CNS Neural/Glial Progenitors as Targets of HIV-1 and Opiates: Effects on Proliferation and Population Dynamics May Alter Behavior Outcomes.

Human immunodeficiency virus (HIV) infected patients with a history of injection opiate abuse have higher incidences of acquired immunodeficiency syndrome (AIDS) and neurological dysfunction. The use of combined anti-retroviral therapy has significantly reduced the prevalence of mortality and progression to AIDS. Due to extended life expectancy, these patients are still at a great risk for HIV-associated neurological disorders and impairment in their later life. Neural progenitor cells (NPCs) play critical roles in brain growth and repair after injury and insult. Pediatric HIV patients whose glial populations are still developing are especially at risk for central nervous system (CNS) damage. Our previous reports suggest that HIV-1 transactivator of transcription (Tat) can directly cause pathology in neural progenitors and oligodendroglia (OLs) (Hauser et al. 2009). Thus, we have hypothesized that NPCs and/or glial progenitors may be targets of HIV proteins ± opiates drugs of abuse. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins Tat or glycoprotein 120 (gp120) with/without morphine. Murine striatal progenitors released increased amount of the beta-chemokines CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES), CCL3/macrophage inflammatory protein-1α (MIP-1α), and CCL4/macrophage inflammatory protein-1β (MIP-1β) after 12 h exposure to HIV-1 Tat, but no to gp120. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti- C-C chemokine receptor type 5 (CCR5) antibody pre-treatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. We also examined effects of Tat and morphine on proliferation and lineage progression of NPCs in vitro and in vivo. In vitro, Tat and morphine independently reduced the proliferation and population of Sox2+ and Olig2+ cells in the absence of cell death. The interactive effects of morphine and either Tat or supernatant from HIV-1SF162 infected monocytes varied depending on outcome measure and time of exposure, but interactive effects occurred primarily on proliferation. In rare instances, viable human progenitors were associated with p24 immunolabeling suggesting that progenitors may be infected, a concept that is still controversial. To investigate effects of Tat and morphine on NPCs in vivo, we used a mouse model in which HIV-1 Tat1-86 is conditionally expressed in astroglia. In vivo results in neonatal striata were similar to those in cell cultures. We extended the experiments into adult mice with inducing Tat expression for 3 month and the effect of sexes was examined in these animals. Intriguingly, males showed more Tat-induced impairment in behavioral tests (rotarod, grip strength, light-dark box) than females. Tat+ males also showed a greater reduction in the proportion of NeuN+ cells and NeuN immunoreactivity in the striatum, accompanied by greater microglial activation (3-nitrotyrosine+/Iba-1+). Unbiased stereological estimation in Nissl staining revealed that the depletion of NeuN immunoreactivity in these mice was not due to neuron cell death or loss, because the total neuron number in striatum and total striatal volume were not affected by long-term Tat induction. Tat exposure appears to selectively reduce levels of NeuN in living neurons, although the reason is not known. Therefore, both the enhanced microglial reactivity and depletion of NeuN levels in males may help to explain sex-specific behavioral outcomes. Sox2+ and Olig2+ cells showed equivalent reduction in their population in both sexes. Overall, our findings show that CNS progenitors, including both undifferentiated NPCs and glial progenitors, are vulnerable to individual or combined effects of HIV-1 or Tat and opiates. Changes in progenitor dynamics may alter the balance of cell populations in both the developing and adult CNS. We speculate that such changes may contribute to the behavioral abnormalities that we observed in Tat+ mice and which appear to model aspects of motor, cognitive and anxiety deficits in HIV-infected patients

Synergistic Effects of Hyaluronate - Epidermal Growth Factor Conjugate Patch on Chronic Wound Healing

The proteolytic microenvironment in the wound area reduces the stability and the half-life of growth factors in vivo, making difficult the topical delivery of growth factors. Here, epidermal growth factor (EGF) was conjugated to hyaluronate (HA) to improve the long-term stability against enzymatic degradation and the therapeutic effect by enhancing the biological interaction with HA receptors on skin cells. After the synthesis of HA-EGF conjugates, they were incorporated into a patch-type formulation for the facile topical application and sustained release of EGF. According to ELISA, the HA-EGF conjugates showed a long-term stability compared with native EGF. Furthermore, HA-EGF conjugates appeared to interact with skin cells through two types of HA and EGF receptors, resulting in a synergistically improved healing effect. Taken together, we could confirm the feasibility of HA-EGF conjugates for the transdermal treatment of chronic wounds.11Ysciescopu

Fractalkine/CX3CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

<p>Abstract</p> <p>Background</p> <p>Fractalkine/CX₃CL1 and its cognate receptor CX₃CR1 are abundantly expressed in the CNS. Fractalkine is an unusual C-X3-C motif chemokine that is important in neuron-microglial communication, a co-receptor for HIV infection, and can be neuroprotective. To assess the effects of fractalkine on opiate-HIV interactive neurotoxicity, wild-type murine striatal neurons were co-cultured with mixed glia from the striata of wild-type or <it>Cx3cr1 </it>knockout mice ± HIV-1 Tat and/or morphine. Time-lapse digital images were continuously recorded at 20 min intervals for up to 72 h using computer-aided microscopy to track the same cells repeatedly.</p> <p>Results</p> <p>Co-exposure to Tat and morphine caused synergistic increases in neuron death, dendritic pruning, and microglial motility as previously reported. Exogenous fractalkine prevented synergistic Tat and morphine-induced dendritic losses and neuron death even though the inflammatory mediator TNF-α remained significantly elevated. Antibody blockade of CX₃CR1 mimicked the toxic effects of morphine plus Tat, but did not add to their toxicity; while fractalkine failed to protect wild-type neurons co-cultured with <it>Cx₃cr1</it>^-/--null glia against morphine and Tat toxicity. Exogenous fractalkine also normalized microglial motility, which is elevated by Tat and morphine co-exposure, presumably limiting microglial surveillance that may lead to toxic effects on neurons. Fractalkine immunofluorescence was expressed in neurons and to a lesser extent by other cell types, whereas CX₃CR1 immunoreactivity or GFP fluorescence in cells cultured from the striatum of <it>Cx3cr1</it>^-/-(<it>Cx3cr1</it>^GFP/GFP) mice were associated with microglia. Immunoblotting shows that fractalkine levels were unchanged following Tat and/or morphine exposure and there was no increase in released fractalkine as determined by ELISA. By contrast, CX₃CR1 protein levels were markedly downregulated.</p> <p>Conclusions</p> <p>The results suggest that deficits in fractalkine-CX₃CR1 signaling contribute to the synergistic neurotoxic effects of opioids and Tat. Importantly, exogenous fractalkine can selectively protect neurons from the injurious effects of chronic opioid-HIV-1 Tat co-exposure, and this suggests a potential therapeutic course for neuroAIDS. Although the cellular mechanisms underlying neuroprotection are not certain, findings that exogenous fractalkine reduces microglial motility and fails to protect neurons co-cultured with <it>Cx3cr1</it>^-/-mixed glia suggest that fractalkine may act by interfering with toxic microglial-neuron interactions.</p

Mapping genomic loci implicates genes and synaptic biology in schizophrenia

Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

A central role for glial CCR5 in directing the neuropathological interactions of HIV-1 Tat and opiates

Abstract Background The collective cognitive and motor deficits known as HIV-associated neurocognitive disorders (HAND) remain high even among HIV+ individuals whose antiretroviral therapy is optimized. HAND is worsened in the context of opiate abuse. The mechanism of exacerbation remains unclear but likely involves chronic immune activation of glial cells resulting from persistent, low-level exposure to the virus and viral proteins. We tested whether signaling through C-C chemokine receptor type 5 (CCR5) contributes to neurotoxic interactions between HIV-1 transactivator of transcription (Tat) and opiates and explored potential mechanisms. Methods Neuronal survival was tracked in neuronal and glial co-cultures over 72 h of treatment with HIV-1 Tat ± morphine using cells from CCR5-deficient and wild-type mice exposed to the CCR5 antagonist maraviroc or exogenously-added BDNF (analyzed by repeated measures ANOVA). Intracellular calcium changes in response to Tat ± morphine ± maraviroc were assessed by ratiometric Fura-2 imaging (analyzed by repeated measures ANOVA). Release of brain-derived neurotrophic factor (BDNF) and its precursor proBDNF from CCR5-deficient and wild-type glia was measured by ELISA (analyzed by two-way ANOVA). Levels of CCR5 and μ-opioid receptor (MOR) were measured by immunoblotting (analyzed by Student’s t test). Results HIV-1 Tat induces neurotoxicity, which is greatly exacerbated by morphine in wild-type cultures expressing CCR5. Loss of CCR5 from glia (but not neurons) eliminated neurotoxicity due to Tat and morphine interactions. Unexpectedly, when CCR5 was lost from glia, morphine appeared to entirely protect neurons from Tat-induced toxicity. Maraviroc pre-treatment similarly eliminated neurotoxicity and attenuated neuronal increases in [Ca2+]i caused by Tat ± morphine. proBDNF/BDNF ratios were increased in conditioned media from Tat ± morphine-treated wild-type glia compared to CCR5-deficient glia. Exogenous BDNF treatments mimicked the pro-survival effect of glial CCR5 deficiency against Tat ± morphine. Conclusions Our results suggest a critical role for glial CCR5 in mediating neurotoxic effects of HIV-1 Tat and morphine interactions on neurons. A shift in the proBDNF/BDNF ratio that favors neurotrophic support may occur when glial CCR5 signaling is blocked. Some neuroprotection occurred only in the presence of morphine, suggesting that loss of CCR5 may fundamentally change signaling through the MOR in glia

Hyaluronate - Epidermal Growth Factor Conjugate for Skin-Wound Healing and Regeneration

Epidermal growth factor (EGF) has been recognized as an excellent wound healing agent due to its therapeutic function stimulating skin cell growth, proliferation and differentiation. However, the transdermal delivery of EGF poses a significant challenge due to its short half-life and lack of efficient formulation. Here, to improve the transdermal delivery efficiency, EGF was conjugated to hyaluronate (HA), which was formulated into a patch-type film for skin wound healing. HA-EGF conjugate was synthesized by coupling reaction between aldehyde-modified HA and N-terminal amine group of EGF to minimize the loss of biological activities. The HA-EGF conjugates exhibited similar biological activities with native EGF as confirmed by ELISA and proliferation tests using murine and human fibroblasts. For the efficient topical delivery, HA-EGF conjugates were incorporated into a matrix film of high molecular weight HA. Two-photon microscopy clearly-visualized more efficient transdermal delivery of HA-EGF conjugates to both normal skin and peripheral tissues around the wound area rather than that of EGF. Optical imaging and ELISA after in vivo transdermal delivery showed that the conjugation of EGF to HA retarded its degradation and extended its residence time in the wound area. Furthermore, in vivo transdermal delivery of HA-EGF conjugate in the patch-type HA film resulted in significantly improved regeneration of skin tissues even into hypodermis.111410sciescopu