Plant genome evolution in the genus Eucalyptus is driven by structural rearrangements that promote sequence divergence.

Genomes have a highly organized architecture (nonrandom organization of functional and nonfunctional genetic elements within chromosomes) that is essential for many biological functions, particularly gene expression and reproduction. Despite the need to conserve genome architecture, a high level of structural variation has been observed within species. As species separate and diverge, genome architecture also diverges, becoming increasingly poorly conserved as divergence time increases. However, within plant genomes, the processes of genome architecture divergence are not well described. Here we use long-read sequencing and de novo assembly of 33 phylogenetically diverse, wild and naturally evolving Eucalyptus species, covering 1-50 million years of diverging genome evolution to measure genome architectural conservation and describe architectural divergence. The investigation of these genomes revealed that following lineage divergence, genome architecture is highly fragmented by rearrangements. As genomes continue to diverge, the accumulation of mutations and the subsequent divergence beyond recognition of rearrangements become the primary driver of genome divergence. The loss of syntenic regions also contribute to genome divergence but at a slower pace than that of rearrangements. We hypothesize that duplications and translocations are potentially the greatest contributors to Eucalyptus genome divergence.

DILATED CARDIOMYOPATHY IN SLENDER-TAILED MEERKATS (SURICATA SURICATTA) UNDER HUMAN CARE.

Meerkats (Suricata suricatta) housed at two accredited zoological institutions in the United States were evaluated via echocardiography, thoracic radiography, and blood biomarkers-taurine and feline N-terminal pro-B-type natriuretic peptide-to determine the prevalence and severity of dilated cardiomyopathy (DCM) in both populations. In total, 24 meerkats were evaluated and 7 were diagnosed with DCM based on the following parameters: left ventricular internal diameter at end diastole > 1.30 cm, left ventricular internal diameter at end systole > 1.10 cm, and a fractional shortening of <18%. Echocardiographic parameters were identified and reported for normal and affected meerkats, whereas thoracic radiographs were not useful for screening for DCM. Meerkats with DCM were treated with pimobendan and/or benazepril and furosemide if indicated. Seven meerkats died during the study period, with the majority exhibiting myocardial fibrosis. Of the blood parameters tested, elevated taurine levels were associated with DCM. Further research is necessary to characterize the etiology of DCM in meerkats.

Limited utility of adding 3T cervical spinal cord MRI to monitor disease activity in multiple sclerosis.

BACKGROUND: Performing routine brain magnetic resonance imaging (MRI) is widely accepted as the standard of care for disease monitoring in multiple sclerosis (MS), but the utility of performing routine spinal cord (SC) MRI for this purpose is still debatable. OBJECTIVE: This study aimed to measure the frequency of new isolated cervical spinal cord lesions (CSLs) in people with MS (pwMS) undergoing routine brain and cervical SC-MRI for disease monitoring and determine the factors associated with the development of new CSLs and their prognostic value. METHODS: We retrospectively identified 1576 pwMS who underwent follow-up 3T brain and cervical SC-MRI over a 9-month period. MRI was reviewed for the presence of new brain lesions (BLs) and CSLs. Clinical records were reviewed for interval relapses between sequential scans and subsequent clinical relapse and disability worsening after the follow-up MRI. RESULTS: In 1285 pwMS (median interval: 13-14 months) who were clinically stable with respect to relapses, 73 (5.7%) had new CSLs, of which 49 (3.8%) had concomitant new BLs and 24 (1.9%) had new isolated CSLs only. New asymptomatic CSLs were associated with ⩾ 3 prior relapses (p = 0.04), no disease-modifying therapy (DMT) use (p = 0.048), and ⩾ 3 new BLs (p < 0.001); ⩾ 3 new BLs (OR: 7.11, 95% CI: 4.3-11.7, p < 0.001) remained independently associated with new CSLs on multivariable analysis. Having new asymptomatic CSLs was not independently associated with subsequent relapse or disability worsening after the follow-up MRI (median follow-up time of 26 months). CONCLUSION: Routine brain and cervical SC-MRI detected new isolated CSLs in only < 2% of clinically stable pwMS. Developing new asymptomatic CSLs was associated with concomitant new BLs and did not confer an independent increased risk of relapse or disability worsening. Performing SC-MRI may not be warranted for routine monitoring in most pwMS, and performing only brain MRI may be sufficient to capture the vast majority of clinically silent disease activity.

Socioemotional Development of Infants and Toddlers During the COVID-19 Pandemic.

Importance: Understanding the impact of the COVID-19 pandemic on children's socioemotional development is critical to plan for ongoing needs in the early intervention and education systems. Objective: To determine if Ages and Stages Questionnaire, Third Edition (ASQ-3) and Ages and Stages Questionnaire Social-Emotional, Second Edition (ASQ:SE-2) scores changed during the COVID-19 pandemic among families served by a nurse-visiting program. Design, Setting, and Participants: This retrospective, cohort study took place from 2015 through 2021 and included 4 cohorts (prepandemic, pandemic 1, pandemic 2, and pandemic 3) with differing pandemic exposure at the time of screening. Analysis was conducted from July 2022 through October 2023. Data from the Nurse-Family Partnership (NFP), a national nurse-visiting program enrolling birthing people during pregnancy and continuing through age 2 years, were used. A total of 60 171 families with a singleton birth at 37 weeks' gestation or longer and at least 1 valid ASQ-3 and/or ASQ:SE-2 screening in the NFP from January 1, 2015, through December 31, 2021, were enrolled. Exposure: COVID-19 pandemic. Main Outcomes and Measures: Outcomes were a positive screening, defined as scores in the refer area on the ASQ-3 at 10 months and 18 months of age and in the ASQ:SE-2 at 12 months and 18 months of age. Multivariable mixed-effects logistic regression models were used to calculate odds ratios (ORs) for positive screening in pandemic cohorts compared with the prepandemic cohort. Covariates included parent age, race and ethnicity, marital status, income, child's biological sex, and multiparity. Results: Of 60 171 families enrolled, pandemic cohorts had fewer teenagers, were more likely to be married, and were multiparous. Compared with the prepandemic cohort, all pandemic cohorts had higher odds of positive screening on the ASQ-SE at 12 months (pandemic 1: OR, 1.35; 95% CI, 1.09-1.66; pandemic 2: OR, 1.60; 95% CI, 1.30-1.96; and pandemic 3: OR, 1.94; 95% CI, 1.61-2.33) and pandemic 2 and 3 had higher odds of a positive screening at 18 months (pandemic 2: OR, 1.61; 95% CI, 1.29-2.00 and pandemic 3: OR, 1.87; 95% CI, 1.50-2.32). On the ASQ-3, pandemic cohorts 2 and 3 were more likely than the prepandemic cohort to screen positive on the communication subscale at 18 months (pandemic 2: OR, 1.39; 95% CI, 1.17-1.64 and pandemic 3: OR, 1.28; 95% CI, 1.07-1.53). Conclusions and Relevance: In this study, exposure to the COVID-19 pandemic, especially during the first year of life, was associated with higher odds of positive ASQ:SE-2 screening, even when adjusting for demographics and family risks. These findings suggest that unmeasured community, family, and child factors that changed as a result of the pandemic contributed to delays in young children's socio-emotional development.

Unsupervised machine learning identifies distinct ALS molecular subtypes in post-mortem motor cortex and blood expression data.

Amyotrophic lateral sclerosis (ALS) displays considerable clinical and genetic heterogeneity. Machine learning approaches have previously been utilised for patient stratification in ALS as they can disentangle complex disease landscapes. However, lack of independent validation in different populations and tissue samples have greatly limited their use in clinical and research settings. We overcame these issues by performing hierarchical clustering on the 5000 most variably expressed autosomal genes from motor cortex expression data of people with sporadic ALS from the KCL BrainBank (N = 112). Three molecular phenotypes linked to ALS pathogenesis were identified: synaptic and neuropeptide signalling, oxidative stress and apoptosis, and neuroinflammation. Cluster validation was achieved by applying linear discriminant analysis models to cases from TargetALS US motor cortex (N = 93), as well as Italian (N = 15) and Dutch (N = 397) blood expression datasets, for which there was a high assignment probability (80-90%) for each molecular subtype. The ALS and motor cortex specificity of the expression signatures were tested by mapping KCL BrainBank controls (N = 59), and occipital cortex (N = 45) and cerebellum (N = 123) samples from TargetALS to each cluster, before constructing case-control and motor cortex-region logistic regression classifiers. We found that the signatures were not only able to distinguish people with ALS from controls (AUC 0.88 ± 0.10), but also reflect the motor cortex-based disease process, as there was perfect discrimination between motor cortex and the other brain regions. Cell types known to be involved in the biological processes of each molecular phenotype were found in higher proportions, reinforcing their biological interpretation. Phenotype analysis revealed distinct cluster-related outcomes in both motor cortex datasets, relating to disease onset and progression-related measures. Our results support the hypothesis that different mechanisms underpin ALS pathogenesis in subgroups of patients and demonstrate potential for the development of personalised treatment approaches. Our method is available for the scientific and clinical community at https://alsgeclustering.er.kcl.ac.uk .

Crop/Plant Modeling Supports Plant Breeding: I. Optimization of Environmental Factors in Accelerating Crop Growth and Development for Speed Breeding.

The environmental conditions in customered speed breeding practice are, to some extent, empirical and, thus, can be further optimized. Crop and plant models have been developed as powerful tools in predicting growth and development under various environments for extensive crop species. To improve speed breeding, crop models can be used to predict the phenotypes resulted from genotype by environment by management at the population level, while plant models can be used to examine 3-dimensional plant architectural development by microenvironments at the organ level. By justifying the simulations via numerous virtual trials using models in testing genotype × environment × management, an optimized combination of environmental factors in achieving desired plant phenotypes can be quickly determined. Artificial intelligence in assisting for optimization is also discussed. We admit that the appropriate modifications on modeling algorithms or adding new modules may be necessary in optimizing speed breeding for specific uses. Overall, this review demonstrates that crop and plant models are promising tools in providing the optimized combinations of environment factors in advancing crop growth and development for speed breeding.

Efficacy and safety of ciclosporin versus methotrexate in the treatment of severe atopic dermatitis in children and young people (TREAT): a multicentre parallel group assessor-blinded clinical trial.

BACKGROUND: Conventional systemic drugs are used to treat children and young people (CYP) with severe atopic dermatitis (AD) worldwide, but no robust randomized controlled trial (RCT) evidence exists regarding their efficacy and safety in this population. While novel therapies have expanded therapeutic options, their high cost means traditional agents remain important, especially in lower-resource settings. OBJECTIVES: To compare the safety and efficacy of ciclosporin (CyA) with methotrexate (MTX) in CYP with severe AD in the TREatment of severe Atopic Eczema Trial (TREAT) trial. METHODS: We conducted a parallel group assessor-blinded RCT in 13 UK and Irish centres. Eligible participants aged 2-16 years and unresponsive to potent topical treatment were randomized to either oral CyA (4 mg kg-1 daily) or MTX (0.4 mg kg-1 weekly) for 36 weeks and followed-up for 24 weeks. Co-primary outcomes were change from baseline to 12 weeks in Objective Severity Scoring of Atopic Dermatitis (o-SCORAD) and time to first significant flare (relapse) after treatment cessation. Secondary outcomes included change in quality of life (QoL) from baseline to 60 weeks; number of participant-reported flares following treatment cessation; proportion of participants achieving ≥ 50% improvement in Eczema Area and Severity Index (EASI 50) and ≥ 75% improvement in EASI (EASI 75); and stratification of outcomes by filaggrin status. RESULTS: In total, 103 participants were randomized (May 2016-February 2019): 52 to CyA and 51 to MTX. CyA showed greater improvement in disease severity by 12 weeks [mean difference in o-SCORAD -5.69, 97.5% confidence interval (CI) -10.81 to -0.57 (P = 0.01)]. More participants achieved ≥ 50% improvement in o-SCORAD (o-SCORAD 50) at 12 weeks in the CyA arm vs. the MTX arm [odds ratio (OR) 2.60, 95% CI 1.23-5.49; P = 0.01]. By 60 weeks MTX was superior (OR 0.33, 95% CI 0.13-0.85; P = 0.02), a trend also seen for ≥ 75% improvement in o-SCORAD (o-SCORAD 75), EASI 50 and EASI 75. Participant-reported flares post-treatment were higher in the CyA arm (OR 3.22, 95% CI 0.42-6.01; P = 0.02). QoL improved with both treatments and was sustained after treatment cessation. Filaggrin status did not affect outcomes. The frequency of adverse events (AEs) was comparable between both treatments. Five (10%) participants on CyA and seven (14%) on MTX experienced a serious AE. CONCLUSIONS: Both CyA and MTX proved effective in CYP with severe AD over 36 weeks. Participants who received CyA showed a more rapid response to treatment, while MTX induced more sustained disease control after discontinuation.

He Looks Like My Father.

Social media has kept us connected in many ways but for Black adults, it can be a harrowing reminder of the treatment of Black bodies. This poem was crafted from focus group data on a study of the effects of vicarious racism on the mental health of Black adults in the wake of George Floyd's death in 2020. This poem uses elements of found poetry and incorporates texts from the narrative (re)telling of Black adults' emotional impact of seeing racially and police-involved killings of Black men. Participants expressed feelings of anger, powerlessness, and sadness and how watching these videos has led to avoidance behavior for the sake of coping. In this poem titled "He Looks like My Father," the participant shares a memory of the last video that they watched on social media in 2014 and why they continue to avoid this type of content. It's traumatizing. It is important to fully reflect on these stories as Black Americans struggle with staying informed and preserving their mental health while being inundated by a continuous feedback loop of Black death. The elements of this poem incorporate repetition and the bolded words are verbatim text from the participant transcript. I acknowledge that my positionality being a Black American woman, with a Black father, and having shared the embodied experience of witnessing Black death via social media influenced the meaning of this poem. As we continue to see a focus on naming racism as a public health threat, this form of vicarious racism is salient and should be explored as health professionals dig deeper into understanding the many ways racism permeates the lives of Black, is a daily stressor, and is a social determinant of mental health equity. These are the narratives from muted lips to unveil your eyes. To view the original version of this poem, see the supplemental material section of this article online.

The power of presentation: How attire, cosmetics, and posture impact the source credibility of women expert witnesses.

Gender stereotypes may negatively affect perceptions of women professionals' credibility, including forensic experts. This study investigated the impact of behavior-based and appearance-based factors on women expert witness's credibility. Jury-eligible adults were shown one of 16 conditions depicting a woman expert which varied based on combinations of three primary independent variables: (1) attire, (2) cosmetic use, and (3) posture. Expert attractiveness and participants' sexist attitudes served as covariates. Results revealed that women experts were seen as marginally more credible when wearing a skirt suit with a closed posture stance than when wearing a pant suit with a closed posture. Secondary analyses indicated expert attractiveness and participant sexist attitudes accounted for the most variability in credibility scores. Credibility of women expert witnesses may be impacted by irrelevant peripheral cues. Findings can inform discussions aimed at mitigating extraneous factors that inadvertently undermine the reception of women expert witness testimony.

Large-scale analyses of CAV1 and CAV2 suggest their expression is higher in post-mortem ALS brain tissue and affects survival.

Introduction: Caveolin-1 and Caveolin-2 (CAV1 and CAV2) are proteins associated with intercellular neurotrophic signalling. There is converging evidence that CAV1 and CAV2 (CAV1/2) genes have a role in amyotrophic lateral sclerosis (ALS). Disease-associated variants have been identified within CAV1/2 enhancers, which reduce gene expression and lead to disruption of membrane lipid rafts. Methods: Using large ALS whole-genome sequencing and post-mortem RNA sequencing datasets (5,987 and 365 tissue samples, respectively), and iPSC-derived motor neurons from 55 individuals, we investigated the role of CAV1/2 expression and enhancer variants in the ALS phenotype. Results: We report a differential expression analysis between ALS cases and controls for CAV1 and CAV2 genes across various post-mortem brain tissues and three independent datasets. CAV1 and CAV2 expression was consistently higher in ALS patients compared to controls, with significant results across the primary motor cortex, lateral motor cortex, and cerebellum. We also identify increased survival among carriers of CAV1/2 enhancer mutations compared to non-carriers within Project MinE and slower progression as measured by the ALSFRS. Carriers showed a median increase in survival of 345 days. Discussion: These results add to an increasing body of evidence linking CAV1 and CAV2 genes to ALS. We propose that carriers of CAV1/2 enhancer mutations may be conceptualised as an ALS subtype who present a less severe ALS phenotype with a longer survival duration and slower progression. Upregulation of CAV1/2 genes in ALS cases may indicate a causal pathway or a compensatory mechanism. Given prior research supporting the beneficial role of CAV1/2 expression in ALS patients, we consider a compensatory mechanism to better fit the available evidence, although further investigation into the biological pathways associated with CAV1/2 is needed to support this conclusion.

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases.

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.

Harnessing Transcriptomic Signals for Amyotrophic Lateral Sclerosis to Identify Novel Drugs and Enhance Risk Prediction.

Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. This study integrates the latest ALS genome-wide association study (GWAS) summary statistics with functional genomic annotations with the aim of providing mechanistic insights into ALS risk loci, inferring drug repurposing opportunities, and enhancing prediction of ALS risk and clinical characteristics. Methods: Genes associated with ALS were identified using GWAS summary statistic methodology including SuSiE SNP-based fine-mapping, and transcriptome- and proteome-wide association study (TWAS/PWAS) analyses. Using several approaches, gene associations were integrated with the DrugTargetor drug-gene interaction database to identify drugs that could be repurposed for the treatment of ALS. Furthermore, ALS gene associations from TWAS were combined with observed blood expression in two external ALS case-control datasets to calculate polytranscriptomic scores and evaluate their utility for prediction of ALS risk and clinical characteristics, including site of onset, age at onset, and survival. Results: SNP-based fine-mapping, TWAS and PWAS identified 117 genes associated with ALS, with TWAS and PWAS providing novel mechanistic insights. Drug repurposing analyses identified five drugs significantly enriched for interactions with ALS associated genes, with directional analyses highlighting α-glucosidase inhibitors may exacerbate ALS pathology. Additionally, drug class enrichment analysis showed calcium channel blockers may reduce ALS risk. Across the two observed expression target samples, ALS polytranscriptomic scores significantly predicted ALS risk (R2 = 4%; p-value = 2.1×10-21). Conclusions: Functionally-informed analyses of ALS GWAS summary statistics identified novel mechanistic insights into ALS aetiology, highlighted several therapeutic research avenues, and enabled statistically significant prediction of ALS risk.

Splenic Rupture Following Colonoscopy.

Splenic rupture following colonoscopy (SRFC) is a rare complication and can have associated mortality if left undiagnosed. Most of the cases reported have been managed operatively. Here, we present a case of a 75-year-old-female who underwent conservative management for SRFC. Splenic rupture should remain a differential in patients presenting with abdominal pain, syncope, and hypotension following colonoscopy. Decisions regarding operative versus conservative management should be guided by the patient's clinical status, hemodynamics, and available resources.

Public health interventions successfully mitigated multiple incursions of SARS-CoV-2 Delta variant in the Australian Capital Territory.

The COVID-19 pandemic has presented a unique opportunity to understand how real-time pathogen genomics can be used for large-scale outbreak investigations. On 12 August 2021, the Australian Capital Territory (ACT) detected an incursion of the SARS-CoV-2 Delta (B.1.617.2) variant. Prior to this date, SARS-CoV-2 had been eliminated locally since 7 July 2020. Several public health interventions were rapidly implemented in response to the incursion, including a territory-wide lockdown and comprehensive contact tracing. The ACT has not previously used pathogen genomics at a population level in an outbreak response; therefore, this incursion also presented an opportunity to investigate the utility of genomic sequencing to support contact tracing efforts in the ACT. Sequencing of >75% of the 1793 laboratory-confirmed cases during the 3 months following the initial notification identified at least 13 independent incursions with onwards spread in the community. Stratification of cases by genomic cluster revealed that distinct cohorts were affected by the different incursions. Two incursions resulted in most of the community transmission during the study period, with persistent transmission in vulnerable sections of the community. Ultimately, both major incursions were successfully mitigated through public health interventions, including COVID-19 vaccines. The high rates of SARS-CoV-2 sequencing in the ACT and the relatively small population size facilitated detailed investigations of the patterns of virus transmission, revealing insights beyond those gathered from traditional contact tracing alone. Genomic sequencing was critical to disentangling complex transmission chains to target interventions appropriately.

Cost-conscious generation of multiplexed short-read DNA libraries for whole-genome sequencing.

Massively parallel, second-generation short-read DNA sequencing has become an integral tool in biology for genomic studies. Offering highly accurate base-pair resolution at the most competitive price, the technology has become widespread. However, high-throughput generation of multiplexed DNA libraries can be costly and cumbersome. Here, we present a cost-conscious protocol for generating multiplexed short-read DNA libraries using a bead-linked transposome from Illumina. We prepare libraries in high-throughput with small reaction volumes that use 1/50th the amount of transposome compared to Illumina DNA Prep tagmentation protocols. By reducing transposome usage and optimising the protocol to circumvent magnetic bead-based clean-ups between steps, we reduce costs, labour time and DNA input requirements. Developing our own dual index primers further reduced costs and enables up to nine 96-well microplate combinations. This facilitates efficient usage of large-scale sequencing platforms, such as the Illumina NovaSeq 6000, which offers up to three terabases of sequencing per S4 flow cell. The protocol presented substantially reduces the cost per library by approximately 1/20th compared to conventional Illumina methods.

Interspecies genome divergence is predominantly due to frequent small scale rearrangements in Eucalyptus.

Synteny, the ordering of sequences within homologous chromosomes, must be maintained within the genomes of sexually reproducing species for the sharing of alleles and production of viable, reproducing offspring. However, when the genomes of closely related species are compared, a loss of synteny is often observed. Unequal homologous recombination is the primary mechanism behind synteny loss, occurring more often in transposon rich regions, and resulting in the formation of chromosomal rearrangements. To examine patterns of synteny among three closely related, interbreeding, and wild Eucalyptus species, we assembled their genomes using long-read DNA sequencing and de novo assembly. We identify syntenic and rearranged regions between these genomes and estimate that ~48% of our genomes remain syntenic while ~36% is rearranged. We observed that rearrangements highly fragment microsynteny. Our results suggest that synteny between these species is primarily lost through small-scale rearrangements, not through sequence loss, gain, or sequence divergence. Further examination of identified rearrangements suggests that rearrangements may be altering the phenotypes of Eucalyptus species. Our study also underscores that the use of single reference genomes in genomic variation studies could lead to reference bias, especially given the scale at which we show potentially adaptive loci have highly diverged, deleted, duplicated and/or rearranged. This study provides an unbiased framework to look at potential speciation and adaptive loci among a rapidly radiating foundation species of woodland trees that are free from selective breeding seen in most crop species.

Comparison Of King's Clinical Staging In Multinational Amyotrophic Lateral Sclerosis Cohorts.

Background: Amyotrophic lateral sclerosis (ALS) shows considerable clinical heterogeneity, which affects clinical trials. A clinical staging system has been proposed for ALS with potential applications in patient care, research, trial design and health economic analyses. The King's system consists of five stages. We have previously shown that progressive clinical stages were reached at predictable proportions through the disease course, but this needs to be validated in other independent samples. Objectives: We aimed to compare King's clinical staging in ALS in four patient groups, located in different regions and countries and using different health care systems from the original study population in South London. Methods: Clinical data were extracted from two European phase 3 randomized controlled trials (MitoTarget and LiCALS) and from two databases predominately from the United States: the PRO-ACT Consortium Database and a database of patients from the PatientsLikeMe website. Clinical stage was estimated using an algorithm, and standardized time to each clinical stage was calculated in deceased patients. Results: 8,796 patients were included, of whom 1,959 had died by the end of follow-up. Stages occurred in the same order as in the original study for all cohorts. Median standardized times to stages (interquartile range) were Stage 2: 0.61 (0.47-0.75), Stage 3: 0.68 (0.56-0.81), Stage 4A: 0.82 (0.71-0.91), Stage 4B: 0.82 (0.69-0.92) and Stage 4 0.80 (0.67-0.91). Discussion: Timings for all stages were similar to those reported in the original study, except Stage 2 which occurred later in the clinical trial databases due to recruitment occurring after diagnosis.

Species-specific basecallers improve actual accuracy of nanopore sequencing in plants.

BACKGROUND: Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii, to train species-specific basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specific basecaller models. We also evaluate accuracy gains from ONT's improved flowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads. RESULTS: Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy's plant-specific model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specific basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specific). CONCLUSIONS: The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specific basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.

RetroSnake: A modular pipeline to detect human endogenous retroviruses in genome sequencing data.

Human endogenous retroviruses (HERVs) integrated into the human genome as a result of ancient exogenous infections and currently comprise ∼8% of our genome. The members of the most recently acquired HERV family, HERV-Ks, still retain the potential to produce viral molecules and have been linked to a wide range of diseases including cancer and neurodegeneration. Although a range of tools for HERV detection in NGS data exist, most of them lack wet lab validation and they do not cover all steps of the analysis. Here, we describe RetroSnake, an end-to-end, modular, computationally efficient, and customizable pipeline for the discovery of HERVs in short-read NGS data. RetroSnake is based on an extensively wet-lab validated protocol, it covers all steps of the analysis from raw data to the generation of annotated results presented as an interactive html file, and it is easy to use by life scientists without substantial computational training. Availability and implementation: The Pipeline and an extensive documentation are available on GitHub.

Physiology and Molecular Breeding in Sustaining Wheat Grain Setting and Quality under Spring Cold Stress.

Spring cold stress (SCS) compromises the reproductive growth of wheat, being a major constraint in achieving high grain yield and quality in winter wheat. To sustain wheat productivity in SCS conditions, breeding cultivars conferring cold tolerance is key. In this review, we examine how grain setting and quality traits are affected by SCS, which may occur at the pre-anthesis stage. We have investigated the physiological and molecular mechanisms involved in floret and spikelet SCS tolerance. It includes the protective enzymes scavenging reactive oxygen species (ROS), hormonal adjustment, and carbohydrate metabolism. Lastly, we explored quantitative trait loci (QTLs) that regulate SCS for identifying candidate genes for breeding. The existing cultivars for SCS tolerance were primarily bred on agronomic and morphophysiological traits and lacked in molecular investigations. Therefore, breeding novel wheat cultivars based on QTLs and associated genes underlying the fundamental resistance mechanism is urgently needed to sustain grain setting and quality under SCS.