scPipe: an extended preprocessing pipeline for comprehensive single-cell ATAC-Seq data integration in R/Bioconductor.

scPipe is a flexible R/Bioconductor package originally developed to analyse platform-independent single-cell RNA-Seq data. To expand its preprocessing capability to accommodate new single-cell technologies, we further developed scPipe to handle single-cell ATAC-Seq and multi-modal (RNA-Seq and ATAC-Seq) data. After executing multiple data cleaning steps to remove duplicated reads, low abundance features and cells of poor quality, a SingleCellExperiment object is created that contains a sparse count matrix with features of interest in the rows and cells in the columns. Quality control information (e.g. counts per cell, features per cell, total number of fragments, fraction of fragments per peak) and any relevant feature annotations are stored as metadata. We demonstrate that scPipe can efficiently identify 'true' cells and provides flexibility for the user to fine-tune the quality control thresholds using various feature and cell-based metrics collected during data preprocessing. Researchers can then take advantage of various downstream single-cell tools available in Bioconductor for further analysis of scATAC-Seq data such as dimensionality reduction, clustering, motif enrichment, differential accessibility and cis-regulatory network analysis. The scPipe package enables a complete beginning-to-end pipeline for single-cell ATAC-Seq and RNA-Seq data analysis in R.

Benchmarking long-read RNA-sequencing analysis tools using in silico mixtures.

The lack of benchmark data sets with inbuilt ground-truth makes it challenging to compare the performance of existing long-read isoform detection and differential expression analysis workflows. Here, we present a benchmark experiment using two human lung adenocarcinoma cell lines that were each profiled in triplicate together with synthetic, spliced, spike-in RNAs (sequins). Samples were deeply sequenced on both Illumina short-read and Oxford Nanopore Technologies long-read platforms. Alongside the ground-truth available via the sequins, we created in silico mixture samples to allow performance assessment in the absence of true positives or true negatives. Our results show that StringTie2 and bambu outperformed other tools from the six isoform detection tools tested, DESeq2, edgeR and limma-voom were best among the five differential transcript expression tools tested and there was no clear front-runner for performing differential transcript usage analysis between the five tools compared, which suggests further methods development is needed for this application.

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Cast selection and non-union rates for acute scaphoid fractures treated conservatively: a systematic review and meta-analysis.

Cast selection for conservatively treated acute scaphoid fractures remains controversial. Cast options include short arm versus long arm, and those that include the thumb or leave it free. We sought to investigate the role of how cast choice affects nonunion rates after conservative management of scaphoid fractures. We searched PubMed, Embase, and Google Scholar from inception through July 14, 2020, according to the Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines. We extracted information of interest, including cast type, and non-union rates at the end of the treatment period. We then performed a meta-analysis using the random-effects model. We identified seven relevant studies. Non-union was observed in 15 out of 156 (9.6%) with short-arm cast and 13 out of the 124 (10.5%) with long-arm cast (OR = 0.79, 95% CI [0.19, 3.26], p = 0.74). Non-union was observed in 18 out of 174 (10.3%) with thumb immobilization cast and 18 out of the 179 (10.1%) without thumb immobilization (OR = 0.97, 95% CI [0.49, 1.94], p = 0.69). In our study, short arm casting was proven non-inferior to long arm casting. Similarly, casts without thumb immobilization were equally as effective as casts with thumb immobilization in terms of non-union rates for acute scaphoid fractures treated non-operatively.

All-trans retinoic acid (ATRA) regulates key genes in the RARG-TOP2B pathway and reduces anthracycline-induced cardiotoxicity.

The effectiveness of anthracycline chemotherapeutics (e.g., doxorubicin) is limited by anthracycline-induced cardiotoxicity (ACT). A nonsynonymous variant (S427L) in the retinoic acid receptor-γ (RARG) gene has been associated with ACT. This variant causes reduced RARG activity, which is hypothesized to lead to increased susceptibility to ACT through reduced activation of the retinoic acid pathway. This study explored the effects of activating the retinoic acid pathway using a RAR-agonist, all-trans retinoic acid (ATRA), in human cardiomyocytes and mice treated with doxorubicin. In human cardiomyocytes, ATRA induced the gene expression of RARs (RARG, RARB) and repressed the expression of topoisomerase II enzyme genes (TOP2A, TOP2B), which encode for the molecular targets of anthracyclines and repressed downstream ACT response genes. Importantly, ATRA enhanced cell survival of human cardiomyocytes exposed to doxorubicin. The protective effect of ATRA was also observed in a mouse model (B6C3F1/J) of ACT, in which ATRA treatment improved heart function compared to doxorubicin-only treated mice. Histological analyses of the heart also indicated that ATRA treatment reduced the pathology associated with ACT. These findings provide additional evidence for the retinoic acid pathway's role in ACT and suggest that the RAR activator ATRA can modulate this pathway to reduce ACT.

Single-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy.

Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.

Traumatic Complex Scaphoid Dislocation With Radial Carpal Disruption: A Case Report.

Background. Scaphoid dislocation with radial carpal disruption constitutes an extremely rare injury, and there are no clear guidelines for treatment. This article reviews a delayed presentation of this injury and its surgical management.

Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a novel fragmented IHHNV EVE sequence.

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such endogenous viral elements and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of endogenous viral elements. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for 1 generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific endogenous viral elements identified an element comprised of a 9,045-bp stretch of repeated, inverted, and jumbled genome fragments of infectious hypodermal and hematopoietic necrosis virus bounded by a repeated 591/590 bp host sequence. As only near complete linear ∼4 kb infectious hypodermal and hematopoietic necrosis virus genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear endogenous viral element types. The existence of joined inverted infectious hypodermal and hematopoietic necrosis virus genome fragments also provides a means by which hairpin double-stranded RNA could be expressed and processed by the shrimp RNA interference machinery.

Benchmarking UMI-based single-cell RNA-seq preprocessing workflows.

BACKGROUND: Single-cell RNA-sequencing (scRNA-seq) technologies and associated analysis methods have rapidly developed in recent years. This includes preprocessing methods, which assign sequencing reads to genes to create count matrices for downstream analysis. While several packaged preprocessing workflows have been developed to provide users with convenient tools for handling this process, how they compare to one another and how they influence downstream analysis have not been well studied. RESULTS: Here, we systematically benchmark the performance of 10 end-to-end preprocessing workflows (Cell Ranger, Optimus, salmon alevin, alevin-fry, kallisto bustools, dropSeqPipe, scPipe, zUMIs, celseq2, and scruff) using datasets yielding different biological complexity levels generated by CEL-Seq2 and 10x Chromium platforms. We compare these workflows in terms of their quantification properties directly and their impact on normalization and clustering by evaluating the performance of different method combinations. While the scRNA-seq preprocessing workflows compared vary in their detection and quantification of genes across datasets, after downstream analysis with performant normalization and clustering methods, almost all combinations produce clustering results that agree well with the known cell type labels that provided the ground truth in our analysis. CONCLUSIONS: In summary, the choice of preprocessing method was found to be less important than other steps in the scRNA-seq analysis process. Our study comprehensively compares common scRNA-seq preprocessing workflows and summarizes their characteristics to guide workflow users.

Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing.

A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.

Transcriptome-wide association study uncovers the role of essential genes in anthracycline-induced cardiotoxicity.

Anthracyclines are highly effective chemotherapeutic agents; however, their clinical utility is limited by severe anthracycline-induced cardiotoxicity (ACT). Genome-wide association studies (GWAS) have uncovered several genetic variants associated with ACT, but the impact of these findings requires further elucidation. We conducted a transcriptome-wide association study (TWAS) using our previous GWAS summary statistics (n = 280 patients) to identify gene expression-related associations with ACT. We identified a genetic association between decreased expression of GDF5 and ACT (Z-score = -4.30, P = 1.70 × 10-5), which was replicated in an independent cohort (n = 845 patients, P = 3.54 × 10-3). Additionally, cell viability of GDF5-silenced human cardiac myocytes was significantly decreased in response to anthracycline treatment. Subsequent gene set enrichment and pathway analyses of the TWAS data revealed that genes essential for survival, cardioprotection and response to anthracyclines, as well as genes involved in ribosomal, spliceosomal and cardiomyopathy pathways are important for the development of ACT.

Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

Fatal perinatal mitochondrial cardiac failure caused by recurrent de novo duplications in the ATAD3 locus.

BACKGROUND: In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The ATAD3 locus is one such region, in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively. METHODS: Whole exome, whole genome and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease. FINDINGS: We report six different de novo duplications in the ATAD3 gene locus causing a distinctive presentation including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently corneal clouding or cataracts and encephalopathy. The recurrent 68 Kb ATAD3 duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The ATAD3 duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue. CONCLUSIONS: ATAD3 duplications appear to act in a dominant-negative manner and the de novo inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience the ATAD3 locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies. FUNDING: Australian NHMRC, US Department of Defense, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance and Australian Mito Foundation.

Acute Ulnar Neurapraxia and Carpal Tunnel Syndrome in the Context of a Distal Radius Fracture.

Distal radius fractures, carpal tunnel syndrome, and ulnar nerve compression are common causes of symptoms that result in patients presenting for hand evaluation. This is a unique case of a distal radius fracture leading to both carpal tunnel syndrome and ulnar nerve compression requiring urgent operative management.

Delayed Hemolytic Transfusion Reaction in a Patient with Sickle Cell Disease: Case Report.

BACKGROUND: Blood transfusion is a key treatment of sickle cell disease (SCD) complications. Delayed hemolytic transfusion reaction (DHTR) is a delayed reaction, that occurs days to weeks following a transfusion, characterized by mild anemia and/or hyperbilirubinemia and is one of the serious complications of blood transfusion. The symptoms of DHTR resemble those of vaso-occlusive crisis secondary to SCD leading to difficulty or delaying in diagnosis of DHTR. DHTR may lead to multiple organ failure and death. CASE REPORT: A 31-year-old female patient with a known case of SCD presented to our ER in King Fahad hospital Hofuf in the Kingdom of Saudi Arabia, with a history of generalized body ache, exertional dyspnoea, headache and easy fatigability for a few days on a background history of episodic hospital admissions for SCD, but she was admitted 3 times over the previous 6 months and received 6 units of packed red blood cells (PRBCs). The last blood transfusion was 18 days earlier. She was sick and her Hb level was 4.5 g/dL with positive Coombs test and positive alloantibodies, diagnosed as DHTR. We treated her with prednisolone tablets 1 mg/kg daily, intravenous immunoglobulins, 0.4 gm/kg daily for 5 days, and rituximab 500 mg IV every week for 4 weeks. Her Hb level raised up to 8.2 g/dL and she was discharged in good condition. CONCLUSION: Identifying risk factors for DHTR by history and presentation is urgently needed in order to risk stratify the transfusion regimen. It is important to avoid additional transfusions in these patients if possible because these may exacerbate the hemolysis and worsen the degree of anemia.

Prevention of adverse drug effects: a pharmacogenomic approach.

PURPOSE OF REVIEW: Adverse drug reactions (ADRs) are a serious burden and can negatively impact patient quality of life. One of these ADRs, anthracycline-induced cardiotoxicity (ACT), occurs in up to 65% of treated patients and can lead to congestive heart failure. Pharmacogenetic studies have helped to reveal the mechanisms of ACT and, consequently, inform current strategies to prevent ACT in the clinic. RECENT FINDINGS: Many pharmacogenetic studies have been conducted for ACT, but few have led to the development of clinical practice guidelines and clinical genetic testing for ACT. This is, in part, because of lack of replication in independent patient cohorts and/or validation of an affected biological pathway. Recent advances in pharmacogenetic studies have been made through the use of novel methods that directly implicate dysregulated genes and perturbed biological pathways in response to anthracycline treatment. SUMMARY: Furthering the understanding of the genetics and altered biological pathways of ACT through these novel methods can inform clinical treatment strategies and enable refinement of current clinical practice guidelines. This can therefore lead to improvement in clinical pharmacogenetic testing for further reduction of the incidence of ACT in pediatric cancer patients taking anthracyclines.

The First Genetic Map for a Psoraleoid Legume (Bituminaria bituminosa) Reveals Highly Conserved Synteny with Phaseoloid Legumes.

We present the first genetic map of tedera (Bituminaria bituminosa (L.) C.H. Stirton), a drought-tolerant forage legume from the Canary Islands with useful pharmaceutical properties. It is also the first genetic map for any species in the tribe Psoraleeae (Fabaceae). The map comprises 2042 genotyping-by-sequencing (GBS) markers distributed across 10 linkage groups, consistent with the haploid chromosome count for this species (n = 10). Sequence tags from the markers were used to find homologous matches in the genome sequences of the closely related species in the Phaseoleae tribe: soybean, common bean, and cowpea. No tedera linkage groups align in their entirety to chromosomes in any of these phaseoloid species, but there are long stretches of collinearity that could be used in tedera research for gene discovery purposes using the better-resourced phaseoloid species. Using Ks analysis of a tedera transcriptome against five legume genomes provides an estimated divergence time of 17.4 million years between tedera and soybean. Genomic information and resources developed here will be invaluable for breeding tedera varieties for forage and pharmaceutical purposes.

Discharge against medical advice in paediatric patients.

OBJECTIVES: Discharge against medical advice (DAMA) from the hospital may negatively impact patients' well-being. The present study aimed to investigate the main reasons for DAMA among parents of children admitted to the paediatric ward of the Ali Ebne Abitaleb Hospital in Zahedan, Iran. METHODS: Participants in this case-control study included 130 children who had been admitted to the hospital's paediatric ward. Participants were divided into two equal groups: (1) those with incomplete treatment and (2) those who stayed in the hospital until the completion of their management and followed regular discharge procedures. A self-administered questionnaire was utilised for data collection. RESULTS: Participants included 130 children aged <1-18 years with an average of 3.3 ± 3.7 years; 51.5% (67) were girls, and 48.5% (63) were boys. The results showed a significant relationship between DAMA and the father's level of education (p < 0.05), length of hospitalization (p < 0.001), and duration of treatment (p = 0.027). No significant correlation was found for other factors (p > 0.05). CONCLUSION: This study found that the key reasons for DAMA were as follows: lack of satisfaction with physicians and hospital staff, family disturbance due to the presence of other children at home, inadequate economic situation, and being away from home. Providing professional education to parents and expressing the benefits and disadvantages of refusing complete treatment may help parents make better decisions.

A single-cell transcriptome atlas of the adult human retina.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.