Researching COVID to enhance recovery (RECOVER) tissue pathology study protocol: Rationale, objectives, and design.

IMPORTANCE: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) tissue pathology study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository. METHODS: RECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Causal inference methods will be employed to investigate associations between risk factors and pathologic outcomes. DISCUSSION: RECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC.

Eleven key measures for monitoring general practice clinical activity during COVID-19: A retrospective cohort study using 48 million adults' primary care records in England through OpenSAFELY.

Background: The COVID-19 pandemic has had a significant impact on delivery of NHS care. We have developed the OpenSAFELY Service Restoration Observatory (SRO) to develop key measures of primary care activity and describe the trends in these measures throughout the COVID-19 pandemic. Methods: With the approval of NHS England, we developed an open source software framework for data management and analysis to describe trends and variation in clinical activity across primary care electronic health record (EHR) data on 48 million adults.We developed SNOMED-CT codelists for key measures of primary care clinical activity such as blood pressure monitoring and asthma reviews, selected by an expert clinical advisory group and conducted a population cohort-based study to describe trends and variation in these measures January 2019-December 2021, and pragmatically classified their level of recovery one year into the pandemic using the percentage change in the median practice level rate. Results: We produced 11 measures reflective of clinical activity in general practice. A substantial drop in activity was observed in all measures at the outset of the COVID-19 pandemic. By April 2021, the median rate had recovered to within 15% of the median rate in April 2019 in six measures. The remaining measures showed a sustained drop, ranging from a 18.5% reduction in medication reviews to a 42.0% reduction in blood pressure monitoring. Three measures continued to show a sustained drop by December 2021. Conclusions: The COVID-19 pandemic was associated with a substantial change in primary care activity across the measures we developed, with recovery in most measures. We delivered an open source software framework to describe trends and variation in clinical activity across an unprecedented scale of primary care data. We will continue to expand the set of key measures to be routinely monitored using our publicly available NHS OpenSAFELY SRO dashboards with near real-time data. Funding: This research used data assets made available as part of the Data and Connectivity National Core Study, led by Health Data Research UK in partnership with the Office for National Statistics and funded by UK Research and Innovation (grant ref MC_PC_20058).The OpenSAFELY Platform is supported by grants from the Wellcome Trust (222097/Z/20/Z); MRC (MR/V015757/1, MC_PC-20059, MR/W016729/1); NIHR (NIHR135559, COV-LT2-0073), and Health Data Research UK (HDRUK2021.000, 2021.0157).

Networks for future healthcare.

The Getting It Right First Time (GIRFT) process is designed to improve the care of patients in the NHS in England through in-depth review of services, benchmarking and presenting a data-driven evidence base to support change. It started as a pilot project targeting unwarranted variation in elective orthopaedic surgery. It rapidly became apparent that the approach of clinically-led deep dives to review the activity in individual orthopaedic units was effective in improving standards of care but also resulted in substantial cost savings that could be reinvested in the clinical service. GIRFT has now expanded to encompass 40 clinical specialties and is funded by NHS England. We describe the ethos of networks and give examples of GIRFT specialty programmes that have made networks a key component of their recommendations.

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

BACKGROUND: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. RESULTS: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. CONCLUSIONS: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

Ruxolitinib vs best available therapy for ET intolerant or resistant to hydroxycarbamide.

Treatments for high-risk essential thrombocythemia (ET) address thrombocytosis, disease-related symptoms, as well as risks of thrombosis, hemorrhage, transformation to myelofibrosis, and leukemia. Patients resistant/intolerant to hydroxycarbamide (HC) have a poor outlook. MAJIC (ISRCTN61925716) is a randomized phase 2 trial of ruxolitinib (JAK1/2 inhibitor) vs best available therapy (BAT) in ET and polycythemia vera patients resistant or intolerant to HC. Here, findings of MAJIC-ET are reported, where the modified intention-to-treat population included 58 and 52 patients randomized to receive ruxolitinib or BAT, respectively. There was no evidence of improvement in complete response within 1 year reported in 27 (46.6%) patients treated with ruxolitinib vs 23 (44.2%) with BAT (P = .40). At 2 years, rates of thrombosis, hemorrhage, and transformation were not significantly different; however, some disease-related symptoms improved in patients receiving ruxolitinib relative to BAT. Molecular responses were uncommon; there were 2 complete molecular responses (CMR) and 1 partial molecular response in CALR-positive ruxolitinib-treated patients. Transformation to myelofibrosis occurred in 1 CMR patient, presumably because of the emergence of a different clone, raising questions about the relevance of CMR in ET patients. Grade 3 and 4 anemia occurred in 19% and 0% of ruxolitinib vs 0% (both grades) in the BAT arm, and grade 3 and 4 thrombocytopenia in 5.2% and 1.7% of ruxolitinib vs 0% (both grades) of BAT-treated patients. Rates of discontinuation or treatment switching did not differ between the 2 trial arms. The MAJIC-ET trial suggests that ruxolitinib is not superior to current second-line treatments for ET. This trial was registered at www.isrctn.com as #ISRCTN61925716.

DRM1 and DRM2 expression regulation: potential role of splice variants in response to stress and environmental factors in Arabidopsis.

The DRM1/ARP gene family is increasingly exhibiting associations with stress conditions at the transcript level. Traditionally correlated with dormancy, increases in transcript levels in response to various treatments have also been reported in various species. As alternative transcript splicing is common in stress conditions, the splice variants of AtDRM1 and AtDRM2 were assessed further in this study. A previously undescribed splice variant of AtDRM1 (AtDRM1.6) is introduced in this work. In silico analyses of predicted protein sequence of all splice variants showed that all variants retain the predicted intrinsically disordered nature. Transcriptional studies of AtDRM1 and AtDRM2 in response to a wide range of abiotic, physical and hormonal treatments showed that AtDRM1.6 is differentially regulated at the transcriptional level compared with other splice variants. Promoter analyses demonstrated AtDRM1 light regulation via the upstream promoter sequence.

Actinidia DRM1--an intrinsically disordered protein whose mRNA expression is inversely correlated with spring budbreak in kiwifruit.

Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia.

Conservation and divergence of four kiwifruit SVP-like MADS-box genes suggest distinct roles in kiwifruit bud dormancy and flowering.

MADS-box genes similar to Arabidopsis SHORT VEGETATIVE PHASE (SVP) have been implicated in the regulation of flowering in annual species and bud dormancy in perennial species. Kiwifruit (Actinidia spp.) are woody perennial vines where bud dormancy and out-growth affect flower development. To determine the role of SVP-like genes in dormancy and flowering of kiwifruit, four MADS-box genes with homology to Arabidopsis SVP, designated SVP1, SVP2, SVP3, and SVP4, have been identified and analysed in kiwifruit and functionally characterized in Arabidopsis. Phylogenetic analysis indicate that these genes fall into different sub-clades within the SVP-like gene group, suggesting distinct functions. Expression was generally confined to vegetative tissues, and increased transcript accumulation in shoot buds over the winter period suggests a role for these genes in bud dormancy. Down-regulation before flower differentiation indicate possible roles as floral repressors. Over-expression and complementation studies in Arabidopsis resulted in a range of floral reversion phenotypes arising from interactions with Arabidopsis MADS-box proteins, but only SVP1 and SVP3 were able to complement the svp mutant. These results suggest that the kiwifruit SVP-like genes may have distinct roles during bud dormancy and flowering.

A rapid transcriptional activation is induced by the dormancy-breaking chemical hydrogen cyanamide in kiwifruit (Actinidia deliciosa) buds.

Budbreak in kiwifruit (Actinidia deliciosa) can be poor in locations that have warm winters with insufficient winter chilling. Kiwifruit vines are often treated with the dormancy-breaking chemical hydrogen cyanamide (HC) to increase and synchronize budbreak. This treatment also offers a tool to understand the processes involved in budbreak. A genomics approach is presented here to increase our understanding of budbreak in kiwifruit. Most genes identified following HC application appear to be associated with responses to stress, but a number of genes appear to be associated with the reactivation of growth. Three patterns of gene expression were identified: Profile 1, an HC-induced transient activation; Profile 2, an HC-induced transient activation followed by a growth-related activation; and Profile 3, HC- and growth-repressed. One group of genes that was rapidly up-regulated in response to HC was the glutathione S-transferase (GST) class of genes, which have been associated with stress and signalling. Previous budbreak studies, in three other species, also report up-regulated GST expression. Phylogenetic analysis of these GSTs showed that they clustered into two sub-clades, suggesting a strong correlation between their expression and budbreak across species.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis.

Erythrocytosis can arise from deregulation of the erythropoietin (Epo) axis resulting from defects in the oxygen-sensing pathway. Epo synthesis is controlled by the hypoxia inducible factor (HIF) complex, composed of an alpha and a beta subunit. There are 2 main alpha subunits, HIF-1 alpha and HIF-2 alpha. Recently, a HIF-2 alpha Gly537Trp mutation was identified in a family with erythrocytosis. This raises the possibility of HIF2A mutations being associated with other cases of erythrocytosis. We now report a subsequent analysis of HIF2A in a cohort of 75 erythrocytosis patients and identify 4 additional patients with novel heterozygous Met535Val and Gly537Arg mutations. All patients presented at a young age with elevated serum Epo. Mutations at Gly-537 account for 4 of 5 HIF2A mutations associated with erythrocytosis. These findings support the importance of HIF-2 alpha in human Epo regulation and warrant investigation of HIF2A in patients with unexplained erythrocytosis.

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 mul of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

What do patients want to happen to their dentures before surgery?

The removal of dentures before having an anaesthetic can be an undignified and distressing event. However, there has been a gradual change in practice and dentures are now often left in place during an anaesthetic. This enquiry into patients' preferences was carried out to find out what patients would like to happen to their dentures, and to see if there were any differences in opinions among age groups. A postal questionnaire was sent out to patients who wore dentures following discharge after surgery. One hundred replies were analysed. There was a spread of opinions across the age groups and by gender. Nurses need to allow patients the choice of what to do in relation to their dentures when going to theatre, although the anaesthetist must make the final decision of whether or not to remove them immediately before the anaesthetic if they feel patient safety could be compromised.

Evidence that continued remission in patients treated for acute leukaemia is dependent upon autologous natural killer cells.

Although it has been known for more than 40 years that allogeneic immune responses cure leukaemias after bone marrow transplantation, autologous leukaemia-specific immunity remains controversial and its impact upon survival has not been established. Here we have tested 25 patients with de novo acute leukaemias, while in remission at completion of their anti-leukaemia therapy, for evidence of autologous cytolytic immunity to their leukaemic cells taken and cryopreserved at disease presentation. We have measured this degree of cell-mediated cytotoxicity in vitro and termed it "leukaemia cytolytic activity" (LCA). Patients whose disease ultimately relapsed had significantly lower LCA than those who remained in remission beyond 2 years (P < 0.001); the absence of LCA when in remission predicted subsequent relapse within 2 years with a sensitivity of 100% and specificity of 77%. LCA was mediated in vitro by CD56+/CD8alpha+/CD3- natural killer cells. We propose that it is this immune response, rather than the chemotherapy per se, which is responsible for continued remission and that measurement of LCA in patients at completion of therapy may be used as an indicator of risk of subsequent relapse. Patients lacking this response will require further treatment, either with an allogeneic donor transplant or an alternative immunotherapeutic strategy.