The functional impact of 1,570 individual amino acid substitutions in human OTC.

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Individuals with neonatal onset appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma, and death, outcomes ameliorated by rapid diagnosis and treatment. Here, we develop a high-throughput functional assay for human OTC and individually measure the impact of 1,570 variants, 84% of all SNV-accessible missense mutations. Comparison to existing clinical significance calls, demonstrated that our assay distinguishes known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. This functional stratification allowed us to identify score ranges corresponding to clinically relevant levels of impairment of OTC activity. Examining the results of our assay in the context of protein structure further allowed us to identify a 13 amino acid domain, the SMG loop, whose function appears to be required in human cells but not in yeast. Finally, inclusion of our data as PS3 evidence under the current ACMG guidelines, in a pilot reclassification of 34 variants with complete loss of activity, would change the classification of 22 from variants of unknown significance to clinically actionable likely pathogenic variants. These results illustrate how large-scale functional assays are especially powerful when applied to rare genetic diseases.

Probing RNA structure and dynamics using nanopore and next generation sequencing.

It has become increasingly evident that the structures RNAs adopt are conformationally dynamic; the various structured states that RNAs sample govern their interactions with other nucleic acids, proteins, and ligands to regulate a myriad of biological processes. Although several biophysical approaches have been developed and used to study the dynamic landscape of structured RNAs, technical limitations have limited their application to all classes of RNA due to variable size and flexibility. Recent advances combining chemical probing experiments with next-generation- and direct sequencing have emerged as an alternative approach to exploring the conformational dynamics of RNA. In this review, we provide a methodological overview of the sequencing-based techniques used to study RNA conformational dynamics. We discuss how different techniques have enabled us to better understand the propensity of RNAs from a variety of different classes to sample multiple conformational states. Finally, we present examples of the ways these techniques have reshaped how we think about RNA structure.

Forensic DNA phenotyping using Oxford Nanopore Sequencing system.

In forensic science, the demand for precision, consistency, and cost-effectiveness has driven the exploration of next-generation sequencing technologies. This study investigates the potential of Oxford Nanopore Sequencing (ONT) Technology for analyzing the HIrisPlex-S panel, a set of 41 single nucleotide polymorphism (SNP) markers used to predict eye, hair, and skin color. Using ONT sequencing, we assessed the accuracy and reliability of ONT-generated data by comparing it with conventional capillary electrophoresis (CE) in 18 samples. The Guppy v6.1 was used as a basecaller, and sample profiles were obtained using Burrows-Wheeler Aligner, Samtools, BCFtools, and Python. Comparing accuracy with CE, we found that 62% of SNPs in ONT-unligated samples were correctly genotyped, with 36% showing allele dropout, and 2% being incorrectly genotyped. In the ONT-ligated samples, 85% of SNPs were correctly genotyped, with 10% showing allele dropout, and 5% being incorrectly genotyped. Our findings indicate that ONT, particularly when combined with ligation, enhances genotyping accuracy and coverage, thereby reducing allele dropouts. However, challenges associated with the technology's error rates and the impact on genotyping accuracy are recognized. Phenotype predictions based on ONT data demonstrate varying degrees of success, with the technology showing high accuracy in several cases. Although ONT technology holds promise in forensic genetics, further optimization and quality control measures are essential to harness its full potential. This study contributes to the ongoing efforts to refine sequence read tuning and improve correction tools in the context of ONT technology's application in forensic genetics.

Clinical and histological comparison of IgA nephritis and renal IgA vasculitis.

BACKGROUND: IgA nephritis (IgAN) and renal IgA vasculitis (IgAV) show renal IgA deposits, but whether these two diseases are distinct entities or a spectrum of the same condition is under debate. In this study, we add perspective by contrasting the clinical course and histological presentation using the Oxford classification and the National Institutes of Health lupus nephritis activity index (LN-AI) and chronicity index (LN-CI) in IgAN and IgAV. METHODS: In this single-center, retrospective study, kidney biopsies of 163 adult patients with IgAN and 60 adult patients with IgAV were compared according to the Oxford MEST-C Score, LN-AI, and LN-CI. At the time of biopsy, clinical presentation was compared in terms of age, arterial hypertension, diabetes mellitus, extrarenal manifestations, as well as estimated glomerular filtration rate, proteinuria, and urine sediment. IgAV patients and all IgAN patients with crescents received immunosuppressive treatment. After biopsy, kidney function was followed until patients reached end-stage renal disease (ESRD) or they died. RESULTS: The clinical course and kidney histology differ in IgAN and IgAV. IgAV patients showed more microhematuria and nephritic sediment, while IgAN patients had a greater history of arterial hypertension, more proteinuria, and a higher risk for ESRD. These clinical differences were associated with histological differences, as kidney biopsies of IgAN patients were characterized by glomerulosclerosis and tubular atrophy, while kidney biopsies of IgAV patients were characterized by endocapillary hypercellularity and crescents. Overall, tubular atrophy and a LN-CI ≥ 4 were associated with a higher risk for ESRD in IgAN and IgAV. CONCLUSION: Our study supports the notion that IgAN and IgAV follow distinct courses, suggesting that they require different treatment strategies. Moreover, we make a point that the Oxford classification and LN-CI can be useful in categorizing and predicting long-term prognosis not only in IgAN, but also in IgAV.

Novel regulatory variant in ABO intronic RUNX1 binding site inducing A3 phenotype.

BACKGROUND AND OBJECTIVES: Mixed-field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations such as in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution. MATERIALS AND METHODS: We used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. RESULTS: Our data revealed a novel heterozygous g.10924C>A variant on the ABO*A allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor's mother, who shared the novel variant and the anti-A specific mixed-field agglutination. CONCLUSION: We discovered a regulatory variant in the 8-bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3 or B3 phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as an emerging strategy for resolving cases with cryptic ABO phenotypes.

Unlocking the genome of the non-sourdough Kazachstania humilis MAW1: insights into inhibitory factors and phenotypic properties.

BACKGROUND: Ascomycetous budding yeasts are ubiquitous environmental microorganisms important in food production and medicine. Due to recent intensive genomic research, the taxonomy of yeast is becoming more organized based on the identification of monophyletic taxa. This includes genera important to humans, such as Kazachstania. Until now, Kazachstania humilis (previously Candida humilis) was regarded as a sourdough-specific yeast. In addition, any antibacterial activity has not been associated with this species. RESULTS: Previously, we isolated a yeast strain that impaired bio-hydrogen production in a dark fermentation bioreactor and inhibited the growth of Gram-positive and Gram-negative bacteria. Here, using next generation sequencing technologies, we sequenced the genome of this strain named K. humilis MAW1. This is the first genome of a K. humilis isolate not originating from a fermented food. We used novel phylogenetic approach employing the 18 S-ITS-D1-D2 region to show the placement of the K. humilis MAW1 among other members of the Kazachstania genus. This strain was examined by global phenotypic profiling, including carbon sources utilized and the influence of stress conditions on growth. Using the well-recognized bacterial model Escherichia coli AB1157, we show that K. humilis MAW1 cultivated in an acidic medium inhibits bacterial growth by the disturbance of cell division, manifested by filament formation. To gain a greater understanding of the inhibitory effect of K. humilis MAW1, we selected 23 yeast proteins with recognized toxic activity against bacteria and used them for Blast searches of the K. humilis MAW1 genome assembly. The resulting panel of genes present in the K. humilis MAW1 genome included those encoding the 1,3-ß-glucan glycosidase and the 1,3-ß-glucan synthesis inhibitor that might disturb the bacterial cell envelope structures. CONCLUSIONS: We characterized a non-sourdough-derived strain of K. humilis, including its genome sequence and physiological aspects. The MAW1, together with other K. humilis strains, shows the new organization of the mating-type locus. The revealed here pH-dependent ability to inhibit bacterial growth has not been previously recognized in this species. Our study contributes to the building of genome sequence-based classification systems; better understanding of K.humilis as a cell factory in fermentation processes and exploring bacteria-yeast interactions in microbial communities.

Nm-Nano: a machine learning framework for transcriptome-wide single-molecule mapping of 2´-O-methylation (Nm) sites in nanopore direct RNA sequencing datasets.

2´-O-methylation (Nm) is one of the most abundant modifications found in both mRNAs and noncoding RNAs. It contributes to many biological processes, such as the normal functioning of tRNA, the protection of mRNA against degradation by the decapping and exoribonuclease (DXO) protein, and the biogenesis and specificity of rRNA. Recent advancements in single-molecule sequencing techniques for long read RNA sequencing data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications from sequencing data. In this study, we propose a bio-computational framework, Nm-Nano, for predicting the presence of Nm sites in direct RNA sequencing data generated from two human cell lines. The Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites: Extreme Gradient Boosting (XGBoost) and Random Forest (RF) with K-mer embedding. Evaluation on benchmark datasets from direct RNA sequecing of HeLa and HEK293 cell lines, demonstrates high accuracy (99% with XGBoost and 92% with RF) in identifying Nm sites. Deploying Nm-Nano on HeLa and HEK293 cell lines reveals genes that are frequently modified with Nm. In HeLa cell lines, 125 genes are identified as frequently Nm-modified, showing enrichment in 30 ontologies related to immune response and cellular processes. In HEK293 cell lines, 61 genes are identified as frequently Nm-modified, with enrichment in processes like glycolysis and protein localization. These findings underscore the diverse regulatory roles of Nm modifications in metabolic pathways, protein degradation, and cellular processes. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.

Parallel in-depth analysis of repeat expansions in ataxia patients by long-read sequencing.

Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on an iterative workflow for quantification of repeat units by PCR-based methods of limited precision. We established and validated clinical nanopore Cas9-targeted sequencing, an amplification-free method for simultaneous analysis of 10 repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR-Cas9, Oxford Nanopore long-read sequencing and a bioinformatics pipeline using the tools STRique and Megalodon for parallel detection of length, sequence, methylation and composition of the repeat loci. Clinical nanopore Cas9-targeted sequencing allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using clinical nanopore Cas9-targeted sequencing we analysed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions on the basis of clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG. Our results highlight the power of clinical nanopore Cas9-targeted sequencing as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.

Imputation strategies for genomic prediction using nanopore sequencing.

BACKGROUND: Genomic prediction describes the use of SNP genotypes to predict complex traits and has been widely applied in humans and agricultural species. Genotyping-by-sequencing, a method which uses low-coverage sequence data paired with genotype imputation, is becoming an increasingly popular SNP genotyping method for genomic prediction. The development of Oxford Nanopore Technologies' (ONT) MinION sequencer has now made genotyping-by-sequencing portable and rapid. Here we evaluate the speed and accuracy of genomic predictions using low-coverage ONT sequence data in a population of cattle using four imputation approaches. We also investigate the effect of SNP reference panel size on imputation performance. RESULTS: SNP array genotypes and ONT sequence data for 62 beef heifers were used to calculate genomic estimated breeding values (GEBVs) from 641 k SNP for four traits. GEBV accuracy was much higher when genome-wide flanking SNP from sequence data were used to help impute the 641 k panel used for genomic predictions. Using the imputation package QUILT, correlations between ONT and low-density SNP array genomic breeding values were greater than 0.91 and up to 0.97 for sequencing coverages as low as 0.1 × using a reference panel of 48 million SNP. Imputation time was significantly reduced by decreasing the number of flanking sequence SNP used in imputation for all methods. When compared to high-density SNP arrays, genotyping accuracy and genomic breeding value correlations at 0.5 × coverage were also found to be higher than those imputed from low-density arrays. CONCLUSIONS: Here we demonstrated accurate genomic prediction is possible with ONT sequence data from sequencing coverages as low as 0.1 × , and imputation time can be as short as 10 min per sample. We also demonstrate that in this population, genotyping-by-sequencing at 0.1 × coverage can be more accurate than imputation from low-density SNP arrays.

Development of Nanopore sequencing-based full-length transcriptome database toward functional genome annotation of the Pacific oyster, Crassostrea gigas.

The Pacific oyster (Crassostrea gigas) is a widely cultivated shellfish in the world, while its transcriptome diversity remains less unexplored due to the limitation of short reads. In this study, we used Oxford Nanopore sequencing to develop the full-length transcriptome database of C. gigas. We identified 77,920 full-length transcripts from 21,523 genes, and uncovered 9668 alternative splicing events and 87,468 alternative polyadenylation sites. Notably, a total of 16,721 novel transcripts were annotated in this work. Furthermore, integrative analysis of 25 publicly available RNA-seq datasets revealed the transcriptome diversity involved in post-transcriptional regulation in C. gigas. We further developed a Drupal based webserver, Cgtdb, which can be used for transcriptome visualization, sequence alignment, and functional genome annotation analyses. This work provides valuable resources and a useful tool for integrative analysis of various transcriptome datasets in C. gigas, which will serve as an essential reference for functional annotation of the oyster genome.

Comparative analysis of PacBio and ONT RNA sequencing methods for Nemopilema Nomurai venom identification.

Recent studies on marine organisms have made use of third-generation sequencing technologies such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). While these specialized bioinformatics tools have different algorithmic designs and performance capabilities, they offer scalability and can be applied to various datasets. We investigated the effectiveness of PacBio and ONT RNA sequencing methods in identifying the venom of the jellyfish species Nemopilema nomurai. We conducted a detailed analysis of the sequencing data from both methods, focusing on key characteristics such as CD, alternative splicing, long-chain noncoding RNA, simple sequence repeat, transcription factor, and functional transcript annotation. Our findings indicate that ONT generally produced higher raw data quality in the transcriptome analysis, while PacBio generated longer read lengths. PacBio was found to be superior in identifying CDs and long-chain noncoding RNA, whereas ONT was more cost-effective for predicting alternative splicing events, simple sequence repeats, and transcription factors. Based on these results, we conclude that PacBio is the most specific and sensitive method for identifying venom components, while ONT is the most cost-effective method for studying venogenesis, cnidocyst (venom gland) development, and transcription of virulence genes in jellyfish. Our study has implications for future sequencing technologies in marine jellyfish, and highlights the power of full-length transcriptome analysis in discovering potential therapeutic targets for jellyfish dermatitis.

Medium-term results of inlay vs. onlay humeral components for reverse shoulder arthroplasty: a New Zealand Joint Registry study.

BACKGROUND: The purpose of this study was to compare medium-term results of inlay and onlay humeral components in reverse shoulder arthroplasty (RSA). Specifically, we report differences in revision rate and functional outcomes between the 2 designs. METHODS: The 3 most used inlay (in-RSA) and onlay (on-RSA) implants by volume from the New Zealand Joint Registry were included in the study. In-RSA was defined as having a humeral tray that recessed within the metaphyseal bone, whereas on-RSA was defined as having a humeral tray that rested on the epiphyseal osteotomy surface. The primary outcome was revision up to 8 years postsurgery. Secondary outcomes included the Oxford Shoulder Score (OSS), implant survival, and revision cause for in-RSA and on-RSA as well as individual prostheses. RESULTS: There were 6707 patients (5736 in-RSA; 971 on-RSA) included in the study. For all causes, in-RSA demonstrated a lower revision rate compared to on-RSA (revision rate/100 component years: in-RSA 0.665, 95% confidence interval [CI] 0.569-0.768; on-RSA 1.010, 95% CI 0.673-1.415). However, the mean 6-month OSS was higher for the on-RSA group (mean difference 2.20, 95% CI 1.37-3.03; P < .001). However, this was not clinically significant. At 5 years, there were no statistically or clinically significant differences between the 2 groups with respect to the OSS. CONCLUSION: The medium-term survival of in-RSA was higher than that of on-RSA. However, functional outcomes at 6 months were better for on-RSA compared to in-RSA. Further follow-up is required to understand the long-term survivorship and functional outcomes between these designs.

Socioeconomic Status Affects Patient-Reported Outcome Measures in Total Hip and Knee Arthroplasty: A Retrospective Dutch Registry Study.

BACKGROUND: To determine the association between socioeconomic status (SES) and patient-reported outcome measures in a Dutch cohort who have undergone total hip arthroplasty (THA) or total knee arthroplasty (TKA). METHODS: A retrospective national registry study of all patients who underwent primary THA or TKA between 2014 and 2020 in the Netherlands was performed. Linear mixed effects regression models were used to assess the association between SES and patient-reported outcome measures for THA and TKA patients separately. The following measures were collected: numeric rating scale for pain, Oxford Hip/Knee Score, Hip/Knee disability and Osteoarthritis Outcome Score, and the EuroQol 5-Dimensions questionnaire. Sex, age, body mass index, American Society of Anesthesiologists classification, Charnley classification, and smoking status were considered as covariates in the models. RESULTS: THA patients (n = 97,443) were on average 70 years old with a body mass index of 27.4 kg/m2, and TKA patients (n = 78,811) were on average 69 years old with a body mass index of 29.7 kg/m2. Preoperatively, patients with a lower SES undergoing THA or TKA reported more severe symptoms and lower health-related quality of life. At 1-year follow-up, they also reported lower scores and less improvement over time compared to patients with a higher SES. CONCLUSIONS: Patients with lower SES report worse symptoms when admitted for surgery and less improvement after surgery. Future research must address potentially mediating factors of the association between SES and symptom reporting such as access to surgery and rehabilitation, subjectivity in reporting, and patient expectation for THA and TKA outcomes.

Comparing patient-reported outcomes of the Oxford Ankle and Foot Questionnaire in children with clubfoot from two different geographic and cultural environments: a cohort study in India and Canada.

PURPOSE: The purpose of this study is to compare the results of the Oxford Ankle and Foot Questionnaire (OxAFQ) in children with clubfoot in Canada and India to assess its ability to predict outcomes and capture patient experiences in different cultural contexts. METHODS: This is a retrospective study of children with clubfoot in India and Canada who completed the OxAFQ. Statistical analyses were implemented on registry-collected data to test for independent predictors of poor outcomes and compare scores between countries, among children and their parents, and in Canada where relapse data was collected and the effect of a relapse on scores. RESULTS: A total of 361 children were included. The mean Indian OxAFQ scores were higher (p < 0.001) in all domains. Pirani score, tenotomy, laterality, and age at presentation were found to be predictive of outcomes between the sites (p < 0.05). OxAFQ scores decreased after relapse for children in Canada (p < 0.05). Canadian children were found to generally have lower OxAFQ scores in all domains compared to their parents (n = 95; Z = -3.178, -3.493, -3.353, and -3.635 for physical, school and play, emotion, and footwear, respectively; p < 0.001). Indian parents and children showed no significant differences in their scores. CONCLUSIONS: A difference was observed in scores between both sites, suggesting there may be differences in how these populations assess personal health outcomes. These findings support the need for cultural validity of patient-reported outcome measures.

Post-traumatic stress disorder is more likely in younger patients undergoing lower limb arthroplasty: impact on function and quality of life.

INTRODUCTION: The aim of this study was to assess whether PTSD was associated with preoperative and/or postoperative joint-specific function and health-related quality of life (HRQoL) in patients undergoing total hip arthroplasty (THA) and total knee arthroplasty (TKA) and whether there were associated preoperative factors. METHODS: A retrospective study was conducted at a single centre using an established arthroplasty database over a 2-year period. Patients undergoing THA and TKA completed pre and 1-year postoperative Oxford hip/knee scores and EuroQoL questionnaire (EQ-5D) to assess joint specific function and HRQoL. Postoperatively, patients completed the self-reported PTSD Checklist for DSM-5 (PCL-5) questionnaire where a score of 31 or greater was used to determine a provisional diagnosis of PTSD. RESULTS: There were 1244 THA and 1356 TKA patients, of which 42 (3.4%) and 54 (4.0%) had a PCL-5 score of ≥ 31, respectively (PTSD groups). Younger age was associated (p < 0.001) with PTSD for both THA (mean difference (MD) 9.9, 95%CI 6.7-13.0) and TKA (MD 4.6, 95%CI 2.2-6.9), which remained significant when adjusting for confounding variables (THA: p < 0.001; TKA: p = 0.020). The preoperative Oxford (THA:MD 4.9, p < 0.001; TKA:MD 5.7, p < 0.001) and EQ-5D scores (THA:MD 0.378, p < 0.001; TKA:MD 0.276, p < 0.001) were significantly worse in the PTSD groups. Age (AUC 73.8%, p < 0.001) and EQ-5D (AUC 72.9%, p < 0.001) were independent factors that were predictive of PTSD in patients undergoing THA and TKA, respectively. When adjusting for confounding variables, PTSD was clinically and statistically significantly (p < 0.001) associated with a lower improvement in the Oxford (THA:MD 9.3; TKA:MD 10.0) and EQ-5D (THA:MD 0.375; TKA:MD 0.293) scores. CONCLUSIONS: One in 25 patients met a provisional PTSD diagnosis; they were younger and had worse preoperative and improvement in postoperative joint specific function and HRQoL. Age and EQ-5D could be used to identify patients at risk.

Simple Talectomy is a Beneficial Surgical Procedure for Talipes Equinovarus and Other Severe Neuromuscular Foot Deformities.

Noninvasive techniques are gold standard to redress Severe Neuromuscular Foot Deformity (SNFD). However, simple talectomy may be considered to obtain a stable, plantigrade, pain-free foot. We present a 10-year follow-up accessing radiological correction rates, functional outcomes, complications, and patient satisfaction. This retrospective case series evaluated talectomies in 2012 to 2022. Simple talectomy was combined with Steinman pin fixation of calcaneus to tibia for approximately 6 weeks. Diagnoses primarily included arthrogryposis multiplex congenita and cerebral palsy. Indications were pain, wounds/pressure marks, severe rigidity, and residual/recurrent deformity. The primary outcome was radiological correction. Tibiotalar angle (TiTa) and tibiocalcaneal angle (TiCa) were measured on mediolateral projections. Secondary outcomes were functional scores of pain/deformity graded as good, fair or poor. Furthermore, validated patient-reported outcome measures, that is, EQ-5D-5L and the Scoliosis Research Society-30 Questionnaire (2 items) assessed health-related quality of life and patient satisfaction. Nineteen talectomies in 11 patients were analyzed. Mean follow-up was 62 months (range 9-112 months). Mean TiTa was 137° (95%CI 128;146). TiCa improved significantly: Mean difference -24° (95%CI -44;-5, p = .02). All feet became plantigrade and pain-free with no skin issues. Functional outcomes were graded as 9/19 good, 10/19 fair and 0/19 poor. Parents/primary caregivers were mainly satisfied. Perceived health was 54 (95%CI 34;75) out of 100 on a visual analogue scale, emphasizing complex medical conditions. In conclusion, simple talectomy is a suitable salvage procedure for SNFD.

A comprehensive analysis of the pre- and postoperative responses to each of the 12 Oxford knee score questions one year following knee arthroplasty.

AIMS: To assess the pre- and postoperative responses to each of the 12 individual Oxford Knee Score (OKS) questions and percentages of those that were better, same or worse after primary knee arthroplasty (KA). METHODS: A single centre retrospective cohort study conducted over a 24-month period which included 3259 patients with completed OKS preoperatively and 1-year after KA. There were 1286 males and 1973 females, with an overall mean age of 70.0 (range 34-94). The mean scores for each question of the OKS were compared between baseline and 1-year. The percentage of patients who reported better, the same or worse postoperative symptoms for each question were calculated and represented on a heatmap. RESULTS: There were significant (p < 0.001) improvements in all 12 questions, all of which demonstrated moderate (Q2, Q7) or large effect sizes. Improvements in individual question responses varied. Symptoms of pain and limping demonstrated the greatest improvement, with 86% of patients enjoying a positive change in their symptoms. Despite this improvement 1067 (41.4%) continued to have mild to severe pain in their knee, and 442 (17.3%) patients limped often to all the time when walking postoperatively. Whereas other questions that did not improve to the same extent for example washing and drying only improved in 53% of patients but only 347 (13.5%) had moderate/extreme trouble or found it impossible to do this postoperatively. Preoperatively four questions (Q1, Q6, Q7, Q8) demonstrated floor effects, postoperatively all questions apart from question 7 (kneeling) demonstrated ceiling effects. CONCLUSION: The mean improvement and outcome at 1-year for each of the 12 questions varied according to the patient's preoperative response. As a clinical tool, the heatmap (improvement, same and worse) will enable communication to patients about their potential change in their knee specific symptoms according to their preoperative responses. LEVEL OF EVIDENCE: Retrospective study, Level III.

A chromosome scale tomato genome built from complementary PacBio and Nanopore sequences alone reveals extensive linkage drag during breeding.

The assembly and scaffolding of plant crop genomes facilitate the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato (Solanum lycopersicum) has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of tobacco mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT Nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We show specific examples of how HiFi and ONT datasets can complement one another to improve assembly contiguity. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all 12 chromosomes were represented as 12 contiguous sequences (N50 = 68.5 Mbp). This chromosome scale assembly did not require scaffolding using an orthogonal data type. The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato genome assemblies that made use of genetic maps and Hi-C for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1-Mbp region of the S. peruvianum genome during tomato breeding. Through marker studies and ONT-based comprehensive haplotyping we show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near-complete genome sequences.

The Complete Genome Resource of Xanthomonas oryzae pv. oryzae CIX2779 Includes the First Sequence of a Plasmid for an African Representative of This Rice Pathogen.

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Effectiveness of Heterologous Coronavirus Disease 2019 (COVID-19) Vaccine Booster Dosing in Brazilian Healthcare Workers, 2021.

BACKGROUND: Little is currently known about vaccine effectiveness (VE) for either 2 doses of Oxford-AstraZeneca (ChAdOx1) viral vector vaccine or CoronaVac (Instituto Butantan) inactivated viral vaccine followed by a third dose of mRNA vaccine (Pfizer/BioNTech) among healthcare workers (HCWs). METHODS: We conducted a retrospective cohort study among HCWs (aged ≥18 years) working in a private healthcare system in Brazil from January to December 2021. VE was defined as 1 - incidence rate ratio (IRR), with IRR determined using Poisson models with the occurrence of laboratory-confirmed coronavirus disease 2019 (COVID-19) infection as the outcome, adjusting for age, sex, and job type. We compared those receiving viral vector or inactivated viral primary series (2 doses) with those who received an mRNA booster. RESULTS: A total of 11 427 HCWs met the inclusion criteria. COVID-19 was confirmed in 31.5% of HCWs receiving 2 doses of CoronaVac vaccine versus 0.9% of HCWs receiving 2 doses of CoronaVac vaccine with mRNA booster (P < .001) and 9.8% of HCWs receiving 2 doses of ChAdOx1 vaccine versus 1% among HCWs receiving 2 doses of ChAdOx1 vaccine with mRNA booster (P < .001). In the adjusted analyses, the estimated VE was 92.0% for 2 CoronaVac vaccines plus mRNA booster and 60.2% for 2 ChAdOx1 vaccines plus mRNA booster, when compared with those with no mRNA booster. Of 246 samples screened for mutations, 191 (77.6%) were Delta variants. CONCLUSIONS: While 2 doses of ChAdOx1 or CoronaVac vaccines prevent COVID-19, the addition of a Pfizer/BioNTech booster provided significantly more protection.