Distinct roles of Bdnf I and Bdnf IV transcript variant expression in hippocampal neurons.

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, dendritic growth, synaptic plasticity, as well as learning and memory. The rodent Bdnf gene contains nine 5' non-coding exons (I-IXa), which are spliced to a common 3' coding exon (IX). Transcription of individual Bdnf variants, which all encode the same BDNF protein, is initiated at unique promoters upstream of each non-coding exon, enabling precise spatiotemporal and activity-dependent regulation of Bdnf expression. Although prior evidence suggests that Bdnf transcripts containing exon I (Bdnf I) or exon IV (Bdnf IV) are uniquely regulated by neuronal activity, the functional significance of different Bdnf transcript variants remains unclear. To investigate functional roles of activity-dependent Bdnf I and IV transcripts, we used a CRISPR activation system in which catalytically dead Cas9 fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs, resulting in transcript-specific Bdnf upregulation. Bdnf I upregulation is associated with gene expression changes linked to dendritic growth, while Bdnf IV upregulation is associated with genes that regulate protein catabolism. Upregulation of Bdnf I, but not Bdnf IV, increased mushroom spine density, volume, length, and head diameter, and also produced more complex dendritic arbors in cultured rat hippocampal neurons. In contrast, upregulation of Bdnf IV, but not Bdnf I, in the rat hippocampus attenuated contextual fear expression. Our data suggest that while Bdnf I and IV are both activity-dependent, BDNF produced from these promoters may serve unique cellular, synaptic, and behavioral functions.

Global guideline for the diagnosis and management of cryptococcosis: an initiative of the ECMM and ISHAM in cooperation with the ASM.

Cryptococcosis is a major worldwide disseminated invasive fungal infection. Cryptococcosis, particularly in its most lethal manifestation of cryptococcal meningitis, accounts for substantial mortality and morbidity. The breadth of the clinical cryptococcosis syndromes, the different patient types at-risk and affected, and the vastly disparate resource settings where clinicians practice pose a complex array of challenges. Expert contributors from diverse regions of the world have collated data, reviewed the evidence, and provided insightful guideline recommendations for health practitioners across the globe. This guideline offers updated practical guidance and implementable recommendations on the clinical approaches, screening, diagnosis, management, and follow-up care of a patient with cryptococcosis and serves as a comprehensive synthesis of current evidence on cryptococcosis. This Review seeks to facilitate optimal clinical decision making on cryptococcosis and addresses the myriad of clinical complications by incorporating data from historical and contemporary clinical trials. This guideline is grounded on a set of core management principles, while acknowledging the practical challenges of antifungal access and resource limitations faced by many clinicians and patients. More than 70 societies internationally have endorsed the content, structure, evidence, recommendation, and pragmatic wisdom of this global cryptococcosis guideline to inform clinicians about the past, present, and future of care for a patient with cryptococcosis.

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial.

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome.

Development of small-molecule Tau-SH3 interaction inhibitors that prevent amyloid-ß toxicity and network hyperexcitability.

Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-ß-induced dysfunction in preclinical models of AD and also prevents amyloid-ß-independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain-containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain-containing proteins ameliorates amyloid-ß-induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-ß-induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer.

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 hr) enriched for inducible transcription factors and later changes (4 hr) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 hr, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 hr after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

Chst9 Marks a Spatially and Transcriptionally Unique Population of Oprm1 -Expressing Neurons in the Nucleus Accumbens.

Opioids produce addictive, analgesic, and euphoric effects via actions at mu opioid receptors (µORs). The µOR is encoded by the Oprm1 gene and is expressed in multiple brain regions that regulate reward and motivation, such as the nucleus accumbens (NAc). Oprm1 expression in NAc medium spiny neurons (MSNs) mediates opioid place preference, seeking, and consumption. However, recent single nucleus RNA sequencing (snRNA-seq) studies in rodent, primate, and human NAc have revealed that multiple subpopulations of NAc neurons express Oprm1 mRNA, making it unclear which populations mediate diverse behaviors resulting from µOR activation. Using published snRNA-seq datasets from the rat NAc, we identified a novel population of MSNs that express the highest levels of Oprm1 of any NAc cell type. Here, we show that this population is selectively marked by expression of Chst9 , a gene encoding a carbohydrate sulfotransferase. To validate this observation and characterize spatial localization of this population in the rat NAc, we performed multiplexed RNAscope fluorescence in situ hybridization studies to detect expression of Oprm1 and Chst9 mRNA along with well-validated markers of MSNs. Notably, Chst9 + neurons exhibited more abundant expression of Oprm1 as compared to other cell types, and formed discrete cellular clusters along the medial and ventral borders of the NAc shell subregion. Moreover, CHST9 mRNA was also found to mark specific MSN populations in published human and primate snRNA-seq studies, indicating that this unique population may be conserved across species. Together, these results identify a spatially and transcriptionally distinct NAc neuron population characterized by the expression of Chst9 . The abundant expression of Oprm1 in this population and the conservation of these cells across species suggests that they may play a key functional role in opioid response and identify this subpopulation as a target for further investigation.

Shared and unique antibody and B cell profiles in HIV-positive and HIV-negative individuals with cryptococcal meningoencephalitis.

Host non-T cell markers to aid in the diagnosis of cryptococcal meningoencephalitis (CM) have not been identified. In this case-control study, we characterized antibody and B cell profiles in HIV-negative and HIV-positive Vietnamese individuals of the Kinh ethnicity recently diagnosed with CM and controls. The study included 60 HIV-negative with no known immunocompromising condition and 60 HIV-positive individuals, with 30 CM cases and 30 controls in each group. Participants were matched by age, sex, HIV serostatus, and CD4 count in the HIV-positive group. Plasma immunoglobulin (Ig) levels, including IgG1, IgG2, IgM, and IgA, Cryptococcus spp. glucuronoxylomannan (GXM)- and laminarin (branched ${\rm{\beta }}$-[1-3]-glucan)-binding IgG, IgM, IgA levels, and peripheral blood B cell subsets were measured. Logistic regression, principal component, and mediation analyses were conducted to assess associations between antibody, B cell levels, and CM. The results showed that GXM-IgG levels were higher and IgG1 and IgG2 were lower in CM cases than controls, regardless of HIV status. In HIV-negative individuals, IgG2 mediated an inverse association between CD19+CD27+CD43+CD5- (B-1b-like) cells and CM. In HIV-positive individuals, lower levels of IgA, laminarin-IgA, and CD19+CD27+IgM+IgD- (IgM+ memory B) cells were each associated with CM. The shared and distinct antibody and B cell profiles identified in HIV-negative and HIV-positive CM cases may inform the identification of non-T-cell markers of CM risk or unsuspected disease, particularly in HIV-negative individuals.

Unlike cryptococcal meningitis (CM) in HIV-positive individuals, there are no known biomarkers of risk in HIV-negative individuals and the diagnosis is often not suspected and delayed. This study identified non-T cells, including antibody and B cell CM-associated profiles that may guide cryptococcal antigen testing in HIV-negative individuals.

Amphetamine disrupts dopamine axon growth in adolescence by a sex-specific mechanism in mice.

Initiating drug use during adolescence increases the risk of developing addiction or other psychopathologies later in life, with long-term outcomes varying according to sex and exact timing of use. The cellular and molecular underpinnings explaining this differential sensitivity to detrimental drug effects remain unexplained. The Netrin-1/DCC guidance cue system segregates cortical and limbic dopamine pathways in adolescence. Here we show that amphetamine, by dysregulating Netrin-1/DCC signaling, triggers ectopic growth of mesolimbic dopamine axons to the prefrontal cortex, only in early-adolescent male mice, underlying a male-specific vulnerability to enduring cognitive deficits. In adolescent females, compensatory changes in Netrin-1 protect against the deleterious consequences of amphetamine on dopamine connectivity and cognitive outcomes. Netrin-1/DCC signaling functions as a molecular switch which can be differentially regulated by the same drug experience as function of an individual's sex and adolescent age, and lead to divergent long-term outcomes associated with vulnerable or resilient phenotypes.

Rise in alanine aminotransferase after HCV treatment is a highly sensitive screen for treatment failure.

Nucleic acid testing to confirm sustained virological response (SVR) after HCV therapy is technical, often expensive, and frequently unavailable where disease prevalence is highest. Alternative surrogate biomarkers merit evaluation. In a short-treatment trial in Vietnam (SEARCH-1; n = 52) we analysed how changes in alanine transaminase (ΔALT) and aspartate transaminase (ΔAST), from end of treatment (EOT) to EOT + 12 weeks, related to SVR, defined as HCV RNA < lower limit of quantification 12 weeks after EOT. In a separate UK trial (STOPHCV1; n = 202), we then tested the hypothesis that any elevation in ALT or AST between EOT and EOT12 is a sensitive screen for treatment failure. In SEARCH-1, among 48 individuals with data, 13 failed to achieve SVR. Median ΔALT and ΔAST were negative in cured patients but elevated when treatment failed [median ΔALT (IQR): -2 IU/L (-6, +2)] versus +17 IU/L (+7.5, +38) (p< 0.001). Amongst treatment failures, 12/13 had increase in ALT and 13/13 had increase in AST after EOT, compared with 12/35 in those cured. In STOPHCV1, 196/202 patients had evaluable data, of which 57 did not achieve SVR. A rise in ALT after EOT was 100% sensitive (95% C.I. [93.7 - 100%]) and 51% specific (42.4 - 59.7%) for detecting treatment failure. ΔAST >0 IU/L was 98.1% (89.9 - 99.9%) sensitive and 35.8% (27.3 - 45.1%) specific. A rise in ALT or AST after HCV therapy is a highly sensitive screen for treatment failure in mild liver disease. This finding could reduce costs and complexity of managing HCV.

Temporally specific gene expression and chromatin remodeling programs regulate a conserved Pdyn enhancer.

Neuronal and behavioral adaptations to novel stimuli are regulated by temporally dynamic waves of transcriptional activity, which shape neuronal function and guide enduring plasticity. Neuronal activation promotes expression of an immediate early gene (IEG) program comprised primarily of activity-dependent transcription factors, which are thought to regulate a second set of late response genes (LRGs). However, while the mechanisms governing IEG activation have been well studied, the molecular interplay between IEGs and LRGs remain poorly characterized. Here, we used transcriptomic and chromatin accessibility profiling to define activity-driven responses in rat striatal neurons. As expected, neuronal depolarization generated robust changes in gene expression, with early changes (1 h) enriched for inducible transcription factors and later changes (4 h) enriched for neuropeptides, synaptic proteins, and ion channels. Remarkably, while depolarization did not induce chromatin remodeling after 1 h, we found broad increases in chromatin accessibility at thousands of sites in the genome at 4 h after neuronal stimulation. These putative regulatory elements were found almost exclusively at non-coding regions of the genome, and harbored consensus motifs for numerous activity-dependent transcription factors such as AP-1. Furthermore, blocking protein synthesis prevented activity-dependent chromatin remodeling, suggesting that IEG proteins are required for this process. Targeted analysis of LRG loci identified a putative enhancer upstream of Pdyn (prodynorphin), a gene encoding an opioid neuropeptide implicated in motivated behavior and neuropsychiatric disease states. CRISPR-based functional assays demonstrated that this enhancer is both necessary and sufficient for Pdyn transcription. This regulatory element is also conserved at the human PDYN locus, where its activation is sufficient to drive PDYN transcription in human cells. These results suggest that IEGs participate in chromatin remodeling at enhancers and identify a conserved enhancer that may act as a therapeutic target for brain disorders involving dysregulation of Pdyn.

Distinct roles of Bdnf I and Bdnf IV transcript variant expression in hippocampal neurons.

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, dendritic growth, synaptic plasticity, as well as learning and memory. The rodent Bdnf gene contains nine 5' non-coding exons (I-IXa), which are spliced to a common 3' coding exon (IX). Transcription of individual Bdnf variants, which all encode the same BDNF protein, is initiated at unique promoters upstream of each non-coding exon, enabling precise spatiotemporal and activity-dependent regulation of Bdnf expression. Although prior evidence suggests that Bdnf transcripts containing exon I (Bdnf I) or exon IV (Bdnf IV) are uniquely regulated by neuronal activity, the functional significance of different Bdnf transcript variants remains unclear. To investigate functional roles of activity-dependent Bdnf I and IV transcripts, we used a CRISPR activation (CRISPRa) system in which catalytically-dead Cas9 (dCas9) fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs (sgRNAs), resulting in transcript-specific Bdnf upregulation. Bdnf I upregulation is associated with gene expression changes linked to dendritic growth, while Bdnf IV upregulation is associated with genes that regulate protein catabolism. Upregulation of Bdnf I, but not Bdnf IV, increased mushroom spine density, volume, length, and head diameter, and also produced more complex dendritic arbors in cultured rat hippocampal neurons. In contrast, upregulation of Bdnf IV, but not Bdnf I, in the rat hippocampus attenuated contextual fear expression. Our data suggest that while Bdnf I and IV are both activity-dependent, BDNF produced from these promoters may serve unique cellular, synaptic, and behavioral functions.

Cross-Platform Synaptic Network Analysis of Human Entorhinal Cortex Identifies TWF2 as a Modulator of Dendritic Spine Length.

Proteomic studies using postmortem human brain tissue samples have yielded robust assessments of the aging and neurodegenerative disease(s) proteomes. While these analyses provide lists of molecular alterations in human conditions, like Alzheimer's disease (AD), identifying individual proteins that affect biological processes remains a challenge. To complicate matters, protein targets may be highly understudied and have limited information on their function. To address these hurdles, we sought to establish a blueprint to aid selection and functional validation of targets from proteomic datasets. A cross-platform pipeline was engineered to focus on synaptic processes in the entorhinal cortex (EC) of human patients, including controls, preclinical AD, and AD cases. Label-free quantification mass spectrometry (MS) data (n = 2260 proteins) was generated on synaptosome fractionated tissue from Brodmann area 28 (BA28; n = 58 samples). In parallel, dendritic spine density and morphology was measured in the same individuals. Weighted gene co-expression network analysis was used to construct a network of protein co-expression modules that were correlated with dendritic spine metrics. Module-trait correlations were used to guide unbiased selection of Twinfilin-2 (TWF2), which was the top hub protein of a module that positively correlated with thin spine length. Using CRISPR-dCas9 activation strategies, we demonstrated that boosting endogenous TWF2 protein levels in primary hippocampal neurons increased thin spine length, thus providing experimental validation for the human network analysis. Collectively, this study describes alterations in dendritic spine density and morphology as well as synaptic proteins and phosphorylated tau from the entorhinal cortex of preclinical and advanced stage AD patients.SIGNIFICANCE STATEMENT Proteomic studies can yield vast lists of molecules that are altered under various experimental or disease conditions. Here, we provide a blueprint to facilitate mechanistic validation of protein targets from human brain proteomic datasets. We conducted a proteomic analysis of human entorhinal cortex (EC) samples spanning cognitively normal and Alzheimer's disease (AD) cases with a comparison of dendritic spine morphology in the same samples. Network integration of proteomics with dendritic spine measurements allowed for unbiased discovery of Twinfilin-2 (TWF2) as a regulator of dendritic spine length. A proof-of-concept experiment in cultured neurons demonstrated that altering Twinfilin-2 protein level induced corresponding changes in dendritic spine length, thus providing experimental validation for the computational framework.

Distinct subpopulations of D1 medium spiny neurons exhibit unique transcriptional responsiveness to cocaine.

Drugs of abuse increase extracellular concentrations of dopamine in the nucleus accumbens (NAc), resulting in transcriptional alterations that drive long-lasting cellular and behavioral adaptations. While decades of research have focused on the transcriptional mechanisms by which drugs of abuse influence neuronal physiology and function, few studies have comprehensively defined NAc cell type heterogeneity in transcriptional responses to drugs of abuse. Here, we used single nucleus RNA-seq (snRNA-seq) to characterize the transcriptome of over 39,000 NAc cells from male and female adult Sprague-Dawley rats following acute or repeated cocaine experience. This dataset identified 16 transcriptionally distinct cell populations, including two populations of medium spiny neurons (MSNs) that express the Drd1 dopamine receptor (D1-MSNs). Critically, while both populations expressed classic marker genes of D1-MSNs, only one population exhibited a robust transcriptional response to cocaine. Validation of population-selective transcripts using RNA in situ hybridization revealed distinct spatial compartmentalization of these D1-MSN populations within the NAc. Finally, analysis of published NAc snRNA-seq datasets from non-human primates and humans demonstrated conservation of MSN subtypes across rat and higher order mammals, and further highlighted cell type-specific transcriptional differences across the NAc and broader striatum. These results highlight the utility in using snRNA-seq to characterize both cell type heterogeneity and cell type-specific responses to cocaine and provides a useful resource for cross-species comparisons of NAc cell composition.

Efficacy of ultra-short, response-guided sofosbuvir and daclatasvir therapy for hepatitis C in a single-arm mechanistic pilot study.

Background: World Health Organization has called for research into predictive factors for selecting persons who could be successfully treated with shorter durations of direct-acting antiviral (DAA) therapy for hepatitis C. We evaluated early virological response as a means of shortening treatment and explored host, viral and pharmacokinetic contributors to treatment outcome. Methods: Duration of sofosbuvir and daclatasvir (SOF/DCV) was determined according to day 2 (D2) virologic response for HCV genotype (gt) 1- or 6-infected adults in Vietnam with mild liver disease. Participants received 4- or 8-week treatment according to whether D2 HCV RNA was above or below 500 IU/ml (standard duration is 12 weeks). Primary endpoint was sustained virological response (SVR12). Those failing therapy were retreated with 12 weeks SOF/DCV. Host IFNL4 genotype and viral sequencing was performed at baseline, with repeat viral sequencing if virological rebound was observed. Levels of SOF, its inactive metabolite GS-331007 and DCV were measured on days 0 and 28. Results: Of 52 adults enrolled, 34 received 4 weeks SOF/DCV, 17 got 8 weeks and 1 withdrew. SVR12 was achieved in 21/34 (62%) treated for 4 weeks, and 17/17 (100%) treated for 8 weeks. Overall, 38/51 (75%) were cured with first-line treatment (mean duration 37 days). Despite a high prevalence of putative NS5A-inhibitor resistance-associated substitutions (RASs), all first-line treatment failures cured after retreatment (13/13). We found no evidence treatment failure was associated with host IFNL4 genotype, viral subtype, baseline RAS, SOF or DCV levels. Conclusions: Shortened SOF/DCV therapy, with retreatment if needed, reduces DAA use in patients with mild liver disease, while maintaining high cure rates. D2 virologic response alone does not adequately predict SVR12 with 4-week treatment. Funding: Funded by the Medical Research Council (Grant MR/P025064/1) and The Global Challenges Research 70 Fund (Wellcome Trust Grant 206/296/Z/17/Z).

Hepatitis C is a blood-borne virus that causes thousands of deaths from liver cirrhosis and liver cancer each year. Antiviral therapies can cure most cases of infection in 12 weeks. Unfortunately, treatment is expensive, and sticking with the regimen for 12 weeks can be difficult. It may be especially challenging for unhoused people or those who use injection drugs and who have high rates of hepatitis C infection. Shorter durations of therapy may make it more accessible, especially for high-risk populations. But studies of shorter antiviral treatment durations have yet to produce high enough cure rates. Finding ways to identify patients who would benefit from shorter therapy is a key goal of the World Health Organization. Potential characteristics that may predict a faster treatment response include low virus levels before initiating treatment, patient genetics, drug resistance mutations in the virus, and higher drug levels in the patient's blood during treatment. For example, previous research showed that a rapid decrease in virus levels in a patient's blood two days after starting antiviral therapy with three drugs predicted patient cures after three weeks of treatment. To test if high cure rates could be achieved in just four weeks of treatment, Flower et al. enrolled 52 patients with hepatitis C in a study to receive the most widely accessible dual antiviral treatment (sofosbuvir and daclatasvir). Participants received four or eight weeks of treatment, depending on the amount of viral RNA in their blood after two days of treatment. The results indicate that a rapid decrease in virus levels in the blood does not adequately predict cure rates with four weeks of two-drug combination therapy. However, eight weeks may be highly effective, regardless of viral levels early in treatment. Thirty-four individuals with low virus levels on the second day of treatment received four weeks of therapy, which cured 21 or 62% of them. All seventeen individuals with higher viral levels on day two were cured after eight weeks of treatment. Twelve weeks of retreatment was sufficient to cure the 13 individuals who did not achieve cure with four weeks of therapy. Even patients with drug resistance genes after the first round of therapy responded to a longer second round. Flower et al. show that patient genetics, virus subtype, drug levels in the patient's blood, and viral drug resistance genes before therapy, were not associated with patient cures after four weeks of treatment. Given that retreatment is safe and effective, larger studies are now needed to determine whether eight weeks of therapy with sofosbuvir and daclatasvir may be enough to cure patients with mild liver disease. More studies are also necessary to identify patients that may benefit from shorter therapy durations. Finding ways to shorten antiviral therapy for hepatitis C could help make treatment more accessible and reduce therapy costs for both individuals and governments.

Distinct subpopulations of D1 medium spiny neurons exhibit unique transcriptional responsiveness to cocaine.

Drugs of abuse increase extracellular concentrations of dopamine in the nucleus accumbens (NAc), resulting in transcriptional alterations that drive long-lasting cellular and behavioral adaptations. While decades of research have focused on the transcriptional mechanisms by which drugs of abuse influence neuronal physiology and function, few studies have comprehensively defined NAc cell type heterogeneity in transcriptional responses to drugs of abuse. Here, we used single nucleus RNA-seq (snRNA-seq) to characterize the transcriptome of over 39,000 NAc cells from male and female adult Sprague-Dawley rats following acute or repeated cocaine experience. This dataset identified 16 transcriptionally distinct cell populations, including two populations of medium spiny neurons (MSNs) that express the Drd1 dopamine receptor (D1-MSNs). Critically, while both populations expressed classic marker genes of D1-MSNs, only one population exhibited a robust transcriptional response to cocaine. Validation of population-selective transcripts using RNA in situ hybridization revealed distinct spatial compartmentalization of these D1-MSN populations within the NAc. Finally, analysis of published NAc snRNA-seq datasets from non-human primates and humans demonstrated conservation of MSN subtypes across rat and higher order mammals, and further highlighted cell type-specific transcriptional differences across the NAc and broader striatum. These results highlight the utility in using snRNA-seq to characterize both cell type heterogeneity and cell type-specific responses to cocaine and provides a useful resource for cross-species comparisons of NAc cell composition.

Non-lethal sampling does not misrepresent trophic level or dietary sources for Sagmariasus verreauxi (eastern rock lobster).

RATIONALE: Isotope analysis can be used to investigate the diets of predators based on assimilation of nitrogen and carbon isotopes from prey. Recent work has shown that tissues taken from legs, antennae or abdomen of lobsters can give different indications of diet, but this has never been evaluated for Sagmariasus verreauxi (eastern rock lobster). Work is now needed to prevent erroneous conclusions being drawn about lobster food webs, and undertaking this work could lead to developing non-lethal sampling methodologies. Non-lethal sampling for lobsters is valuable both ethically and for areas of conservation significance such as marine reserves. METHOD: We evaluated this by dissecting 76 lobsters and comparing δ13 C and δ15 N isotope values in antennae, leg and abdomen tissue from the same individuals ranging from 104 to 137 mm carapace length. Stable isotope values were determined using a Europa EA GSL elemental analyser coupled with Hydra 20-20 Isoprime IRMS. RESULTS: We found the abdomen δ13 C values to be lower than other tissues by 0.3 ± 0.2‰ for antennae tissue and 0.1 ± 0.2‰ δ13 C for leg tissues, whereas for δ15 N, no significant difference between tissues was observed. There was no significant effect of lobster size or sex, though we did observe interactions between month and tissue type, indicating that differences may be seasonal. Importantly, the detected range of isotopic variability between tissues is within the range of uncertainty used for discrimination factors in isotopic Bayesian modelling of 0‰-1.0‰ for δ13 C and 3.0‰-4.0‰ for δ15 N. CONCLUSIONS: We show that S. verreauxi can be sampled non-lethally with mathematical corrections applied for δ13 C, whereas any tissue is suitable for δ15 N. Our results indicate that a walking leg is most favourable and would also be the least intrusive for the lobster. The application of non-lethal sampling provides avenues for the contribution of citizen science to understanding lobster food webs and to undertake fieldwork in ecologically sensitive areas such as marine reserves.

Global estimates and determinants of antituberculosis drug pharmacokinetics in children and adolescents: a systematic review and individual patient data meta-analysis.

BACKGROUND: Suboptimal exposure to antituberculosis (anti-TB) drugs has been associated with unfavourable treatment outcomes. We aimed to investigate estimates and determinants of first-line anti-TB drug pharmacokinetics in children and adolescents at a global level. METHODS: We systematically searched MEDLINE, Embase and Web of Science (1990-2021) for pharmacokinetic studies of first-line anti-TB drugs in children and adolescents. Individual patient data were obtained from authors of eligible studies. Summary estimates of total/extrapolated area under the plasma concentration-time curve from 0 to 24 h post-dose (AUC0-24) and peak plasma concentration (C max) were assessed with random-effects models, normalised with current World Health Organization-recommended paediatric doses. Determinants of AUC0-24 and C max were assessed with linear mixed-effects models. RESULTS: Of 55 eligible studies, individual patient data were available for 39 (71%), including 1628 participants from 12 countries. Geometric means of steady-state AUC0-24 were summarised for isoniazid (18.7 (95% CI 15.5-22.6) h·mg·L-1), rifampicin (34.4 (95% CI 29.4-40.3) h·mg·L-1), pyrazinamide (375.0 (95% CI 339.9-413.7) h·mg·L-1) and ethambutol (8.0 (95% CI 6.4-10.0) h·mg·L-1). Our multivariate models indicated that younger age (especially <2 years) and HIV-positive status were associated with lower AUC0-24 for all first-line anti-TB drugs, while severe malnutrition was associated with lower AUC0-24 for isoniazid and pyrazinamide. N-acetyltransferase 2 rapid acetylators had lower isoniazid AUC0-24 and slow acetylators had higher isoniazid AUC0-24 than intermediate acetylators. Determinants of C max were generally similar to those for AUC0-24. CONCLUSIONS: This study provides the most comprehensive estimates of plasma exposures to first-line anti-TB drugs in children and adolescents. Key determinants of drug exposures were identified. These may be relevant for population-specific dose adjustment or individualised therapeutic drug monitoring.