Identification and characterization of recent retrovirus in Rhinolophus ferrumequinum bats.

An investigation into retrovirus was conducted in six species of bats (Myotis aurascens, Myotis petax, Myotis macrodactylus, Miniopterus fuliginosus, Rhinolophus ferrumequinum, and Pipistrellus abramus) inhabiting South Korea. Exogenous retroviruses (XRVs) were detected in the tissue samples of R. ferrumequinum individuals by PCR assay. Proviruses were identified in all tissue samples through viral quantification using a digital PCR assay per organ (lung, intestine, heart, brain, wing, kidney, and liver), with viral loads varying greatly between each organ. In phylogenetic analysis based on the whole genome, the Korean bat retroviruses and the R. ferrumequinum retrovirus (RfRV) strain formed a new clade distinct from the Gammaretrovirus clade. The phylogenetic results determined these viruses to be RfRV-like viruses. In the Simplot comparison, Korean RfRV-like viruses exhibited relatively strong fluctuated patterns in the latter part of the envelope gene area compared to other gene areas. Several point mutations within this region (6,878-7,774 bp) of these viruses were observed compared to the RfRV sequence. One Korean RfRV-like virus (named Y4b strain) was successfully recovered in the Raw 264.7 cell line, and virus particles replicated in the cells were confirmed by transmission electron microscopy. RfRVs (or RfRV-like viruses) have been spreading since their first discovery in 2012, and the Korean RfRV-like viruses were assumed to be XRVs that evolved from RfRV.IMPORTANCER. ferrumequinum retrovirus (RfRV)-like viruses were identified in greater horseshoe bats in South Korea. These RfRV-like viruses were considered exogenous retroviruses (XRVs) that emerged from RfRV. Varying amounts of provirus detected in different organs suggest ongoing viral activity, replication, and de novo integration in certain organs. Additionally, the successful recovery of the virus in the Raw 264.7 cell line provides strong evidence supporting their status as XRVs. These viruses have now been identified in South Korea and, more recently, in Kenya since RfRV was discovered in China in 2012, indicating that RfRVs (or RfRV-like viruses) have spread worldwide.

A Panoramic View of Cell Population Dynamics in Mammalian Aging.

To elucidate the aging-associated cellular population dynamics throughout the body, here we present PanSci, a single-cell transcriptome atlas profiling over 20 million cells from 623 mouse tissue samples, encompassing a range of organs across different life stages, sexes, and genotypes. This comprehensive dataset allowed us to identify more than 3,000 unique cellular states and catalog over 200 distinct aging-associated cell populations experiencing significant depletion or expansion. Our panoramic analysis uncovered temporally structured, organ- and lineage-specific shifts of cellular dynamics during lifespan progression. Moreover, we investigated aging-associated alterations in immune cell populations, revealing both widespread shifts and organ-specific changes. We further explored the regulatory roles of the immune system on aging and pinpointed specific age-related cell population expansions that are lymphocyte-dependent. The breadth and depth of our 'cell-omics' methodology not only enhance our comprehension of cellular aging but also lay the groundwork for exploring the complex regulatory networks among varied cell types in the context of aging and aging-associated diseases.

Smartphone and Wearable Device-Based Digital Phenotyping to Understand Substance use and its Syndemics.

Digital phenotyping is a process that allows researchers to leverage smartphone and wearable data to explore how technology use relates to behavioral health outcomes. In this Research Concepts article, we provide background on prior research that has employed digital phenotyping; the fundamentals of how digital phenotyping works, using examples from participant data; the application of digital phenotyping in the context of substance use and its syndemics; and the ethical, legal and social implications of digital phenotyping. We discuss applications for digital phenotyping in medical toxicology, as well as potential uses for digital phenotyping in future research. We also highlight the importance of obtaining ground truth annotation in order to identify and establish digital phenotypes of key behaviors of interest. Finally, there are many potential roles for medical toxicologists to leverage digital phenotyping both in research and in the future as a clinical tool to better understand the contextual features associated with drug poisoning and overdose. This article demonstrates how medical toxicologists and researchers can progress through phases of a research trajectory using digital phenotyping to better understand behavior and its association with smartphone usage.

The salience of structural barriers and behavioral health problems to ART adherence in people receiving HIV primary care in South Africa.

Multilevel factors (individual and structural) influence adherence to antiretroviral therapy, particularly in high HIV prevalence areas such as South Africa. The present study examined the relative importance of structural barriers to HIV care and behavioral health factors, depression and alcohol use, in Khayelitsha, Cape Town, South Africa. People receiving HIV care in six primary care clinics in Khayelitsha (N = 194) completed the Center for Epidemiologic Studies Depression Scale, the Alcohol Use Disorders Identification Test, the Structural Barriers to Medication Taking questionnaire, and a qualitative rating of past-two-week adherence. Correlations were employed to examine associations among these variables, and hierarchical regression analysis was used to examine the unique effects of structural barriers over and above depression and alcohol use as predictors of adherence. Participants were primarily Black South African (99%) women (83%), and 41 years old on average. All four variables were significantly correlated. The hierarchical regression analysis showed that among behavioral health predictors, alcohol use alone significantly predicted ART adherence (b = -.032, p = .002). When structural barriers was added to the model, it was the only significant unique predictor of ART adherence (b = -1.58, p < .001). Findings highlight the need to consider structural vulnerabilities in HIV care in South Africa when developing behavioral health interventions.

Acceptance of digital phenotyping linked to a digital pill system to measure PrEP adherence among men who have sex with men with substance use.

Once-daily oral HIV pre-exposure prophylaxis (PrEP) is an effective strategy to prevent HIV, but is highly dependent on adherence. Men who have sex with men (MSM) who use substances face unique challenges maintaining PrEP adherence. Digital pill systems (DPS) allow for real-time adherence measurement through ingestible sensors. Integration of DPS technology with other digital health tools, such as digital phenotyping, may improve understanding of nonadherence triggers and development of personalized adherence interventions based on ingestion behavior. This study explored the willingness of MSM with substance use to share digital phenotypic data and interact with ancillary systems in the context of DPS-measured PrEP adherence. Adult MSM on PrEP with substance use were recruited through a social networking app. Participants were introduced to DPS technology and completed an assessment to measure willingness to participate in DPS-based PrEP adherence research, contribute digital phenotyping data, and interact with ancillary systems in the context of DPS-based research. Medical mistrust, daily worry about PrEP adherence, and substance use were also assessed. Participants who identified as cisgender male and were willing to participate in DPS-based research (N = 131) were included in this subsample analysis. Most were White (76.3%) and non-Hispanic (77.9%). Participants who reported daily PrEP adherence worry had 3.7 times greater odds (95% CI: 1.03, 13.4) of willingness to share biometric data via a wearable device paired to the DPS. Participants with daily PrEP adherence worry were more likely to be willing to share smartphone data (p = 0.006) and receive text messages surrounding their daily activities (p = 0.003), compared to those with less worry. MSM with substance use disorder, who worried about PrEP adherence, were willing to use DPS technology and share data required for digital phenotyping in the context of PrEP adherence measurement. Efforts to address medical mistrust can increase advantages of this technology for HIV prevention.

Real-World Implementation Challenges Associated with a Digital Pill System to Measure Adherence to HIV Pre-Exposure Prophylaxis from Two Studies of Men Who Have Sex With Men.

Once-daily oral pre-exposure prophylaxis (PrEP) is highly effective for HIV prevention, but its efficacy is dependent on adherence, which can be challenging for men who have sex with men (MSM) with substance use. Digital pill systems (DPS) represent a novel tool for directly measuring adherence through ingestible radiofrequency sensors that confirm ingestions in real-time. We examined operational challenges across two studies involving DPS to measure PrEP adherence. While most participants successfully operated the system, a number of technological and sociobehavioral challenges requiring intervention were identified across both studies. Technological issues were both system- and participant-related, and were primarily addressed with technical updates and participant re-education, while sociobehavioral issues, including health and housing changes and issues with technology access, warranted innovative solutions. Future research leveraging DPS technology should develop robust supportive infrastructure and mitigation procedures to promptly identify and resolve operational issues to optimize the potential benefits of DPS use.

Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain.

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

Preliminary feasibility of a wrist-worn receiver to measure medication adherence via an ingestible radiofrequency sensor.

Adherence to medications is a complex task that requires complex biobehavioral support. To better provide tools to assist with medication adherence, digital pills provide an option to directly measure medication taking behaviors. These systems comprise a gelatin capsule with radiofrequency emitter, a wearable Reader that collects the radio signal and a smartphone app that collects ingestion data displays it for patients and clinicians. These systems are feasible in measuring adherence in the real-world, even in stigmatized diseases like HIV treatment adherence. While the current iteration of the digital pill system utilizes a wearable Reader worn like a necklace, preliminary feedback demonstrated that a miniaturized system that was worn on the wrist could be more functional in the real-world. This paper therefore describes the development and preliminary field testing of a wrist-borne wearable Reader to facilitate acquisition of oral HIV pre-exposure prophylaxis (PrEP) adherence data among individual prescribed PrEP.

A global view of aging and Alzheimer's pathogenesis-associated cell population dynamics and molecular signatures in human and mouse brains.

Conventional methods fall short in unraveling the dynamics of rare cell types related to aging and diseases. Here we introduce EasySci, an advanced single-cell combinatorial indexing strategy for exploring age-dependent cellular dynamics in the mammalian brain. Profiling approximately 1.5 million single-cell transcriptomes and 400,000 chromatin accessibility profiles across diverse mouse brains, we identified over 300 cell subtypes, uncovering their molecular characteristics and spatial locations. This comprehensive view elucidates rare cell types expanded or depleted upon aging. We also investigated cell-type-specific responses to genetic alterations linked to Alzheimer's disease, identifying associated rare cell types. Additionally, by profiling 118,240 human brain single-cell transcriptomes, we discerned cell- and region-specific transcriptomic changes tied to Alzheimer's pathogenesis. In conclusion, this research offers a valuable resource for probing cell-type-specific dynamics in both normal and pathological aging.

Sex Differences in the Cognitive Performance of a South African Cohort of People With HIV and Comorbid Major Depressive Disorder.

Women with HIV (WWH) may be more vulnerable to cognitive impairment than men with HIV (MWH), which may be explained by the direct effects of HIV or by sociodemographic and psychiatric characteristics. We recruited 105 people with HIV (PWH; 76 women) with incomplete antiretroviral therapy adherence, comorbid major depressive disorder, and socioeconomically disadvantaged backgrounds. Participants completed neuropsychological testing and measures gathering sociodemographic, medical, and psychiatric information. We compared WWH and MWH cognitive performance using unadjusted and adjusted regressions, and within each respective group, we explored predictors of cognitive performance. Results showed no significant between-sex differences in cognitive performance, both globally and within domains. Fewer years of education (ß = 0.94), illiteracy (ß = 4.55), and greater food insecurity (ß = -0.28) predicted lower cognitive performance in WWH but not MWH. We conclude that sex differences in PWH are likely due to sample characteristics representing broader inequalities, rather than true biological differences.

Infernape uncovers cell type-specific and spatially resolved alternative polyadenylation in the brain.

Differential polyadenylation sites (PAs) critically regulate gene expression, but their cell type-specific usage and spatial distribution in the brain have not been systematically characterized. Here, we present Infernape, which infers and quantifies PA usage from single-cell and spatial transcriptomic data and show its application in the mouse brain. Infernape uncovers alternative intronic PAs and 3'-UTR lengthening during cortical neurogenesis. Progenitor-neuron comparisons in the excitatory and inhibitory neuron lineages show overlapping PA changes in embryonic brains, suggesting that the neural proliferation-differentiation axis plays a prominent role. In the adult mouse brain, we uncover cell type-specific PAs and visualize such events using spatial transcriptomic data. Over two dozen neurodevelopmental disorder-associated genes such as Csnk2a1 and Mecp2 show differential PAs during brain development. This study presents Infernape to identify PAs from scRNA-seq and spatial data, and highlights the role of alternative PAs in neuronal gene regulation.

Single-cell DNA methylation and 3D genome architecture in the human brain.

Delineating the gene-regulatory programs underlying complex cell types is fundamental for understanding brain function in health and disease. Here, we comprehensively examined human brain cell epigenomes by probing DNA methylation and chromatin conformation at single-cell resolution in 517 thousand cells (399 thousand neurons and 118 thousand non-neurons) from 46 regions of three adult male brains. We identified 188 cell types and characterized their molecular signatures. Integrative analyses revealed concordant changes in DNA methylation, chromatin accessibility, chromatin organization, and gene expression across cell types, cortical areas, and basal ganglia structures. We further developed single-cell methylation barcodes that reliably predict brain cell types using the methylation status of select genomic sites. This multimodal epigenomic brain cell atlas provides new insights into the complexity of cell-type-specific gene regulation in adult human brains.

"Trajectories of treatment response in a cognitive-behavioral therapy intervention for depression and adherence in persons with HIV in South Africa".

BACKGROUND: People with HIV (PHW) are at greater risk of depression than the general population. Insight into the time-to-treatment-response and predictors of response to psychotherapy may improve implementation in primary care. METHODS: We assessed depression treatment response among 80 participants in a trial of cognitive-behavioral therapy for adherence and depression (CBT-AD) for PWH with MDD and suboptimal antiretroviral therapy (ART) adherence. Participants self-reported depressive symptoms (CESD) at each therapy session. Clinicians assessed participants' depression (HAMD), along with potential predictors of response, every four months for one year. Latent class analyses examined classes of responders for the active and the post-treatment phases. Regression analyses identified predictors of class membership for each phase. RESULTS: During the active treatment phase (CESD) we identified an early response (at session 2 and with continued trajectory of improvement) and a non-response group. There were also two classes during post-treatment (HAM-D): early responders (4-month) and late responders (12-month). Distress aversion was associated with lower likelihood of early response to CBT-AD (aOR = 0.74, 95%CI[0.56-0.90], p = .009), and social support was associated with increased likelihood of early response (aOR = 2.24, 95%CI[1.07-5.46], p = .045). LIMITATIONS: Self-reported depression during the treatment phase may have resulted from social desirability bias. CONCLUSIONS: Most participants responded to CBT-AD early during treatment (89 %) and had sustained improvements in depression by 4 months (80 %). Distress aversion was a risk factor for late response, and social support was protective. Future research is needed to assess the optimal dose of CBT-AD in resource limited settings.

Dissecting key regulators of transcriptome kinetics through scalable single-cell RNA profiling of pooled CRISPR screens.

We present a combinatorial indexing method, PerturbSci-Kinetics, for capturing whole transcriptomes, nascent transcriptomes and single guide RNA (sgRNA) identities across hundreds of genetic perturbations at the single-cell level. Profiling a pooled CRISPR screen targeting various biological processes, we show the gene expression regulation during RNA synthesis, processing and degradation, miRNA biogenesis and mitochondrial mRNA processing, systematically decoding the genome-wide regulatory network that underlies RNA temporal dynamics at scale.

Tracking cell-type-specific temporal dynamics in human and mouse brains.

Progenitor cells are critical in preserving organismal homeostasis, yet their diversity and dynamics in the aged brain remain underexplored. We introduced TrackerSci, a single-cell genomic method that combines newborn cell labeling and combinatorial indexing to characterize the transcriptome and chromatin landscape of proliferating progenitor cells in vivo. Using TrackerSci, we investigated the dynamics of newborn cells in mouse brains across various ages and in a mouse model of Alzheimer's disease. Our dataset revealed diverse progenitor cell types in the brain and their epigenetic signatures. We further quantified aging-associated shifts in cell-type-specific proliferation and differentiation and deciphered the associated molecular programs. Extending our study to the progenitor cells in the aged human brain, we identified conserved genetic signatures across species and pinpointed region-specific cellular dynamics, such as the reduced oligodendrogenesis in the cerebellum. We anticipate that TrackerSci will be broadly applicable to unveil cell-type-specific temporal dynamics in diverse systems.

Deep spatial profiling of Venezuelan equine encephalitis virus reveals increased genetic diversity amidst neuroinflammation and cell death during brain infection.

Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance.

Single Cell Multi-Omics of an iPSC Model of Human Sinoatrial Node Development Reveals Genetic Determinants of Heart Rate and Arrhythmia Susceptibility.

Cellular heterogeneity within the sinoatrial node (SAN) is functionally important but has been difficult to model in vitro , presenting a major obstacle to studies of heart rate regulation and arrhythmias. Here we describe a scalable method to derive sinoatrial node pacemaker cardiomyocytes (PCs) from human induced pluripotent stem cells that recapitulates differentiation into distinct PC subtypes, including SAN Head, SAN Tail, transitional zone cells, and sinus venosus myocardium. Single cell (sc) RNA-sequencing, sc-ATAC-sequencing, and trajectory analyses were used to define epigenetic and transcriptomic signatures of each cell type, and to identify novel transcriptional pathways important for PC subtype differentiation. Integration of our multi-omics datasets with genome wide association studies uncovered cell type-specific regulatory elements that associated with heart rate regulation and susceptibility to atrial fibrillation. Taken together, these datasets validate a novel, robust, and realistic in vitro platform that will enable deeper mechanistic exploration of human cardiac automaticity and arrhythmia.

PCR-Based Detection and Genetic Characterization of Parainfluenza Virus 5 Detected in Pigs in Korea from 2016 to 2018.

This study applied a molecular-based method to detect parainfluenza virus 5 (PIV5) collected from 2016 to 2018 in nine provinces of Republic of Korea. We demonstrated that PIV5 was detectable in both serum and pooled organs at an average positive rate of 1.78% (99/5566). Among these, the complete genome sequence of 15,246 nucleotides was obtained for 12 field strains. Three out of the 12 strains had the lowest genetic identity (96.20-96.68%) among the 21 porcine PIV5 genomes collected in Germany, China, India, and Republic of Korea from 1998 to 2017. By analyzing a large collection of complete genome sequences of the structural protein-coding F and HN genes, this study proposed a classification of PIV5 into two lineages, 1 and 2, and identified that group 2.2.2 within sub-lineage 2.2 was substantially divergent. The evolution of two structural protein-coding genes was largely under purifying selection. A few codons (6/9 for the F gene, 7/8 for the HN gene) had elevated dN/dS values, which were loaded on internal branches and were predicted to be related to beneficial trait(s) of the virus.

Single-cell DNA Methylome and 3D Multi-omic Atlas of the Adult Mouse Brain.

Cytosine DNA methylation is essential in brain development and has been implicated in various neurological disorders. A comprehensive understanding of DNA methylation diversity across the entire brain in the context of the brain's 3D spatial organization is essential for building a complete molecular atlas of brain cell types and understanding their gene regulatory landscapes. To this end, we employed optimized single-nucleus methylome (snmC-seq3) and multi-omic (snm3C-seq1) sequencing technologies to generate 301,626 methylomes and 176,003 chromatin conformation/methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell type taxonomy that contains 4,673 cell groups and 261 cross-modality-annotated subclasses. We identified millions of differentially methylated regions (DMRs) across the genome, representing potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide multiplexed error-robust fluorescence in situ hybridization (MERFISH2) data validated the association of this spatial epigenetic diversity with transcription and allowed the mapping of the DNA methylation and topology information into anatomical structures more precisely than our dissections. Furthermore, multi-scale chromatin conformation diversities occur in important neuronal genes, highly associated with DNA methylation and transcription changes. Brain-wide cell type comparison allowed us to build a regulatory model for each gene, linking transcription factors, DMRs, chromatin contacts, and downstream genes to establish regulatory networks. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a companion whole-brain SMART-seq3 dataset. Our study establishes the first brain-wide, single-cell resolution DNA methylome and 3D multi-omic atlas, providing an unparalleled resource for comprehending the mouse brain's cellular-spatial and regulatory genome diversity.