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The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4 + T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4 + T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) play important roles in the control of HIV-1 infection. Here, we performed RNA-seq profiling of miRNAs and mRNAs expressed in CD4 + T lymphocytes upon HIV-1 infection. Our results reveal significant alterations in miRNA and mRNA expression profiles in infected relative to uninfected cells. One of the miRNAs markedly downregulated in infected cells is miRNA-26a. Among the putative targets of miRNA-26a are CD59 receptor transcripts, which are significantly upregulated in infected CD4 + T cells. The addition of miRNA-26a mimics to CD4 + T cells reduces CD59 at both the mRNA and surface protein levels, validating CD59 as a miRNA-26a target. Consistent with the reported inhibitory role of CD59 in complement-mediated lysis (CML), knocking out CD59 in CD4 + T cells renders both HIV-1-infected cells and progeny virions more prone to antibody-dependent CML (ADCML). The addition of miRNA-26a mimics to infected cells leads to enhanced sensitivity of progeny virions to ADCML, a condition linked to a reduction in CD59 packaging into released virions. Lastly, HIV-1-mediated downregulation of miRNA-26a expression is shown to be dependent on integrated HIV-1 expression but does not involve viral accessory proteins. Overall, these results highlight a novel mechanism by which HIV-1 limits ADCML by upregulating CD59 expression via miRNA-26a downmodulation.

Environmental circumstances shaping soil microbial communities have been studied extensively. However, due to disparate study designs, it has been difficult to resolve whether a globally consistent set of predictors exists, or context-dependency prevails. Here, we used a network of 18 grassland sites (11 of those containing regional plant productivity gradients) to examine (i) if similar abiotic or biotic factors predict both large-scale (across sites) and regional-scale (within sites) patterns in bacterial and fungal community composition, and (ii) if microbial community composition differs consistently at two levels of regional plant productivity (low vs. high). Our results revealed that bacteria were associated with particular soil properties (such as base saturation) and both bacteria and fungi were associated with plant community composition across sites and within the majority of sites. Moreover, a discernible microbial community signal emerged, clearly distinguishing high and low-productivity soils across different grasslands independent of their location in the world. Hence, regional productivity differences may be typified by characteristic soil microbial communities across the grassland biome. These results could encourage future research aiming to predict the general effects of global changes on soil microbial community composition in grasslands and to discriminate fertile from infertile systems using generally applicable microbial indicators., (© 2023 John Wiley & Sons Ltd.)

Importance: In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response., Competing Interests: The authors declare no conflict of interest.

Structured morning rounds have been used to improve communication, provide learning opportunities, and support patient care in various healthcare settings. The leadership team in an inner-city Inpatient Mental Health Unit identified a gap in the structure of morning rounds; to remedy this, a standardized reporting tool and structured morning rounding process were implemented. This short communication reports on an evaluation of staff...s perceptions on the outcomes of using a standardized reporting tool and structured rounding process to improve efficiency and communication regarding patient care among an interprofessional team. Feedback was provided on logistics, attendance, supporting factors that ensured ease of use for the new structure, and benefits of the program. While this evaluation focuses on the opinions of nurses, it serves as an example for leadership in various healthcare units on the benefits of a structured rounding program, and key factors that contribute to making the process successful. This short communication also provides an example for an efficient communication tool that can be adapted to meet the needs of various groups of healthcare disciplines., (Copyright © 2022. Published by Elsevier Inc.)

Activated-to-memory transitioning CD4 + T cells display elevated expression of the HIV-1 co-receptor CCR5 and are more prone to HIV-1 latent infection. Here, we show that p53-regulated miRNA-103 downmodulates CCR5 levels in CD4 + T lymphocytes. We reveal that miRNA-103 mimics, as well as Nutlin-3, an inhibitor of Mdm2-mediated p53 degradation, decrease CCR5-dependent HIV-1 infection. Using a dual-reporter virus, we subsequently validate that in transitioning CD4 + T cells, Nutlin-3 treatment decreases the frequency of both productively and latently infected cells via upregulation of miRNA-103. Importantly, we provide evidence that CD4 + T cells from HIV-1 elite controllers express less CCR5 than those from antiretroviral therapy-naïve progressors, an effect linked to a significant increase in miRNA-103 levels. By contributing to the control of CCR5 expression in CD4 + T cells, miRNA-103 is likely to play a key role in countering the establishment of latent HIV-1 reservoirs in vivo ., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

A key aspect in the safety testing of metal nanoparticles (NPs) is the measurement of their dissolution and of the true particle uptake in organisms. Here, based on the tendency of Ag-NP to dissolve and Au-NP to be inert in the environment, we exposed the earthworm Eisenia fetida to Au core-Ag shell NPs (Au@Ag-NPs, Ag-NPs with a Au core) and to both single and combined exposures of non-coated Au-NPs, Ag-NPs, Ag + and Au + ions in natural soil. Our hypothesis was that the Ag shell would partially or completely dissolve from the Au@Ag-NPs and that the Au core would thereby behave as a tracer of particulate uptake. Au and Ag concentrations were quantified in all the soils, in soil extract and in organisms by inductively coupled plasma mass spectrometry (ICP-MS). The earthworm exposed to Au@Ag-NPs, and to all the combinations of Ag and Au, were analyzed by single particle inductively coupled plasma time-of-flight mass spectrometry (spICP-TOFMS) to allow the quantification of the metals that were truly part of a bimetallic particle. Results showed that only 5% of the total metal amounts in the earthworm were in the bimetallic particulate form and that the Ag shell increased in thickness, suggesting that biotransformation processes took place at the surface of the NPs. Additionally, the co-exposure to both metal ions led to a different uptake pattern compared to the single metal exposures. The study unequivocally confirmed that dissolution is the primary mechanism driving the uptake of (dissolving) metal NPs in earthworms. Therefore, the assessment of the uptake of metal nanoparticles is conservatively covered by the assessment of the uptake of their ionic counterpart., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Résumé La thérapie antirétrovirale (TAR) inhibe la réplication du VIH-1 mais n'est pas curative. Pendant la TAR, le génome intégré du VIH-1 persiste principalement dans les lymphocytes T mémoires CD4+ ainsi que dans d'autres cellules immunitaires, notamment les cellules myéloïdes comme les macrophages. La majorité de ces cellules ne produisent pas de particules virales infectieuses et constituent le réservoir latent. D'importants progrès ont été réalisés dans l'identification des facteurs qui contribuent à l'établissement et au maintien du réservoir latent qui demeure le principal obstacle à l'éradication du VIH-1. Dans cette revue, nous mettrons en relief le rôle des microARN dans le développement des réservoirs viraux vu que ceux-ci sont d'importants modulateurs de l'expression génique, ciblant des facteurs de transcription ainsi que d'autres effecteurs nécessaires à l'infection productive du VIH-1. Certains microARN ciblent même directement les transcrits viraux. Nous soulignerons les grandes questions en suspens sur la participation active des microARN de l'hôte aux mécanismes de persistance virale et notamment ceux régissant la latence virale. Finalement, compte tenu des stratégies actuelles qui ne permettent toujours pas de réduire efficacement les réservoirs viraux, les perspectives quant à l'utilisation des microARN comme approche pour contrer la persistance des réservoirs latents seront discutées.

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O 2 , 5% CO 2 ), hypoxically (2% O 2 , 5% CO 2 ) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.

The remote distractor effect (RDE) is a well-known and robust phenomenon whereby latencies of saccades are increased when a distractor is presented simultaneously along with the saccade target. Studies of the RDE in patients with a loss of vision in one visual field (hemianopia) following damage to primary visual cortex have provided conflicting results. Rafal, Smith, Krantz, Cohen, and Brennan (1990) reported a naso-temporal asymmetry in the RDE in patients with hemianopias, with a greater influence of distractors presented in their blind temporal visual field. This asymmetry was not observed in typically sighted controls. By contrast, Walker, Mannan, Maurer, Pambakian, and Kennard (2000) observed no effect of distractors presented to either the blind nasal or blind temporal hemifield of hemianopes, but the naso-temporal asymmetry was observed in typically sighted controls. The present study addressed one potential methodological differences between the two studies by investigating the inhibitory effect of a distractor on saccade latency in neurotypical participants. Here participants were tested monocularly and the effect of a nasal/temporal hemifield distractor on saccade latency observed in the presence or absence of peripheral placeholders. Our results showed a naso-temporal asymmetry in the magnitude of the RDE in the no placeholder condition, with a greater RDE when the distractor was presented in the temporal visual field. However, in the placeholder condition the opposite asymmetry was observed, that is an increased RDE when the distractor was presented in the nasal visual field. Our results suggest that the presence/absence of a placeholder might be the critical factor explaining the discrepancy between Rafal et al. (1990) and Walker et al. (2000) in participants without visual field loss. The current results can be interpreted in terms of additional inhibitory or attentional processes that bias selection towards stimuli in the nasal hemifield in the presence of placeholders, still, the mechanisms underlying these effects remain unclear., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

HIV-1 encodes several accessory proteins-Nef, Vif, Vpr, and Vpu-whose functions are to modulate the cellular environment to favor immune evasion and viral replication. While Vpr was shown to mediate a G 2 /M cell cycle arrest and provide a replicative advantage during infection of myeloid cells, the mechanisms underlying these functions remain unclear. In this study, we defined HIV-1 Vpr proximity interaction network using the BioID proximity labeling approach and identified 352 potential Vpr partners/targets, including several complexes, such as the cell cycle-regulatory anaphase-promoting complex/cyclosome (APC/C). Herein, we demonstrate that both the wild type and cell cycle-defective mutants of Vpr induce the degradation of APC1, an essential APC/C scaffolding protein, and show that this activity relies on the recruitment of DCAF1 by Vpr and the presence of a functional proteasome. Vpr forms a complex with APC1, and the APC/C coactivators Cdh1 and Cdc20 are associated with these complexes. Interestingly, we found that Vpr encoded by the prototypic HIV-1 NL4.3 does not interact efficiently with APC1 and is unable to mediate its degradation as a result of a N28S-G41N amino acid substitution. In contrast, we show that APC1 degradation is a conserved feature of several primary Vpr variants from transmitted/founder virus. Functionally, Vpr-mediated APC1 degradation did not impact the ability of the protein to induce a G 2 cell cycle arrest during infection of CD4 + T cells or enhance HIV-1 replication in macrophages, suggesting that this conserved activity may be important for other aspects of HIV-1 pathogenesis. IMPORTANCE The function of the Vpr accessory protein during HIV-1 infection remains poorly defined. Several cellular targets of Vpr were previously identified, but their individual degradation does not fully explain the ability of Vpr to impair the cell cycle or promote HIV-1 replication in macrophages. Here, we used the unbiased proximity labeling approach, called BioID, to further define the Vpr proximity interaction network and identified several potentially new Vpr partners/targets. We validated our approach by focusing on a cell cycle master regulator, the APC/C complex, and demonstrated that Vpr mediated the degradation of a critical scaffolding component of APC/C called APC1. Furthermore, we showed that targeting of APC/C by Vpr did not impact the known activity of Vpr. Since degradation of APC1 is a conserved feature of several primary variants of Vpr, it is likely that the interplay between Vpr and APC/C governs other aspects of HIV-1 pathogenesis.

Aims: The Rural Hospital Medicine Training Programme (RHMTP) was established in 2008 to develop New Zealand's rural hospital medical workforce. This study evaluates the RHMTP's first 10-year outcomes., Methods: A mixed-methods descriptive study. Database interrogation of: the Royal New Zealand College of General Practitioners records; University of Otago's e-Vision; the Medical Council of New Zealand's register of doctors. A survey of trainees who had graduated or withdrew from the programme. Survey questions included: current scope and place of employment; undergraduate rural experience; and trainee experiences., Results: From 2009-2018, 98 doctors entered the RHMTP: 29 graduated, 20 withdrew and 49 are active registrars. Of the graduates, more than half (17/29) also completed GP training. Overall survey response rate: 80% (39/49). Graduate response rate: 97% (28/29). 92% (24/26) of currently practising graduates are working in rural New Zealand, mostly (22/24) in rural hospitals. Trainees value the RHMTP's flexibility and breadth of clinical exposure. The main challenges relate to a lack of alignment of training requirements and funding., Conclusions: In its first decade, the RHMTP has been successful in generating a rural hospital workforce and the programme is steadily growing. Attention to existing barriers is needed to ensure the RHMTP can reach its potential to benefit all of New Zealand's rural communities., Competing Interests: Katharina Blattner and Garry Nixon have teaching roles in the RHMTP’s academic component. Patrick McHugh has been involved in RHMTP governance. Rory Miller is a graduate of the RHMTP and is involved in teaching of the RHMTPs academic component. Joel Pirini is a current trainee of the RHMTP.

Macrophages are a target of human immunodeficiency virus type 1 (HIV-1) and may serve as a viral reservoir during antiretroviral therapy (ART). Their susceptibility to HIV-1 infection is subject to variations from permissiveness to resistance depending on their origin, tissue localization, and polarization profile. This is in part due to the expression of regulatory microRNAs. Here, we identify two microRNA paralogs, microRNA 103 (miR-103) and miR-107, as regulators of CCR5 expression that are upregulated in noninfected bystander cells of HIV-1-infected-monocyte-derived macrophage (MDM) cultures. Transfection of microRNA 103 mimics in MDMs reduced CCR5 expression levels and inhibited CCR5-dependent HIV-1 entry, whereas the corresponding antagomirs enhanced virus spread in HIV-infected MDMs. Treatment of MDMs with interleukin-1β (IL-1β) enhanced microRNA 103 expression, a condition that we found contributed to the reduction of CCR5 mRNA in IL-1β-exposed MDMs. Interestingly, we show that the induction of miR-103/107 expression is part of a tumor suppressor p53 response triggered by secreted IL-1β that renders macrophages refractory to HIV-1 entry. In a more physiological context, the levels of microRNAs 103 and 107 were found enriched in tissue-resident colon macrophages of healthy donors and alveolar macrophages of individuals under antiretroviral therapy, conceivably contributing to their relative resistance to HIV-1 infection. Overall, these findings highlight the role of p53 in enforcing proinflammatory antiviral responses in macrophages, at least in part, through miR-103/107-mediated downmodulation of CCR5 expression and HIV-1 entry. IMPORTANCE Macrophages are heterogeneous immune cells that display varying susceptibilities to HIV-1 infection, in part due to the expression of small noncoding microRNAs involved in the posttranscriptional regulation of gene expression and silencing. Here, we identify microRNAs 103 and 107 as important p53-regulated effectors of the antiviral response triggered by the proinflammatory cytokine IL-1β in macrophages. These microRNAs, which are enriched in colon macrophages of healthy donors and alveolar macrophages of HIV-infected individuals under antiretroviral therapy, act as inhibitors of HIV-1 entry through their capacity to downregulate the CCR5 coreceptor. These results highlight the important role played by miR-103/107 in modulating CCR5 expression and HIV-1 entry in macrophages. They further underscore a distinct function of the tumor suppressor p53 in enforcing proinflammatory antiviral responses in macrophages, thus providing insight into a cellular pathway that could be targeted to limit the establishment of viral reservoirs in these cells., (Copyright © 2020 Lodge et al.)

Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors. IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis., (Copyright © 2020 Xu et al.)

HIV-associated neurocognitive disorders (HAND) is a syndrome defined by neurocognitive deficits that are driven by viral neurotoxins, cytokines, free radicals, and proteases expressed in the brain. This neurological disease has also been linked to activation of Protease-Activated Receptors 1 and 2 (PAR1,2). These receptors are highly expressed in the central nervous system and are upregulated in HAND. Secretory basic-amino-acid-specific Proprotein Convertases (PCs), which cleave precursor proteins at basic residues, are also induced in HAND. They are vital for many biological processes including HIV-1 entry into cells. The cytoprotective role of Furin, PC5, and PACE4 has been linked to the presence of a potential PC-cleavage site R 41 XXXXR 46 ↓ in PAR1. Furthermore, Furin binds PAR1 and both are trapped in the trans-Golgi-network (TGN) as inactive proteins, likely due to the intermediary trafficking role of phospho-Furin acidic cluster sorting protein 1 (PACS1). Nothing is known about PAR2 and its possible recognition by PCs at its putative R 31 XXXXR 36 ↓ processing site. The present study implicates PACS1 in the retrograde trafficking of PAR1 to the TGN and demonstrates that the cytosolic extreme C-terminal tail of PAR1 contains an acidic phosphorylatable PACS1-sensitive domain. We further show the requirement of Asn 47 in PAR1 for its Furin-dependent TGN localization. Our data revealed that Furin is the only convertase that efficiently cleaves PAR2 at Arg 36 ↓. N-glycosylation of PAR2 at Asn 30 reduces the efficacy, but enhances selectivity of the Furin cleavage. Finally, in co-cultures comprised of human neuroblastoma SK-N-SH cells (stably expressing PAR1/2 and/or Furin) and HIV-1-infected primary macrophages, we demonstrate that the expression of Furin enhances neuronal cell viability in the context of PAR1- or PAR2-induced neuronal cytotoxicity. The present study provides insights into early stages of HIV-1 induced neuronal injury and the protective role of Furin in neurons co-expressing PAR1 and/or PAR2, as observed in HAND.

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20-30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pygmy perches (Percichthyidae) are a group of poorly dispersing freshwater fishes that have a puzzling biogeographic disjunction across southern Australia. Current understanding of pygmy perch phylogenetic relationships suggests past east-west migrations across a vast expanse of now arid habitat in central southern Australia, a region lacking contemporary rivers. Pygmy perches also represent a threatened group with confusing taxonomy and potentially cryptic species diversity. Here, we present the first study of the evolutionary history of pygmy perches based on genome-wide information. Data from 13 991 ddRAD loci and a concatenated sequence of 1 075 734 bp were generated for all currently described and potentially cryptic species. Phylogenetic relationships, biogeographic history and cryptic diversification were inferred using a framework that combines phylogenomics, species delimitation and estimation of divergence times. The genome-wide phylogeny clarified the biogeographic history of pygmy perches, demonstrating multiple east-west events of divergence within the group across the Australian continent. These results also resolved discordance between nuclear and mitochondrial data from a previous study. In addition, we propose three cryptic species within a southwestern species complex. The finding of potentially new species demonstrates that pygmy perches may be even more susceptible to ecological and demographic threats than previously thought. Our results have substantial implications for improving conservation legislation of pygmy perch lineages, especially in southwestern Western Australia., Competing Interests: The authors declare no competing interests.

To support bradykinin (BK) B 2 receptor (B 2 R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B 2 Rs (immunoblots, epifluorescence microscopy). APEX2-(NG) 15 -MK is a bona fide agonist of the rat, but not of the human B 2 R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3',5,5'-tetramethylbenzidine (luminescence or colourimetric B 2 R detection, cell well plate format). APEX2-(NG) 15 -MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B 2 R in the concentration range of 50-600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B 2 R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B 2 R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

Human immunodeficiency virus type-1 (HIV-1) infection of monocyte/macrophages is modulated by the levels of entry receptors cluster of differentiation 4 (CD4) and C-C chemokine receptor type 5 (CCR5), as well as by host antiviral restriction factors, which mediate several post-entry blocks. We recently identified two microRNAs, miR-221 and miR-222, which limit HIV-1 entry during infection of monocyte-derived macrophages (MDMs) by down-regulating CD4 expression. Interestingly, CD4 is also down-regulated during the differentiation of monocytes into macrophages. In this study, we compared microRNA expression profiles in primary monocytes and macrophages by RNAseq and found that miR-221/miR-222 are enhanced in macrophages. We took advantage of the monocytic THP-1 cell line that, once differentiated, is poorly susceptible to HIV-1. Accordingly, we found that CD4 levels are very low in THP-1 differentiated cells and that this down-regulation of the virus receptor is the result of miR-221/miR-222 up-regulation during differentiation. We thus established a THP-1 cell line stably expressing a modified CD4 (THP-1-CD4 R ) that is not modulated by miR-221/miR-222. We show that in contrast to parental THP-1, this line is productively infected by HIV-1 following differentiation, sustaining efficient HIV-1 CD4-dependent replication and spread. This new THP-1-CD4 R cell line represents a useful tool for the study of HIV-1-macrophage interactions particularly in contexts where spreading of viral infection is necessary., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to publish the results.

Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tissue localization. Their susceptibility to HIV-1 is subject to variations from permissiveness to resistance, owing in part to regulatory microRNAs. Here, we used RNA sequencing (RNA-seq) to examine the expression of >400 microRNAs in productively infected and bystander cells of HIV-1-exposed macrophage cultures. Two microRNAs upregulated in bystander macrophages, miR-221 and miR-222, were identified as negative regulators of CD4 expression and CD4-mediated HIV-1 entry. Both microRNAs were enhanced by tumor necrosis factor alpha (TNF-α), an inhibitor of CD4 expression. MiR-221/miR-222 inhibitors recovered HIV-1 entry in TNF-α-treated macrophages by enhancing CD4 expression and increased HIV-1 replication and spread in macrophages by countering TNF-α-enhanced miR-221/miR-222 expression in bystander cells. In line with these findings, HIV-1-resistant intestinal myeloid cells express higher levels of miR-221 than peripheral blood monocytes. Thus, miR-221/miR-222 act as effectors of the antiviral host response activated during macrophage infection that restrict HIV-1 entry., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

It has been proposed that macrophages could serve as long-lived compartments for HIV-1 infection under in vivo situations because these cells are resistant to the virus-mediated cytopathic effect, produce progeny virus over extended periods of time and are localized in tissues that are often less accessible by treatment. Comprehensive experimental studies are thus needed to characterize the HIV-1-induced modulation of host genes in these myeloid lineage cells. To shed light on this important issue, we performed comparative analyses of mRNA expression levels of host genes in uninfected bystander and HIV-1-infected human macrophages using an infectious reporter virus construct coupled with a large-scale RNA sequencing approach. We observed a rapid differential expression of several host factors in the productively infected macrophage population including genes regulating DNA replication factors and chromatin remodeling. A siRNA-mediated screening study to functionally identify host determinants involved in HIV-1 biology has provided new information on the virus molecular regulation in macrophages.

Unlabelled: This study aims to determine risk factors of opioid overdose among the Colorado Medicaid population. A retrospective nested case-control study was undertaken. Medicaid beneficiaries who had ≥1 medical claim for an emergency department visit or a hospitalization associated with an opioid overdose from July 2009 to June 2014 were defined as cases. Controls were selected using a nearest neighbor matching without replacement. The matched controls were selected on the basis of age, sex, and opioid prescription. One case was matched with three controls. Multivariate conditional logistic regression was used to compare risk factors. A total of 816 cases with 2,448 controls were included. Six factors were associated with opioid overdose: mean morphine dose equivalent (>50 mg/d; odds ratio [OR] = 1.986 [95% confidence interval [CI], 1.509-2.614]), methadone use (switching opioid to methadone vs. no methadone use; OR = 7.230 [95% CI, 2.346-22.286]), drug/alcohol abuse (OR = 3.104 [95% CI, 2.195-4.388]), other psychiatric illness (OR = 1.730 [95% CI, 1.307-2.291]), benzodiazepine use (OR = 2.005 [95% CI, 1.516-2.652]), and the number of pharmacies used by the beneficiary (≥4 pharmacies vs. 1 pharmacy; OR = 1.514 [95% CI, 1.003-2.286]). In conclusion, several factors are associated with opioid overdose. States and communities should ensure the availability of at-home intranasal naloxone for overdose rescue on the basis of the presence of risk factors., Perspective: This article presents the risk factors of opioid overdose among the Colorado Medicaid population. On the basis of study findings, Colorado Medicaid is currently working with physicians, hospitals, and other health system stakeholders to continue to develop policies to identify and assist this subset of our population. One such policy will be to provide at-home intranasal naloxone for overdose rescue., (Copyright © 2016 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Tredennick et al. criticize one of our statistical analyses and emphasize the low explanatory power of models relating productivity to diversity. These criticisms do not detract from our key findings, including evidence consistent with the unimodal constraint relationship predicted by the humped-back model and evidence of scale sensitivities in the form and strength of the relationship., (Copyright © 2016, American Association for the Advancement of Science.)

The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg46↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The search for predictions of species diversity across environmental gradients has challenged ecologists for decades. The humped-back model (HBM) suggests that plant diversity peaks at intermediate productivity; at low productivity few species can tolerate the environmental stresses, and at high productivity a few highly competitive species dominate. Over time the HBM has become increasingly controversial, and recent studies claim to have refuted it. Here, by using data from coordinated surveys conducted throughout grasslands worldwide and comprising a wide range of site productivities, we provide evidence in support of the HBM pattern at both global and regional extents. The relationships described here provide a foundation for further research into the local, landscape, and historical factors that maintain biodiversity., (Copyright © 2015, American Association for the Advancement of Science.)

Background: Prostate cancer is the most common cancer in males in the UK and affects around 105 men for every 100,000. The role of radiotherapy in the management of prostate cancer significantly changed over the last few decades with developments in brachytherapy, external beam radiotherapy (EBRT), intensity-modulated radiotherapy (IMRT), and image-guided radiotherapy (IGRT). One of the challenging factors of radiotherapy treatment of localized prostate cancer is the development of acute and late genitourinary and gastrointestinal toxicities. The recent European guidelines suggest that there is no consensus regarding the timing of high-dose rate (HDR) brachytherapy and EBRT. The schedules vary in different institutions where an HDR boost can be given either before or after EBRT. Few centers deliver HDR in between the fractions of EBRT., Objective: Assessment of acute genitourinary and gastrointestinal toxicities at various time points to better understand if the order in which treatment modality is delivered (ie, HDR brachytherapy or EBRT first) has an effect on the toxicity profile., Methods: Timing of HDR brachytherapy with EBRT in Prostate CAncer (THEPCA) is a single-center, open, randomized controlled feasibility trial in patients with intermediate and high-risk localized prostate cancer. A group of 50 patients aged 18 years old and over with histological diagnosis of prostate cancer (stages T1b-T3BNOMO), will be randomized to one of two treatment arms (ratio 1:1), following explanation of the study and informed consent. Patients in both arms of the study will be treated with HDR brachytherapy and EBRT, however, the order in which they receive the treatments will vary. In Arm A, patients will receive HDR brachytherapy before EBRT. In Arm B (control arm), patients will receive EBRT before HDR brachytherapy. Study outcomes will look at prospective assessment of genitourinary and gastrointestinal toxicities. The primary endpoint will be grade 3 genitourinary toxicity and the secondary endpoints will be all other grades of genitourinary toxicities (grades 1 and 2), gastrointestinal toxicities (grades 1 to 4), prostate-specific antigen (PSA) recurrence-free survival, overall survival, and quality of life., Results: Results from this feasibility trial will be available in mid-2016., Conclusions: If the results from this feasibility trial show evidence that the sequence of treatment modality does affect the patients' toxicity profiles, then funding would be sought to conduct a large, multicenter, randomized controlled trial., Trial Registration: International Standard Randomized Controlled Trial Number (ISRCTN): 15835424; http://www.isrctn.com/ISRCTN15835424 (Archived by WebCite at http://www.webcitation.org/6Xz7jfg1u).