Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity.

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

Artificial Intelligence-Guided Segmentation and Path Planning Software for Transthoracic Lung Biopsy.

PURPOSE: To validate the sensitivity and specificity of a 3-dimensional (3D) convolutional neural network (CNN) artificial intelligence (AI) software for lung lesion detection and to establish concordance between AI-generated needle paths and those used in actual biopsy procedures. MATERIALS AND METHODS: This was a retrospective study using computed tomography (CT) scans from 3 hospitals. Inclusion criteria were scans with 1-5 nodules of diameter ≥5 mm; exclusion criteria were poor-quality scans or those with nodules measuring <5mm in diameter. In the lesion detection phase, 2,147 nodules from 219 scans were used to develop and train the deep learning 3D-CNN to detect lesions. The 3D-CNN was validated with 235 scans (354 lesions) for sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) analysis. In the path planning phase, Bayesian optimization was used to propose possible needle trajectories for lesion biopsy while avoiding vital structures. Software-proposed needle trajectories were compared with actual biopsy path trajectories from intraprocedural CT scans in 150 patients, with a match defined as an angular deviation of <5° between the 2 trajectories. RESULTS: The model achieved an overall AUC of 97.4% (95% CI, 96.3%-98.2%) for lesion detection, with mean sensitivity of 93.5% and mean specificity of 93.2%. Among the software-proposed needle trajectories, 85.3% were feasible, with 82% matching actual paths and similar performance between supine and prone/oblique patient orientations (P = .311). The mean angular deviation between matching trajectories was 2.30° (SD ± 1.22); the mean path deviation was 2.94 mm (SD ± 1.60). CONCLUSIONS: Segmentation, lesion detection, and path planning for CT-guided lung biopsy using an AI-guided software showed promising results. Future integration with automated robotic systems may pave the way toward fully automated biopsy procedures.

Landscape of Epstein-Barr virus gene expression and perturbations in cancer.

Epstein-Barr virus (EBV) is the causative agent for multiple neoplastic diseases of epithelial and lymphocytic origin1-3. The heterogeneity of the viral elements expressed and the mechanisms by which these coding and non-coding genes maintain cancer cell properties in vivo remain elusive4,5. Here we conducted a multi-modal transcriptomic analysis of EBV-associated neoplasms and identified that the ubiquitously expressed RPMS1 non-coding RNAs support cancer cell properties by disruption of the interferon response. Our map of EBV expression shows a variable, but pervasive expression of BNLF2 discerned from the overlapping LMP1 RNA in bulk sequencing data. Using long-read single-molecule sequencing, we identified three new viral elements within the RPMS1 gene. Furthermore, single-cell sequencing datasets allowed for the separation of cancer cells and healthy cells from the same tissue biopsy and the characterization of a microenvironment containing interferon gamma excreted by EBV-stimulated T-lymphocytes. In comparison with healthy epithelium, EBV-transformed cancer cells exhibited increased proliferation and inhibited immune response induced by the RPMS1-encoded microRNAs. Our atlas of EBV expression shows that the EBV-transformed cancer cells express high levels of non-coding RNAs originating from RPMS1 and that the oncogenic properties are maintained by RPMS1 microRNAs. Through bioinformatic disentanglement of single cells from cancer tissues we identified a positive feedback loop where EBV-activated immune cells stimulate cancer cells to proliferate, which in turn undergo viral reactivation and trigger an immune response.

Misogynistic Extremism: A Scoping Review.

In recent years, the concept of "misogynistic extremism" has emerged as a subject of interest among scholars, governments, law enforcement personnel, and the media. Yet a consistent understanding of how misogynistic extremism is defined and conceptualized has not yet emerged. Varying epistemological orientations may contribute to the current conceptual muddle of this topic, reflecting long-standing and on-going challenges with the conceptualization of its individual components. To address the potential impact of misogynistic extremism (i.e., violent attacks), a more precise understanding of what this phenomenon entails is needed. To summarize the existing knowledge base on the nature of misogynistic extremism, this scoping review analyzed publications within English-language peer-reviewed and gray literature sources. Seven electronic databases and citation indexes were systematically searched using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews (PRISMA-ScR) checklist and charted using the 2020 PRISMA flow diagram. Inclusion criteria included English peer-reviewed articles and relevant gray literature publications, which contained the term "misogynistic extremism" and other closely related terms. No date restrictions were imposed. The search strategy initially yielded 475 publications. After exclusion of ineligible articles, 40 publications remained for synthesis. We found that misogynistic extremism is most frequently conceptualized in the context of misogynistic incels, male supremacism, far-right extremism, terrorism, and the black pill ideology. Policy recommendations include increased education among law enforcement and Countering and Preventing Violent Extremism experts on male supremacist violence and encouraging legal and educational mechanisms to bolster gender equality. Violence stemming from misogynistic worldviews must be addressed by directly acknowledging and challenging socially embedded systems of oppression such as white supremacy and cisheteropatriarchy.

NKG2A gene variant predicts outcome of immunotherapy in AML and modulates the repertoire and function of NK cells.

BACKGROUND: The natural killer (NK) complex (NKC) harbors multiple genes such as KLRC1 (encoding NKG2A) and KLRK1 (encoding NKG2D) that are central to regulation of NK cell function. We aimed at determining to what extent NKC haplotypes impact on NK cell repertoire and function, and whether such gene variants impact on outcome of IL-2-based immunotherapy in acute myeloid leukemia (AML). METHODS: Genotype status of NKG2D rs1049174 and NKG2A rs1983526 was determined using the TaqMan-Allelic discrimination approach. To dissect the impact of single nucloetide polymorphim (SNP) on NK cell function, we engineered the K562 cell line with CRISPR to be killed in a highly NKG2D-dependent fashion. NK cells were assayed for degranulation, intracellular cytokine production and cytotoxicity using flow cytometry. RESULTS: In AML patients receiving immunotherapy, the NKG2A gene variant, rs1983526, was associated with superior leukemia-free survival and overall survival. We observed that superior NK degranulation from individuals with the high-cytotoxicity NKG2D variant was explained by presence of a larger, highly responsive NKG2A+ subset. Notably, NK cells from donors homozygous for a favorable allele encoding NKG2A mounted stronger cytokine responses when challenged with leukemic cells, and NK cells from AML patients with this genotype displayed higher accumulation of granzyme B during histamine dihydrochloride/IL-2 immunotherapy. Additionally, among AML patients, the NKG2A SNP defined a subset of patients with HLA-B-21 TT with a strikingly favorable outcome. CONCLUSIONS: The study results imply that a dimorphism in the NKG2A gene is associated with enhanced NK cell effector function and improved outcome of IL-2-based immunotherapy in AML.

Phantom and Animal Study of a Robot-Assisted, CT-Guided Targeting System using Image-Only Navigation for Stereotactic Needle Insertion without Positional Sensors.

PURPOSE: To evaluate the feasibility and accuracy of a robotic system to integrate and map computed tomography (CT) and robotic coordinates, followed by automatic trajectory execution by a robotic arm. The system was hypothesized to achieve a targeting error of <5 mm without significant influence from variations in angulation or depth. MATERIALS AND METHODS: An experimental study was conducted using a robotic system (Automated Needle Targeting device for CT [ANT-C]) for needle insertions into a phantom model on both moving patient table and moving gantry CT scanners. Eight spherical markers were registered as targets for 90 insertions at different trajectories. After a single ANT-C registration, the closed-loop software targeted multiple markers via the insertion of robotically aligned 18-gauge needles. Accuracy (distance from the needle tip to the target) was assessed by postinsertion CT scans. Similar procedures were repeated to guide 10 needle insertions into a porcine lung. A regression analysis was performed to test the effect of needle angulation and insertion depth on the accuracy of insertion. RESULTS: In the phantom model, all needle insertions (median trajectory depth, 64.8 mm; range, 46.1-153 mm) were successfully performed in single attempts. The overall accuracy was 1.36 mm ± 0.53, which did not differ between the 2 types of CT scanners (1.39 mm ± 0.54 [moving patient table CT] vs 1.33 mm ± 0.52 [moving gantry CT]; P = .54) and was not significantly affected by the needle angulation and insertion depth. The accuracy for the porcine model was 9.09 mm ± 4.21. CONCLUSIONS: Robot-assisted needle insertion using the ANT-C robotic device was feasible and accurate for targeting multiple markers in a phantom model.

Association of Gut Microbiota With Metabolism in Rainbow Trout Under Acute Heat Stress.

Global warming is one of the most common environmental challenges faced by cold-water fish farming. Heat stress seriously affects the feeding, growth, immunity, and disease resistance of fish. These changes are closely related to the destruction of intestinal barrier function, the change of intestinal microbiota, and metabolic dysfunction. However, the causal relationship between the phenotypic effects of heat stress as well as intestinal and metabolic functions of fish is unknown. In the current study, the optimal growth temperature (16°C) of rainbow trout was used as the control group, while the fish treated at 22.5°C, 23.5°C, and 24.5°C for 24 h, respectively, were the treatment groups. The 16S rRNA gene sequencing analysis showed that with the increase in temperature, the relative abundance and diversity of intestinal microbiota decreased significantly, while the number of Mycoplasma, Firmicutes, and Tenericutes increased significantly. Non-targeted metabolomics analysis by liquid chromatography-mass spectrometry analysis and correlation analysis showed that the changes of metabolites related to amino acids, vitamins, and short-chain fatty acids in serum of rainbow trout under acute heat stress were strongly correlated with the decrease of relative abundance of various intestinal microbiota, especially Morganella, Enterobacter, Lactobacillus, Lawsonia, and Cloacibacterium. In addition, we also found that acute heat stress seriously affected the intestinal structure and barrier function, and also caused the pathological damage of epithelial cells. These results indicate that the gut microbiome of acute heat-stressed rainbow trout could mediate metabolite transfer through the gut barrier by affecting its integrity. Significant changes in gut morphology, permeability, antioxidant capacity, and pro-inflammatory cytokine levels were observed. Therefore, it is necessary to explore the changes of intestinal microbiota under heat stress to help understand the regulatory mechanism of heat stress and protect the intestinal health of rainbow trout from the negative effects of rising water temperature.

Nowhere to Hide: Online Rumor Detection Based on Retweeting Graph Neural Networks.

Online rumor detection is crucial for a healthier online environment. Traditional methods mainly rely on content understanding. However, these contents can be easily adjusted to avoid such supervision and are insufficient to improve the detection result. Compared with the content, information propagation patterns are more informative to support further performance promotion. Unfortunately, learning the propagation patterns is difficult, since the retweeting tree is more topologically complicated than linear sequences or binary trees. In light of this, we propose a novel rumor detection framework based on structure-aware retweeting graph neural networks. To capture the propagation patterns, we first design a novel conversion method to transform the complex retweeting tree as more tractable binary tree without losing the reconstruction information. Then, we serialize the retweeting tree as a corpus of meta-tree paths, where each meta-tree can preserve a basic substructure. A deep neural network is then designed to integrate all meta-trees and to generate the global structural embeddings. Furthermore, we propose to integrate content, users, and propagation patterns to enhance more reliable performance. To this end, we propose a novel self-attention-based retweeting neural network to learn individual features from both content and users. We then fuse the node-level features with our global structural embeddings via a mutual attention unit. In this way, we can generate more comprehensive representations for rumor detection. Extensive evaluations on two real-world datasets show remarkable superiorities of our model compared with existing methods.

Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics.

Infection in the central nervous system is a severe condition associated with high morbidity and mortality. Despite ample testing, the majority of encephalitis and meningitis cases remain undiagnosed. Metagenomic sequencing of cerebrospinal fluid has emerged as an unbiased approach to identify rare microbes and novel pathogens. However, several major hurdles remain, including establishment of individual limits of detection, removal of false positives and implementation of universal controls. Twenty-one cerebrospinal fluid samples, in which a known pathogen had been positively identified by available clinical techniques, were subjected to metagenomic DNA sequencing. Fourteen samples contained minute levels of Epstein-Barr virus. The detection threshold for each sample was calculated by using the total leukocyte content in the sample and environmental contaminants found in the bioinformatic classifiers. Virus sequences were detected in all ten samples, in which more than one read was expected according to the calculations. Conversely, no viral reads were detected in seven out of eight samples, in which less than one read was expected according to the calculations. False positive pathogens of computational or environmental origin were readily identified, by using a commonly available cell control. For bacteria, additional filters including a comparison between classifiers removed the remaining false positives and alleviated pathogen identification. Here we show a generalizable method for identification of pathogen species using DNA metagenomic sequencing. The choice of bioinformatic method mainly affected the efficiency of pathogen identification, but not the sensitivity of detection. Identification of pathogens requires multiple filtering steps including read distribution, sequence diversity and complementary verification of pathogen reads.

RNA-Seq Analysis of the Key Long Noncoding RNAs and mRNAs Related to the Regulation of Acute Heat Stress in Rainbow Trout.

As the global climate warms, more creatures are threatened by high temperatures, especially cold-water fish such as rainbow trout. Evidence has demonstrated that long noncoding RNAs (lncRNAs) play a pivotal role in regulating heat stress in animals, but we have little understanding of this regulatory mechanism. The present study aimed to identify potential key lncRNAs involved in regulating acute heat stress in rainbow trout. lncRNA and mRNA expression profiles of rainbow trout head kidney were analyzed via high-throughput RNA sequencing, which exhibited that 1256 lncRNAs (802 up-regulation, 454 down-regulation) and 604 mRNAs (353 up-regulation, 251 down-regulation) were differentially expressed. These differentially expressed genes were confirmed to be primarily associated with immune regulation, apoptosis, and metabolic process signaling pathways through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and coding-noncoding co-expression network analysis. These results suggested that 18 key lncRNA-mRNA pairs are essential in regulating acute heat stress in rainbow trout. Overall, these analyses showed the effects of heat stress on various physiological functions in rainbow trout at the transcriptome level, providing a theoretical basis for improving the production and breeding of rainbow trout and the selection of new heat-resistant varieties.

FLAME: long-read bioinformatics tool for comprehensive spliceome characterization.

Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.

Case history on Epstein-Barr Virus-associated smooth muscle tumor (EBV-SMT) of cranio-cervical junction in an immunocompetent patient.

Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) is a rare mesenchymal tumor that almost exclusively occurs in immunocompromised hosts. Here, we report a 75-year-old Taiwanese woman without definite immune-deficient history presenting with progressive occipital neuralgia, low cranial nerve deficits (CN9-12) and cervical (C1-C5) radiculopathy. Magnetic resonance imaging revealed a 4.5*4.0*6.7 cm infiltrating mass occupying posterior skull base and C1-C2 vertebra and C1-5 epidural extension with bone destruction and vertebral artery (VA) encasement. There was also a synchronous 2.7 cm tonsillar tumor. A two-stage operation for cranio-cervical tumor excision and stabilization was performed. Tumor was confirmed directly arising from VA intraoperatively. Pathology reported a spindle cell neoplasm and the diagnosis of EBV-SMT was confirmed by EBER (EBV-encoded small RNA) in situ hybridization. An immune survey and reconstruction should be conducted for patient with EBV-SMT. A near-total resection of tumor may be beneficial for local control, however, the role of surgical resection in treating CNS EBV-SMT remains to be determined.

A primary nasopharyngeal three-dimensional air-liquid interface cell culture model of the pseudostratified epithelium reveals differential donor- and cell type-specific susceptibility to Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC.

The effect of acute heat stress on the innate immune function of rainbow trout based on the transcriptome.

Heat stress is a condition in which the body's homeostasis is disturbed as a result of the rise in water temperature, resulting in the decline or even death of growth, immunity, and other functions. The mechanisms directing this response are not fully understood. To better characterize the effects of acute heat stress on the innate immune function of rainbow trout, we identified differentially regulated messenger RNA (mRNA) and non-coding RNA (ncRNA) in rainbow trout exposed to acute heat stress. Next-generation RNA sequencing and comprehensive bioinformatics analysis were conducted to characterize the transcriptome profiles, including mRNA, microRNA (miRNA), and long non-coding RNA (lncRNA). The head kidney of rainbow trout were exposed to acute heat stress at 22.5 °C for 24 h. A total of 2605 lncRNAs, 214 miRNAs, and 5608 mRNAs were identified as differentially regulated. Among these expressed genes differentially, 45 lncRNAs and 2 target genes, as well as 38 miRNAs and 14 target genes were significantly enriched in the innate immune response of rainbow trout. LncRNA is used as competitive endogenous RNA (ceRNA) to construct the ceRNA-miRNA-mRNA interaction network. Enrichment analysis of the Kyoto encyclopedia of genes and genomes (KEGG) of ceRNA, the differentially expressed genes related to the innate immune function of rainbow trout, were significantly enriched in the signaling pathway mediated by mitogen-activated protein kinase (MAPK). Overall, these analyses showed the effects of heat stress on the innate immune function in rainbow trout at the transcriptome level, providing a theoretical basis to improve the production and breeding of rainbow trout and the selection of new heat-resistant varieties.

Role of surgery in treating epstein-barr virus-associated smooth muscle tumor (EBV-SMT) with central nervous system invasion: A systemic review from 1997 to 2019.

Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) is a rare mesenchymal tumor occurred almost exclusively in immunocompromised hosts. This article provides a systematic review of literature under PRISMA guideline on clinical features, treatment modalities, roles of surgical intervention, and outcomes of all 65 reported EBV-SMTs with central nervous system (CNS) invasion. Over 95% of reported cases were immunocompromised, while human immunodeficiency virus infection and post-organ transplantation were the most commonly associated underlying causes (near 90%). Despite a heterogeneous follow-up period, a 1-year survival rate of 76.0% and 5-year survival rate of 59.6% may support the indolent and non-deadly nature of EBV-SMT even with CNS invasion. Immune survey and reconstruction should be conducted for every patient with CNS EBV-SMT. Surgical resection is mostly adopted as primary treatment to obtain diagnosis and relieve compressive effect. A total resection of tumor may be beneficial if tumor was symptomatic and had intracranial invasion.

Automated Needle Targeting with X-ray (ANT-X) - Robot-assisted device for percutaneous nephrolithotomy (PCNL) with its first successful use in human.

INTRODUCTION: To make percutaneous access easier in PCNL, we developed Automated Needle Targeting with X-ray (ANT-X). METHOD: ANT-X uses an image registration software with a closed loop feedback system to autoalign the puncture needle to the desired calyx using the bullseye technique. We tried percutaneous punctures on a live pig model and compared the results with free-hand technique. We then performed our first PCNL in a human subject with the aid of ANT-X. Our patient was a 48 year-old gentleman with a 1.4cm left lower pole stone. RESULTS: Initial results for live animal trial showed radiation exposure for robot-assisted arm during puncture was reduced by 26% compared to the free-hand technique (8.2mGy vs 11.2mGy). In the human trial, obtaining percutaneous access was successful at first attempt. CONCLUSION: ANT-X system can help surgeons feel confident and potentially reduce complications, hence enabling more surgeons to adopt this procedure.

The development and validation of a prototype mobility tracker for assessing the life space mobility and activity participation of older adults.

BACKGROUND: There is increasing interest in examining the life space mobility and activity participation of older adults in the community using sensor technology. Objective data from these technologies may overcome the limitations of self-reported surveys especially in older adults with age-associated cognitive impairment. This paper describes the development and validation of a prototype hybrid mobility tracker for assessing life space mobility and out-of-home activities amongst 33 community-ambulant older adults in Singapore. METHODS: A hybrid mobility tracker was developed by combining a passive Global Positioning System logger, tri-axial accelerometer and radio-frequency identification. Objective measures of life space, derived from 1 week of tracking data using Geographic Information Systems, were the maximum Euclidean distance from home (Max Euclid) and the area of the minimum convex polygon surrounding all GPS waypoints (MCP area). Out-of-home activities were quantified by visually identifying the total number of activity nodes, or places where participants spent ≥5 min, from mobility tracks. Self-reported measure of life space in 4 weeks was obtained using the University of Alabama at Birmingham Study of Life Space Assessment (UAB-LSA) questionnaire. Self-reported out-of-home activities were recorded daily in a travel diary for 1 week. Bivariate correlations were used to examine convergent validity between objective and subjective measures of life space and out-of-home activities. RESULTS: The mean age of participants was 69.2 ± 7.1 years. The mean UAB-LSA total score was 79.1 ± 17.4. The median (range) Max Euclid was 2.44 km (0.26-7.50) per day, and the median (range) MCP area was 3.31 km2 (0.03-34.23) per day. The UAB-LSA total score had good correlation with Max Euclid (r = 0.51, p = 0.002), and moderate correlation with MCP area (r = 0.46, p = 0.007). The median (range) total number of activity nodes measured by tracker of 20 (8-47) per week had a good correlation with the total activity count recorded in the travel diaries of 15 (6-40) per week (r = 0.52, p = 0.002). CONCLUSIONS: The tracking system developed to understand out-of-home travel was feasible and reliable. Comparisons with the UAB-LSA and travel diaries showed that it provided reliable and valid spatiotemporal data to assess the life space mobility and activity participation of older adults.

Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Integration of HBV DNA into the human genome may contribute to oncogenesis and to the production of the hepatitis B surface antigen (HBsAg). Whether integrations contribute to HBsAg levels in the blood is poorly known. Here, we characterize the HBV RNA profile of HBV integrations in liver tissue in patients with chronic HBV infection, with or without concurrent hepatitis D infection, by transcriptome deep sequencing. Transcriptomes were determined in liver tissue by deep sequencing providing 200 million reads per sample. Integration points were identified using a bioinformatic pipeline. Explanted liver tissue from five patients with end-stage liver disease caused by HBV or HBV/HDV was studied along with publicly available transcriptomes from 21 patients. Almost all HBV RNA profiles were devoid of reads in the core and the 3' redundancy (nt 1830-1927) regions, and contained a large number of chimeric viral/human reads. Hence, HBV transcripts from integrated HBV DNA rather than from covalently closed circular HBV DNA (cccDNA) predominated in late-stage HBV infection, in particular in cases with hepatitis D virus co-infection. The findings support the suggestion that integrated HBV DNA can be a significant source of HBsAg in humans.

High Frequency of Either Altered Pre-Core StartCodon or Weakened Kozak Sequence in the CorePromoter Region in Hepatitis B Virus A1 Strainsfrom Rwanda.

Hepatitis B virus (HBV) is endemic in Rwanda and is a major etiologic agent for chronic liver disease in the country. In a previous analysis of HBV strains from Rwanda, the S genes of most strains segregated into one single clade of subgenotype, A1. More than half (55%) of the anti-HBe positive individuals were viremic. In this study, 23 complete HBV genomes and the core promoter region (CP) from 18 additional strains were sequenced. Phylogenetic analysis of complete genomes confirmed that most Rwandan strain formed a single unique clade, within subgenotype A1. Strains from 17 of 22 (77%) anti-HBe positive HBV carriers had either mutated the precore start codon (9 strains with either CUG, ACG, UUG, or AAG) or mutations in the Kozak sequence preceding the pre-core start codon (8 strains). These mutually exclusive mutations were also identified in subgenotypes A1 (70/266; 26%), A2 (12/255; 5%), and A3 (26/49; 53%) sequences from the GenBank. The results showed that previous, rarely described HBV variants, expressing little or no HBeAg, are selected in anti-HBe positive subgenotype Al carriers from Rwanda and that mutations reducing HBeAg synthesis might be unique for a particular HBV clade, not just for a specific genotype or subgenotype.

Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.