Chemical Genetic Identification of PKC Epsilon Substrates in Mouse Brain.

PKC epsilon (PKCε) plays important roles in behavioral responses to alcohol and in anxiety-like behavior in rodents, making it a potential drug target for reducing alcohol consumption and anxiety. Identifying signals downstream of PKCε could reveal additional targets and strategies for interfering with PKCε signaling. We used a chemical genetic screen combined with mass spectrometry to identify direct substrates of PKCε in mouse brain and validated findings for 39 of them using peptide arrays and in vitro kinase assays. Prioritizing substrates with several public databases such as LINCS-L1000, STRING, GeneFriends, and GeneMAINA predicted interactions between these putative substrates and PKCε and identified substrates associated with alcohol-related behaviors, actions of benzodiazepines, and chronic stress. The 39 substrates could be broadly classified in three functional categories: cytoskeletal regulation, morphogenesis, and synaptic function. These results provide a list of brain PKCε substrates, many of which are novel, for future investigation to determine the role of PKCε signaling in alcohol responses, anxiety, responses to stress, and other related behaviors.

Warming, but not infection with Borrelia burgdorferi, increases off-host winter activity in the ectoparasite, Ixodes scapularis.

Warming winters will change patterns of behaviour in temperate and polar arthropods, but we know little about the drivers of winter activity in animals such as ticks. Any changes in behaviour are likely to arise from a combination of both abiotic (e.g. temperature) and biotic (e.g. infection) drivers, and will have important consequences for survival and species interactions. Blacklegged ticks, Ixodes scapularis, have invaded Atlantic Canada and high proportions (30-50%) are infected with the bacteria causing Lyme disease, Borrelia burgdorferi. Infection is correlated with increased overwintering survival of adult females, and ticks are increasingly active in the winter, but it is unclear if infection is associated with activity. Further, we know little about how temperature drives the frequency of winter activity. Here, we exposed wild-caught, adult, female Ixodes scapularis ticks to three different winter temperature regimes (constant low temperatures, increased warming, and increased warming + variability) to determine the thermal and infection conditions that promote or suppress activity. We used automated behaviour monitors to track daily activity in individual ticks and repeated the study with fresh ticks over three years. Following exposure to winter conditions we determined whether ticks were infected with the bacteria B. burgdorferi and if infection was responsible for any patterns in winter activity. Warming conditions promoted increased activity throughout the overwintering period but infection with B. burgdorferi had no impact on the frequency or overall number of ticks active throughout the winter. Individual ticks varied in their levels of activity throughout the winter, such that some were largely dormant for several weeks, while others were active almost daily; however, we do not yet know the drivers behind this individual variation in behaviour. Overall, warming winters will heighten the risk of tick-host encounters.

Determinants of Bladder Care at Night in a Subacute Ward for Aged Patients: An Observational Study.

PURPOSE: The purpose of this study was to evaluate how bladder care at night correlates to patients' mobility status. DESIGN: This was an observational study. SUBJECTS AND SETTING: The sample comprises 63 aged care subacute patients who were observed over 3 consecutive nights in an inpatient subacute aged care ward located in Melbourne, Australia. Half of the participants were female; their mean age was 82.0 (standard deviation 8.2) years. Data were collected from December 2020 to March 2021. METHODS: Outcome variables included demographic and pertinent clinical characteristics, type and timing of toileting and/or bladder care provided, and mobility classification. Descriptive statistics were used to summarize the frequency of nocturnal bladder care provided each night and as a mean over the 3 nights. The relationship between the type of care units provided and other variables was explored using a 1-way analysis of variance; values ≤.05 were deemed statistically significant. RESULTS: Findings indicate that 27% (n = 17), 41% (n = 26), and 48% (n = 30) of participants required assistance to move in bed, sit up in bed, and stand from sitting, respectively. Care episodes for both incontinence and assistance with voiding were significantly associated with functional mobility in bed ( F = 5.52, P < .001; F = 2.14, P = .02) and with ambulation independence ( F = 3.52, P = .001; F = 2.04, P = .03) but not with age or ambulation distance. CONCLUSIONS: Care provided for urinary incontinence during the night was related to the need for physical support of ambulation and poor mobility in bed. Targeted input from a multidisciplinary team is warranted to facilitate change of practice at night.

RNA Solutions: Synthesizing Information to Support Transcriptomics (RNASSIST).

MOTIVATION: Transcriptomics is a common approach to identify changes in gene expression induced by a disease state. Standard transcriptomic analyses consider differentially expressed genes (DEGs) as indicative of disease states so only a few genes would be treated as signals when the effect size is small, such as in brain tissue. For tissue with small effect sizes, if the DEGs do not belong to a pathway known to be involved in the disease, there would be little left in the transcriptome for researchers to follow up with. RESULTS: We developed RNA Solutions: Synthesizing Information to Support Transcriptomics (RNASSIST), a new approach to identify hidden signals in transcriptomic data by linking differential expression and co-expression networks using machine learning. We applied our approach to RNA-seq data of post-mortem brains that compared the Alcohol Use Disorder (AUD) group with the control group. Many of the candidate genes are not differentially expressed so would likely be ignored by standard transcriptomic analysis pipelines. Through multiple validation strategies, we concluded that these RNASSIST-identified genes likely play a significant role in AUD. AVAILABILITY AND IMPLEMENTATION: The RNASSIST algorithm is available at https://github.com/netrias/rnassist and both the software and the data used in RNASSIST are available at https://figshare.com/articles/software/RNAssist_Software_and_Data/16617250. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

From perplexing to predictive: are we ready to forecast insect disease susceptibility in a warming world?

Insects are critical to our ecosystems, but we do not fully understand their future in our warming world. Rising temperatures are affecting insect physiology in myriad ways, including changes to their immune systems and the ability to fight infection. Whether predicted changes in temperature will contribute to insect mortality or success, and the role of disease in their future survival, remains unclear. Although heat can enhance immunity by activating the integrated defense system (e.g. via the production of protective molecules such as heat-shock proteins) and accelerating enzyme activity, heat can also compromise the immune system through energetic-resource trade-offs and damage. The responses to heat are highly variable among species. The reasons for this variability are poorly known, and we are lagging in our understanding of how and why the immune system responds to changes in temperature. In this Commentary, we highlight the variation in insect immune responses to heat and the likely underlying mechanisms. We suggest that we are currently limited in our ability to predict the effects of rising temperatures on insect immunity and disease susceptibility, largely owing to incomplete information, coupled with a lack of tools for data integration. Moreover, existing data are concentrated on a relatively small number of insect Orders. We provide suggestions for a path towards making more accurate predictions, which will require studies with realistic temperature exposures and housing design, and a greater understanding of both the thermal biology of the immune system and connections between immunity and the physiological responses to heat.

Blood and brain gene expression signatures of chronic intermittent ethanol consumption in mice.

Alcohol Use Disorder (AUD) is a chronic, relapsing syndrome diagnosed by a heterogeneous set of behavioral signs and symptoms. There are no laboratory tests that provide direct objective evidence for diagnosis. Microarray and RNA-Seq technologies enable genome-wide transcriptome profiling at low costs and provide an opportunity to identify biomarkers to facilitate diagnosis, prognosis, and treatment of patients. However, access to brain tissue in living patients is not possible. Blood contains cellular and extracellular RNAs that provide disease-relevant information for some brain diseases. We hypothesized that blood gene expression profiles can be used to diagnose AUD. We profiled brain (prefrontal cortex, amygdala, and hypothalamus) and blood gene expression levels in C57BL/6J mice using RNA-seq one week after chronic intermittent ethanol (CIE) exposure, a mouse model of alcohol dependence. We found a high degree of preservation (rho range: [0.50, 0.67]) between blood and brain transcript levels. There was small overlap between blood and brain DEGs, and considerable overlap of gene networks perturbed after CIE related to cell-cell signaling (e.g., GABA and glutamate receptor signaling), immune responses (e.g., antigen presentation), and protein processing / mitochondrial functioning (e.g., ubiquitination, oxidative phosphorylation). Blood gene expression data were used to train classifiers (logistic regression, random forest, and partial least squares discriminant analysis), which were highly accurate at predicting alcohol dependence status (maximum AUC: 90.1%). These results suggest that gene expression profiles from peripheral blood samples contain a biological signature of alcohol dependence that can discriminate between CIE and Air subjects.

"I Can't Take This Shitty Quarantine Anymore": Sexual Behavior and PrEP Use Among Young Men Who Have Sex with Men and Transgender Women in Brazil During the COVID-19 Pandemic.

This study analyzes how the COVID-19 pandemic affected sexual behavior and use of HIV prevention methods among young transgender women (YTGW) and young gay, bisexual and other men who have sex with men (YGBMSM) participating in an HIV pre-exposure prophylaxis (PrEP) demonstration study in Brazil. Online interviews with 39 participants aged 15-22 years old were conducted between September and November 2020 and analyzed based on social constructionism and human rights-based approaches to health. The pandemic disrupted interviewees' routines, negatively affecting their life conditions. Among those who did not have a steady partner, social distance measures led to temporary interruption of sexual encounters and increased sexting and solo sex. Conversely, for those who had a steady relationship such measures contributed to increasing sexual practices and intimacy. Participants who had sexual encounters during the pandemic reported having continued to use PrEP. However, home confinement with family, lack of privacy, loss of daily routines and changes in housing impaired PrEP adherence and attendance at follow-up consultations. These results highlight the importance of maintaining HIV-service delivery for these groups during a public health crisis, as well as to address the structural drivers of the epidemic with comprehensive HIV prevention policies and by ensuring access of YGBMSM and YTGW to social protection policies.

Global assessment of existing HIV and key population stigma indicators: A data mapping exercise to inform country-level stigma measurement.

BACKGROUND: Stigma is an established barrier to the provision and uptake of HIV prevention, diagnostic, and treatment services. Despite consensus on the importance of addressing stigma, there are currently no country-level summary measures to characterize stigma and track progress in reducing stigma around the globe. This data mapping exercise aimed to assess the potential for existing data to be used to summarize and track stigma, including discrimination, related to HIV status, or key population membership at the country level. METHODS AND FINDINGS: This study assessed existing indicators of stigma related to living with HIV or belonging to 1 of 4 key populations including gay men and other men who have sex with men, sex workers, people who use drugs, and transgender persons. UNAIDS Strategic Information Department led an initial drafting of possible domains, subdomains, and indicators, and a 3-week e-consultation was held to provide feedback. From the e-consultation, 44 indicators were proposed for HIV stigma; 14 for sexual minority stigma (including sexual behavior or orientation) related to men who have sex with men; 12 for sex work stigma; 10 for drug use stigma; and 17 for gender identity stigma related to transgender persons. We conducted a global data mapping exercise to identify and describe the availability and quality of stigma data across countries with the following sources: UNAIDS National Commitments and Policies Instrument (NCPI) database; Multiple Indicator Cluster Surveys (MICS); Demographic and Health Surveys (DHS); People Living with HIV Stigma Index surveys; HIV Key Populations Data Repository; Integrated Biological and Behavioral Surveys (IBBS); and network databases. Data extraction was conducted between August and November 2020. Indicators were evaluated based on the following: if an existing data source could be identified; the number of countries for which data were available for the indicator at present and in the future; variation in the indicator across countries; and considerations of data quality or accuracy. This mapping exercise resulted in the identification of 24 HIV stigma indicators and 10 key population indicators as having potential to be used at present in the creation of valid summary measures of stigma at the country level. These indicators may allow assessment of legal, societal, and behavioral manifestations of stigma across population groups and settings. Study limitations include potential selection bias due to available data sources to the research team and other biases due to the exploratory nature of this data mapping process. CONCLUSIONS: Based on the current state of data available, several indicators have the potential to characterize the level and nature of stigma affecting people living with HIV and key populations across countries and across time. This exercise revealed challenges for an empirical process reliant on existing data to determine how to weight and best combine indicators into indices. However, results for this study can be combined with participatory processes to inform summary measure development and set data collection priorities going forward.

The gene cortex controls mimicry and crypsis in butterflies and moths.

The wing patterns of butterflies and moths (Lepidoptera) are diverse and striking examples of evolutionary diversification by natural selection. Lepidopteran wing colour patterns are a key innovation, consisting of arrays of coloured scales. We still lack a general understanding of how these patterns are controlled and whether this control shows any commonality across the 160,000 moth and 17,000 butterfly species. Here, we use fine-scale mapping with population genomics and gene expression analyses to identify a gene, cortex, that regulates pattern switches in multiple species across the mimetic radiation in Heliconius butterflies. cortex belongs to a fast-evolving subfamily of the otherwise highly conserved fizzy family of cell-cycle regulators, suggesting that it probably regulates pigmentation patterning by regulating scale cell development. In parallel with findings in the peppered moth (Biston betularia), our results suggest that this mechanism is common within Lepidoptera and that cortex has become a major target for natural selection acting on colour and pattern variation in this group of insects.

Conceptualisations of masculinity and sexual development among boys and young men in Korogocho slum in Kenya.

Youth and adolescence are times when young men negotiate their identity in relation to social and cultural expectations of being a man, with enduring implications for sexual health and wellbeing. This study explored how boys aged 10-14 years living in Korogocho slum in Nairobi, Kenya conceptualised masculinity, their perceptions of how masculinities are performed, and the linkage between conceptualisations of masculinity and sexual development. Three bases of gender socialisation were identified: (1) verbal messaging (mainly from parents and teachers); (2) observing the behaviours of older men in the community; and (3) information received from mainstream and social media. Masculinity conceptualisations focussed on financial stability, family life and responsibility, physical attributes, character and religion. Two contrasting portrayals of masculinity emerged in the form of idealised and dominant masculinities. A close linkage was found between masculinity conceptualisations and sexual development. Findings are important for programmes that aim to transform harmful gender norms and signal the need for longitudinal research exploring how gender beliefs may change over time.

Thermal Variability and Plasticity Drive the Outcome of a Host-Pathogen Interaction.

Variable, changing climates may affect each participant in a biotic interaction differently. We explored the effects of temperature and plasticity on the outcome of a host-pathogen interaction to try to predict the outcomes of infection under fluctuating temperatures. We infected Gryllus veletis crickets with the entomopathogenic fungus Metarhizium brunneum under constant (6°, 12°, 18°, or 25°C) or fluctuating (from 6° to 18°C or from 6° to 25°C) temperatures. We also acclimated crickets and fungi to constant or fluctuating conditions. Crickets acclimated to fluctuating conditions survived best under constant conditions if paired with warm-acclimated fungus. Overall, matches and mismatches in thermal performance, driven by acclimation, determined host survival. Mismatched performance also determined differences in survival under different fluctuating thermal regimes: crickets survived best when fluctuating temperatures favored their performance (from 6° to 25°C), compared with fluctuations that favored fungus performance (from 6° to 18°C). Thus, we could predict the outcome of infection under fluctuating temperatures by averaging relative host-pathogen performance under constant temperatures, suggesting that it may be possible to predict responses to fluctuating temperatures for at least some biotic interactions.

Detection of heliothine sex pheromone components in the Australian budworm moth, Helicoverpa punctigera: electrophysiology, neuroanatomy, and behavior.

This study examined electrophysiological responses of the Australian budworm moth Helicoverpa punctigera, to heliothine sex pheromone components, via single sensillum recordings (SSR), and examined male neuroanatomy using confocal microscopy and 3D imaging tools. We found that male H. punctigera have three distinct regions of the macroglomerular complex (MGC) in the antennal lobe. Male antennae have only two functional types of sensilla trichoidea (A and C) and type A sensilla contain an olfactory sensory neuron (OSN) that responds to the major sex pheromone component (Z)-11-hexadecenal (Z11-16:Ald) with axons projecting to the cumulus of the macroglomerular complex (MGC) in the antennal lobe. Type C sensilla contained large-spiking receptor neurons which responded primarily to (Z)-9-tetradecenal (Z9-14:Ald) and to a lesser degree to (Z)-11-hexadecenol (Z11-16:OH). These were co-compartmentalized with small-spiking receptor neurons in type C sensilla which responded strongly to Z9-14:Ald and (Z)-9-hexadecenal (Z9-16:Ald), and to a lesser degree to (Z)-11-hexadecenyl acetate (Z11-16:OAc) and Z11-16:OH. Axons from the two co-localized neurons in Type C sensilla projected to the two small MGC units, the dorsomedial anterior and dorsomedial posterior, respectively. In wind tunnel assays, the addition of Z9-16:Ald to an otherwise attractive blend completely shut down male H. punctigera upwind flight.

Results-based financing to increase uptake of skilled delivery services in The Gambia: using the 'three delays' model to interpret midline evaluation findings.

BACKGROUND: Delays in accessing skilled delivery services are a major contributor to high maternal mortality in resource-limited settings. In 2015, the government of The Gambia initiated a results-based financing intervention that sought to increase uptake of skilled delivery. We performed a midline evaluation to determine the impact of the intervention and explore causes of delays. METHODS: A mixed methods design was used to measure changes in uptake of skilled delivery and explore underlying reasons, with communities randomly assigned to four arms: (1) community-based intervention, (2) facility-based intervention, (3) community- and facility-based intervention, and (4) control. We obtained quantitative data from household surveys conducted at baseline (n = 1423) and midline (n = 1573). Qualitative data came from semi-structured interviews (baseline n = 20; midline n = 20) and focus group discussions (baseline n = 27; midline n = 39) with a range of stakeholders. Multivariable linear regression models were estimated using pooled data from baseline and midline. Qualitative data were recorded, transcribed, translated and thematically analyzed. RESULTS: No increase was found in uptake of skilled delivery services between baseline and midline. However, relative to the control group, significant increases in referral to health facilities for delivery were found in areas receiving the community-based intervention (beta = 0.078, p < 0.10) and areas receiving both the community-based and facility-based interventions (beta = 0.198, p < 0.05). There was also an increase in accompaniment to health facilities for delivery in areas receiving only community-based interventions (beta = 0.095, p < 0.05). Transportation to health facilities for delivery increased in areas with both interventions (beta = 0.102, p < 0.05). Qualitative data indicate that delays in the decision to seek institutional delivery usually occurred when women had limited knowledge of delivery indications. Delays in reaching a health facility typically occurred due to transportation-related challenges. Although health workers noted shortages in supplies and equipment, women reported being supported by staff and experiencing minimal delays in receiving skilled delivery care once at the facility. CONCLUSIONS: Focusing efforts on informing the decision to seek care and overcoming transportation barriers can reduce delays in care-seeking among pregnant women and facilitate efforts to increase uptake of skilled delivery services through results-based financing mechanisms.

New Roles for the Nucleolus in Health and Disease.

Over the last decade, our appreciation of the importance of the nucleolus for cellular function has progressed from the ordinary to the extraordinary. We no longer think of the nucleolus as simply the site of ribosome production, or a dynamic subnuclear body noted by pathologists for its changes in size and shape with malignancy. Instead, the nucleolus has emerged as a key controller of many cellular processes that are fundamental to normal cell homeostasis and the target for dysregulation in many human diseases; in some cases, independent of its functions in ribosome biogenesis. These extra-nucleolar or new functions, which we term "non-canonical" to distinguish them from the more traditional role of the nucleolus in ribosome synthesis, are the focus of this review. In particular, we explore how these non-canonical functions may provide novel insights into human disease and in some cases new targets for therapeutic development.

Expanding the role of paralegals: supporting realization of the right to health for vulnerable communities.

BACKGROUND: All around the world, the paralegal program prepares members of marginalized communities to face the legal system. Having a common background with their clients and being capable of conducting flexible work, paralegals' role moves beyond enlarging the beneficiaries of legal aid to addressing intersectional issues around health-related rights. This study assesses the health and other impacts of paralegals recruited by Lembaga Bantuan Hukum Masyarakat (LBHM), a human rights organization which provides legal assistance and operates in Jakarta, Indonesia. The positive results these paralegals can bring in the specific context can contribute to the development of community-based paralegals elsewhere. METHODS: This mixed methods research was carried out in 2016-2018. In 2016, a quantitative survey was administered to LBHM-trained paralegals and a sub-set of paralegals who completed the survey were then also interviewed. Quantitative data were analysed using SPSS, and, for the qualitative data, thematic analysis was conducted. RESULTS: The paralegals make important contributions to health-related rights in four distinct ways. Firstly, most of the paralegals checked their clients' health in all stages of detention, especially regarding their drug dependency status and checking for signs of torture. Secondly, paralegals help clients to be more aware of their health-related rights, especially regarding their rights to obtain health services inside detention. Thirdly, paralegals can ensure that their clients obtain health services by taking medicines directly to the clients or encouraging the law enforcement agencies to refer the clients to health services. Lastly, in drug cases, paralegals help their clients to obtain alternative sentences besides imprisonment. CONCLUSIONS: These four contributions verify the positive impacts paralegals, recruited from marginalized communities, can deliver for community members facing criminal justice processes. The shifting role of paralegals from merely an intermediary between clients and lawyers to champions of the health-related rights of their clients can happen as a result of adequate training, support, and networks with other agents in criminal justice system.

'We are not infertile': challenges and limitations faced by women in same-sex relationships when seeking conception services in São Paulo, Brazil.

The use of reproductive technologies has expanded beyond cases of infertility, and opportunities for individuals of different sexual orientations to use such technologies for conception have increased. The authors examined the challenges and limitations faced by women in same-sex relationships seeking conception services in São Paulo, Brazil. They interviewed 16 women in same-sex relationships who conceived children using reproductive technologies. Access to public health services for conception remains limited and exclusive to infertile heterosexual couples. Women in same-sex relationships are forced to select between costly medical treatments in the private sector, or self-insemination with sperm from a known donor. In this study, individuals reported that they experienced adverse effects of treatments; were treated with technologies for infertility when they were, in fact, fertile; and were exposed to unnecessary and invasive interventions. Growing political conservatism and the reduction of investment in public health services are factors that can directly affect sexual and reproductive health, including that of lesbians. Public policies that enable same-sex couples to conceive have yet to be developed in many countries, and a clearer articulation of the right to evidence-informed, rights-based care, consistent with national and international guidelines is a much-needed next step.

Next-generation flexible formats of VNAR domains expand the drug platform's utility and developability.

Therapeutic mAbs have delivered several blockbuster drugs in oncology and autoimmune inflammatory disease. Revenue for mAbs continues to rise, even in the face of competition from a growing portfolio of biosimilars. Despite this success, there are still limitations associated with the use of mAbs as therapeutic molecules. With a molecular mass of 150 kDa, a two-chain structure and complex glycosylation these challenges include a high cost of goods, limited delivery options, and poor solid tumour penetration. There remains an urgency to create alternatives to antibody scaffolds in a bid to circumvent these limitations, while maintaining or improving the therapeutic success of conventional mAb formats. Smaller, less complex binders, with increased domain valency, multi-specific/paratopic targeting, tuneable serum half-life and low inherent immunogenicity are a few of the characteristics being explored by the next generation of biologic molecules. One novel 'antibody-like' binder that has naturally evolved over 450 million years is the variable new antigen receptor (VNAR) identified as a key component of the adaptive immune system of sharks. At only 11 kDa, these single-domain structures are the smallest IgG-like proteins in the animal kingdom and provide an excellent platform for molecular engineering and biologics drug discovery. VNAR attributes include high affinity for target, ease of expression, stability, solubility, multi-specificity, and increased potential for solid tissue penetration. This review article documents the recent drug developmental milestones achieved for therapeutic VNARs and highlights the first reported evidence of the efficacy of these domains in clinically relevant models of disease.

Eco-immunology in the cold: the role of immunity in shaping the overwintering survival of ectotherms.

The effect of temperature on physiology mediates many of the challenges that ectotherms face under climate change. Ectotherm immunity is thermally sensitive and, as such, environmental change is likely to have complex effects on survival, disease resistance and transmission. The effects of temperature on immunity will be particularly profound in winter because cold and overwintering are important triggers and regulators of ectotherm immune activity. Low temperatures can both suppress and activate immune responses independent of parasites, which suggests that temperature not only affects the rate of immune responses but also provides information that allows overwintering ectotherms to balance investment in immunity and other physiological processes that underlie winter survival. Changing winter temperatures are now shifting ectotherm immunity, as well as the demand for energy conservation and protection against parasites. Whether an ectotherm can survive the winter will thus depend on whether new immune phenotypes will shift to match the conditions of the new environment, or leave ectotherms vulnerable to infection or energy depletion. Here, we synthesise patterns of overwintering immunity in ectotherms and examine how new winter conditions might affect ectotherm immunity. We then explore whether it is possible to predict the effects of changing winter conditions on ectotherm vulnerability to the direct and indirect effects of parasites.