injection of a liposome-encapsulated proapoptotic drug. This manipulation abrogated CNS and hormonal indices of HPA activation under immune challenge conditions (interleukin-1) that activated prostanoid synthesis only in PVCs, while enhancing these responses to stimuli (lipopolysaccharide) that engaged prostanoid production by ECs as well. Thus, PVCs provide both prostanoid-mediated drive to the HPA axis and an anti-inflammatory action that constrains endothelial and overall CNS responses to inflammatory insults. [Copyright &y& Elsevier]

Summary: Remembering experiences that lead to reward is essential for survival. The hippocampus is required for forming and storing memories of events and places, but the mechanisms that associate specific experiences with rewarding outcomes are not understood. Event memory storage is thought to depend on the reactivation of previous experiences during hippocampal sharp wave ripples (SWRs). We used a sequence switching task that allowed us to examine the interaction between SWRs and reward. We compared SWR activity after animals traversed spatial trajectories and either received or did not receive a reward. Here, we show that rat hippocampal CA3 principal cells are significantly more active during SWRs following receipt of reward. This SWR activity was further enhanced during learning and reactivated coherent elements of the paths associated with the reward location. This enhanced reactivation in response to reward could be a mechanism to bind rewarding outcomes to the experiences that precede them. [Copyright &y& Elsevier]

Summary: Multipotential naive CD4+ T cells differentiate into distinct lineages including T helper 1 (Th1), Th2, Th17, and inducible T regulatory (iTreg) cells. The remarkable diversity of CD4+ T cells begs the question whether the observed changes reflect terminal differentiation with heritable epigenetic modifications or plasticity in T cell responses. We generated genome-wide histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) trimethylation maps in naive, Th1, Th2, Th17, iTreg, and natural Treg (nTreg) cells. We found that although modifications of signature-cytokine genes (Ifng, Il4, and Il17) partially conform to the expectation of lineage commitment, genes encoding transcription factors like Tbx21 exhibit a broad spectrum of epigenetic states, consistent with our demonstration of T-bet and interferon-γ induction in nTreg cells. Our data suggest an epigenetic mechanism underlying the specificity and plasticity of effector and regulatory T cells and also provide a framework for understanding complexity of CD4+ T helper cell differentiation. [Copyright &y& Elsevier]

Summary: The ability of macrophages to clear pathogens and elicit a sustained immune response is regulated by various cytokines, including interferon-γ (IFN-γ). To investigate the molecular mechanisms by which IFN-γ modulates phagosome functions, we profiled the changes in composition, abundance, and phosphorylation of phagosome proteins in resting and activated macrophages by using quantitative proteomics and bioinformatics approaches. We identified 2415 phagosome proteins together with 2975 unique phosphorylation sites with a high level of sensitivity. Using network analyses, we determined that IFN-γ delays phagosomal acquisition of lysosomal hydrolases and peptidases for the gain of antigen presentation. Furthermore, this gain in antigen presentation is dependent on phagosomal networks of the actin cytoskeleton and vesicle-trafficking proteins, as well as Src kinases and calpain proteases. Major histocompatibility complex class I antigen-presentation assays validated the molecular participation of these networks in the enhanced capacity of IFN-γ-activated macrophages to crosspresent exogenous antigens to CD8+ T cells. [Copyright &y& Elsevier]

Summary: In budding yeast, the highly conserved small GTPase Cdc42 localizes to the cortex at a cell pole and orchestrates the trafficking and deposition of cell surface materials required for building a bud or mating projection (shmoo). Using a combination of quantitative imaging and mathematical modeling, we elucidate mechanisms of dynamic recycling of Cdc42 that balance diffusion. Rdi1, a guanine nucleotide dissociation inhibitor (GDI), mediates a fast recycling pathway, while actin patch-mediated endocytosis accounts for a slower one. These recycling mechanisms are restricted to the same region of the nascent bud, as both are coupled to the Cdc42 GTPase cycle. We find that a single dynamic parameter, the rate of internalization inside the window of polarized delivery, is tuned to give rise to distinct shapes of Cdc42 distributions that correlate with distinct morphogenetic fates, such as the formation of a round bud or a pointed shmoo. [Copyright &y& Elsevier]

Summary: The discovery of pathways and regulatory networks whose perturbation contributes to neoplastic transformation remains a fundamental challenge for cancer biology. We show that such pathway perturbations, and the cis-regulatory elements through which they operate, can be efficiently extracted from global gene expression profiles. Our approach utilizes information-theoretic analysis of expression levels, pathways, and genomic sequences. Analysis across a diverse set of human cancers reveals the majority of previously known cancer pathways. Through de novo motif discovery we associate these pathways with transcription-factor binding sites and miRNA targets, including those of E2F, NF-Y, p53, and let-7. Follow-up experiments confirmed that these predictions correspond to functional in vivo regulatory interactions. Strikingly, the majority of the perturbations, associated with putative cis-regulatory elements, fall outside of known cancer pathways. Our study provides a systems-level dissection of regulatory perturbations in cancer—an essential component of a rational strategy for therapeutic intervention and drug-target discovery. [Copyright &y& Elsevier]

Summary: Many sensory systems (e.g., vision and hearing) show a response that is proportional to the fold-change in the stimulus relative to the background, a feature related to Weber''s Law. Recent experiments suggest such a fold-change detection feature in signaling systems in cells: a response that depends on the fold-change in the input signal, and not on its absolute level. It is therefore of interest to find molecular mechanisms of gene regulation that can provide such fold-change detection. Here, we demonstrate theoretically that fold-change detection can be generated by one of the most common network motifs in transcription networks, the incoherent feedforward loop (I1-FFL), in which an activator regulates both a gene and a repressor of the gene. The fold-change detection feature of the I1-FFL applies to the entire shape of the response, including its amplitude and duration, and is valid for a wide range of biochemical parameters. [Copyright &y& Elsevier]

Summary: Signal-transduction cascades are usually studied on cell averages, masking variability between individual cells. To address this, we studied in individual cells the dynamic response of ERK2, a well-characterized MAPK signaling protein, which enters the nucleus upon stimulation. Using fluorescent tagging at the endogenous chromosomal locus, we found that cells show wide basal variation in ERK2 nuclear levels. Upon EGF stimulation, cells show (1) a fold-change response, where peak nuclear accumulation of ERK2 is proportional to basal level in each cell; and (2) exact adaptation in nuclear levels of ERK2, returning to original basal level of each cell. The timing of ERK2 dynamics is more precise between cells than its amplitude. We further found that in some cells ERK2 exhibits a second pulse of nuclear entry, smaller than the first. The present study suggests that this signaling system compensates for natural biological noise: despite large variation in nuclear basal levels, ERK2''s fold dynamics is similar between cells. [Copyright &y& Elsevier]