eScholarship@UMMS

BACKGROUND: Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.

METHOD: The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.

RESULTS: Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.

CONCLUSIONS: B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.

Dendritic cells (DCs) regulate innate and acquired immunity through their roles as antigen-presenting cells. Specific subsets of mature DCs, including monocyte-derived and lymphoid-derived DCs, can be distinguished based on distinct immunophenotypes and functional properties. The leukocyte integrin, CD11c, is considered a specific marker for DCs and it is expressed by all DC subsets. We created a strain of mice in which DCs and their progenitors could be lineage traced based on activity of the CD11c proximal promoter. Surprisingly, we observed levels of CD11c promoter activity that were similar in DCs and in other mature leukocytes, including monocytes, granulocytes, and lymphocytes. We sought to identify DNA elements and transcription factors that regulate DC-associated expression of CD11c. The ets transcription factor, PU.1, is a key regulator of DC development, and expression of PU.1 varies in different DC subsets. GM-CSF increased monocyte-derived DCs in mice and from mouse bone marrow cultured in vitro, but it did not increase CD8(+) lymphoid-derived DCs or B220(+) plasmacytoid DCs. FLT3L increased both monocyte-derived DCs and lymphoid-derived DCs from mouse bone marrow cultured in vitro. GM-CSF increased the 5.3 Kb CD11c proximal promoter activity in monocyte-derived DCs and CD8(+) lymphoid-derived DCs, but not in B220(+) plasmacytoid DCs. In contrast, FLT3L increased the CD11c proximal promoter activity in both monocyte-derived DCs and B220(+) plasmacytoid DCs. We used shRNA gene knockdown and chromatin immunoprecipitation to demonstrate that PU.1 is required for the effects of GM-CSF or FLT3L on monocyte-derived DCs. We conclude that both GM-CSF and FLT3L act through PU.1 to activate the 5.3 Kb CD11c proximal promoter in DCs and to induce differentiation of monocyte-derived DCs. We also confirm that the CD11c proximal promoter is not sufficient to direct lineage specificity of CD11c expression, and that additional DNA elements are required for lineage-specific CD11c expression.

Breast cancers that are "triple-negative" for the clinical markers ESR1, PGR, and HER2 typically belong to the Basal-like molecular subtype. Defective Rb, p53, and Brca1 pathways are each associated with triple-negative and Basal-like subtypes. Our mouse genetic studies demonstrate that the combined inactivation of Rb and p53 pathways is sufficient to suppress the physiological cell death of mammary involution. Furthermore, concomitant inactivation of all three pathways in mammary epithelium has an additive effect on tumor latency and predisposes highly penetrant, metastatic adenocarcinomas. The tumors are poorly differentiated and have histologic features that are common among human Brca1-mutated tumors, including heterogeneous morphology, metaplasia, and necrosis. Gene expression analyses demonstrate that the tumors share attributes of both Basal-like and Claudin-low signatures, two molecular subtypes encompassed by the broader, triple-negative class defined by clinical markers.

Binary cell fate decisions allow the production of distinct sister neurons from an intermediate precursor. Neurons are further diversified based on the birth order of intermediate precursors. Here we examined the interplay between binary cell fate and birth-order-dependent temporal fate in the Drosophila lateral antennal lobe (lAL) neuronal lineage. Single-cell mapping of the lAL lineage by twin-spot mosaic analysis with repressible cell markers (ts-MARCM) revealed that projection neurons (PNs) and local interneurons (LNs) are made in pairs through binary fate decisions. Forty-five types of PNs innervating distinct brain regions arise in a stereotyped sequence; however, the PNs with similar morphologies are not necessarily born in a contiguous window. The LNs are morphologically less diverse than the PNs, and the sequential morphogenetic changes in the two pairs occur independently. Sanpodo-dependent Notch activity promotes and patterns the LN fates. By contrast, Notch diversifies PN temporal fates in a Sanpodo-dispensable manner. These pleiotropic Notch actions underlie the differential temporal fate specification of twin neurons produced by common precursors within a lineage, possibly by modulating postmitotic neurons' responses to Notch-independent transcriptional cascades.

Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5-1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans.

Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53-) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.

Each fall, eastern North American monarch butterflies in their northern range undergo a long-distance migration south to their overwintering grounds in Mexico. Migrants use a time-compensated sun compass to determine directionality during the migration. This compass system uses information extracted from sun-derived skylight cues that is compensated for time of day and ultimately transformed into the appropriate motor commands. The central complex (CX) is likely the site of the actual sun compass, because neurons in this brain region are tuned to specific skylight cues. To help illuminate the neural basis of sun compass navigation, we examined the neuronal composition of the CX and its associated brain regions. We generated a standardized version of the sun compass neuropils, providing reference volumes, as well as a common frame of reference for the registration of neuron morphologies. Volumetric comparisons between migratory and nonmigratory monarchs substantiated the proposed involvement of the CX and related brain areas in migratory behavior. Through registration of more than 55 neurons of 34 cell types, we were able to delineate the major input pathways to the CX, output pathways, and intrinsic neurons. Comparison of these neural elements with those of other species, especially the desert locust, revealed a surprising degree of conservation. From these interspecies data, we have established key components of a conserved core network of the CX, likely complemented by species-specific neurons, which together may comprise the neural substrates underlying the computations performed by the CX.

(c) 2012 Wiley Periodicals, Inc.

To navigate during their long-distance migration, monarch butterflies (Danaus plexippus) use a time-compensated sun compass. The sun compass timing elements reside in light-entrained circadian clocks in the antennae. Here we show that either antenna is sufficient for proper time compensation. However, migrants with either antenna painted black (to block light entrainment) and the other painted clear (to permit light entrainment) display disoriented group flight. Remarkably, when the black-painted antenna is removed, re-flown migrants with a single, clear-painted antenna exhibit proper orientation behaviour. Molecular correlates of clock function reveal that period and timeless expression is highly rhythmic in brains and clear-painted antennae, while rhythmic clock gene expression is disrupted in black-painted antennae. Our work shows that clock outputs from each antenna are processed and integrated together in the monarch time-compensated sun compass circuit. This dual timing system is a novel example of the regulation of a brain-driven behaviour by paired organs.

The monarch butterfly (Danaus plexippus) is emerging as a model organism to study the mechanisms of circadian clocks and animal navigation, and the genetic underpinnings of long-distance migration. The initial assembly of the monarch genome was released in 2011, and the biological interpretation of the genome focused on the butterfly's migration biology. To make the extensive data associated with the genome accessible to the general biological and lepidopteran communities, we established MonarchBase (available at http://monarchbase.umassmed.edu). The database is an open-access, web-available portal that integrates all available data associated with the monarch butterfly genome. Moreover, MonarchBase provides access to an updated version of genome assembly (v3) upon which all data integration is based. These include genes with systematic annotation, as well as other molecular resources, such as brain expressed sequence tags, migration expression profiles and microRNAs. MonarchBase utilizes a variety of retrieving methods to access data conveniently and for integrating biological interpretations.

Appropriate termination of the phototransduction cascade is critical for photoreceptors to achieve high temporal resolution and to prevent excessive Ca(2+)-induced cell toxicity. Using a genetic screen to identify defective photoresponse mutants in Drosophila, we isolated and identified a novel Galpha(q) mutant allele, which has defects in both activation and deactivation. We revealed that G(q) modulates the termination of the light response and that metarhodopsin/G(q) interaction affects subsequent arrestin-rhodopsin (Arr2-Rh1) binding, which mediates the deactivation of metarhodopsin. We further showed that the Galpha(q) mutant undergoes light-dependent retinal degeneration, which is due to the slow accumulation of stable Arr2-Rh1 complexes. Our study revealed the roles of G(q) in mediating photoresponse termination and in preventing retinal degeneration. This pathway may represent a general rapid feedback regulation of G protein-coupled receptor signaling.

Inwardly rectifying potassium channels (Kir) are a special subset of potassium selective ion channels which pass potassium more easily into rather than out of the cell. These channels mediate a variety of cellular functions, including control of membrane resting potential, maintenance of potassium homeostasis and regulation of cellular metabolism. Given the existence of fifteen Kir genes in mammals, current genetic studies using mutant animals that lack a single channel may have missed many important physiological functions of these channels due to gene redundancy. This issue can be circumvented by using a simple model organism like Drosophila, whose genome encodes only 3 Kir proteins. The sophisticated genetic approaches of Drosophila may also provide powerful tools to identify additional regulation mechanisms of Kir channels. Here we provide an overview of the progress made in elucidating the function of Drosophila Kir channels. The knowledge of Drosophila Kir channels may lead us to uncover novel functions and regulation mechanisms of human Kir channels and help on pathological studies of related diseases.

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.

A daily body temperature rhythm (BTR) is critical for the maintenance of homeostasis in mammals. Whereas mammals use internal energy to regulate body temperature, ectotherms typically regulate body temperature behaviorally [1]. Some ectotherms maintain homeostasis via a daily temperature preference rhythm (TPR) [2], but the underlying mechanisms are largely unknown. Here, we show that Drosophila exhibit a daily circadian clock-dependent TPR that resembles mammalian BTR. Pacemaker neurons critical for locomotor activity are not necessary for TPR; instead, the dorsal neuron 2 s (DN2s), whose function was previously unknown, is sufficient. This indicates that TPR, like BTR, is controlled independently from locomotor activity. Therefore, the mechanisms controlling temperature fluctuations in fly TPR and mammalian BTR may share parallel features. Taken together, our results reveal the existence of a novel DN2-based circadian neural circuit that specifically regulates TPR; thus, understanding the mechanisms of TPR will shed new light on the function and neural control of circadian rhythms.

The animal circadian pacemaker is composed of two transcriptional feedback loops, which regulate electrical activity in circadian neurons. Surprisingly, a new study reports that electrical activity can reprogram circadian transcription, and identifies CREB proteins as candidates for this reprograming.

Circadian rhythms are generated by well-conserved interlocked transcriptional feedback loops in animals. In Drosophila, the dimeric transcription factor CLOCK/CYCLE (CLK/CYC) promotes period (per), timeless (tim), vrille (vri), and PAR-domain protein 1 (Pdp1) transcription. PER and TIM negatively feed back on CLK/CYC transcriptional activity, whereas VRI and PDP1 negatively and positively regulate Clk transcription, respectively. Here, we show that the alpha isoform of the Drosophila FOS homolog KAYAK (KAY) is required for normal circadian behavior. KAY-alpha downregulation in circadian pacemaker neurons increases period length by 1.5 h. This behavioral phenotype is correlated with decreased expression of several circadian proteins. The strongest effects are on CLK and the neuropeptide PIGMENT DISPERSING FACTOR, which are both under VRI and PDP1 control. Consistently, KAY-alpha can bind to VRI and inhibit its interaction with the Clk promoter. Interestingly, KAY-alpha can also repress CLK activity. Hence, in flies with low KAY-alpha levels, CLK derepression would partially compensate for increased VRI repression, thus attenuating the consequences of KAY-alpha downregulation on CLK targets. We propose that the double role of KAY-alpha in the two transcriptional loops controlling Drosophila circadian behavior brings precision and stability to their oscillations.

Glial cells are crucial regulators of synapse formation, elimination, and plasticity [1, 2]. In vitro studies have begun to identify glial-derived synaptogenic factors [1], but neuron-glia signaling events during synapse formation in vivo remain poorly defined. The coordinated development of pre- and postsynaptic compartments at the Drosophila neuromuscular junction (NMJ) depends on a muscle-secreted retrograde signal, the TGF-beta/BMP Glass bottom boat (Gbb) [3, 4]. Muscle-derived Gbb activates the TGF-beta receptors Wishful thinking (Wit) and either Saxophone (Sax) or Thick veins (Tkv) in motor neurons [3, 4]. This induces phosphorylation of Mad (P-Mad) in motor neurons, its translocation into the nucleus with a co-Smad, and activation of transcriptional programs controlling presynaptic bouton growth [5]. Here we show that NMJ glia release the TGF-beta ligand Maverick (Mav), which likely activates the muscle activin-type receptor Punt to potently modulate Gbb-dependent retrograde signaling and synaptic growth. Loss of glial Mav results in strikingly reduced P-Mad at NMJs, decreased Gbb transcription in muscle, and in turn reduced muscle-to-motor neuron retrograde TGF-beta/BMP signaling. We propose that by controlling Gbb release from muscle, glial cells fine tune the ability of motor neurons to extend new synaptic boutons in correlation to muscle growth. Our work identifies a novel glia-derived synaptogenic factor by which glia modulate synapse formation in vivo.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWC(OFF) and one induced AWC(ON), through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWC(ON)/1AWC(OFF) decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWC(ON) fate, in contrast to the autonomous role of calcium in AWCs to promote the AWC(OFF) fate. In addition, calcium in specific non-AWCs promotes AWC(ON) side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWC(OFF) and non-autonomously promoting AWC(ON).

Discusses the Function and Outcomes Research for Comparative Effectiveness in TJR (FORCE-TJR) research program for total joint replacement funded by the Agency for Healthcare Research and Quality. Led by a team of researchers at the University of Massachusetts Medical School in cooperation with a national network of surgeons, FORCE-TJR assembled a consortium of orthopedic practices to serve as a research laboratory to generate comparative effectiveness research to guide surgeon and patient decisions. The FORCE-TJR has a national scope, is representative of US practices, includes longitudinal patient-reported outcomes, and has the ability to measure implant failure as well as important clinical outcomes and complications.