High-resolution HLA genotyping in inclusion body myositis refines 8.1 ancestral haplotype association to DRB1*03:01:01 and highlights pathogenic role of arginine-74 of DRß1 chain.

OBJECTIVES: Inclusion body myositis (IBM) is a progressive inflammatory-degenerative muscle disease of older individuals, with some patients producing anti-cytosolic 5'-nucleotidase 1A (NT5C1A, aka cN1A) antibodies. Human Leukocyte Antigens (HLA) is the highest genetic risk factor for developing IBM. In this study, we aimed to further define the contribution of HLA alleles to IBM and the production of anti-cN1A antibodies. METHODS: We HLA haplotyped a Western Australian cohort of 113 Caucasian IBM patients and 112 ethnically matched controls using Illumina next-generation sequencing. Allele frequency analysis and amino acid alignments were performed using the Genentech/MiDAS bioinformatics package. Allele frequencies were compared using Fisher's exact test. Age at onset analysis was performed using the ggstatsplot package. All analysis was carried out in RStudio version 1.4.1717. RESULTS: Our findings validated the independent association of HLA-DRB1*03:01:01 with IBM and attributed the risk to an arginine residue in position 74 within the DRß1 protein. Conversely, DRB4*01:01:01 and DQA1*01:02:01 were found to have protective effects; the carriers of DRB1*03:01:01 that did not possess these alleles had a fourteenfold increased risk of developing IBM over the general Caucasian population. Furthermore, patients with the abovementioned genotype developed symptoms on average five years earlier than patients without. We did not find any HLA associations with anti-cN1A antibody production. CONCLUSIONS: High-resolution HLA sequencing more precisely characterised the alleles associated with IBM and defined a haplotype linked to earlier disease onset. Identification of the critical amino acid residue by advanced biostatistical analysis of immunogenetics data offers mechanistic insights and future directions into uncovering IBM aetiopathogenesis.

Combatting SARS-CoV-2 With Digital Contact Tracing and Notification: Navigating Six Points of Failure.

Digital contact tracing and notification were initially hailed as promising strategies to combat SARS-CoV-2; however, in most jurisdictions, they did not live up to their promise. To avert a given transmission event, both parties must have adopted the technology, it must detect the contact, the primary case must be promptly diagnosed, notifications must be triggered, and the secondary case must change their behavior to avoid the focal tertiary transmission event. If we approximate these as independent events, achieving a 26% reduction in the effective reproduction number Rt would require an 80% success rate at each of these 6 points of failure. Here, we review the 6 failure rates experienced by a variety of digital contact tracing and contact notification schemes, including Singapore's TraceTogether, India's Aarogya Setu, and leading implementations of the Google Apple Exposure Notification system. This leads to a number of recommendations, for example, that the narrative be framed in terms of user autonomy rather than user privacy, and that tracing/notification apps be multifunctional and integrated with testing, manual contact tracing, and the gathering of critical scientific data.

The safety of repeated ketamine dosing in paediatrics: A systematic review.

BACKGROUND: Ketamine is emerging as an effective, rapidly acting antidepressant in adult research. Hypothetical concerns about its long-term safety and impact on the developing brain are limiting its research in children. However, a wealth of paediatric safety and dosing data exists for ketamine, given its extensive use globally as an anaesthetic, analgesic and sedative agent. AIMS: To evaluate the safety of repeat dosing of ketamine in children. METHODS: A systematic search of EMBASE, MEDLINE, PsycINFO, Cochrane Library and PubMed from inception to 13 April 2023 was conducted. Included studies were those reporting adverse events when ketamine was given repeatedly to children aged 5-18, for any condition. No language restrictions were applied. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guideline and Joanna Briggs Institute Critical Appraisal Tools Checklist for study quality assessment were used. The review process was performed independently by two reviewers. RESULTS: Five observational studies (87 patients) were included. The maximum number of doses per patient was 42, over a maximum of 4 months. There were no serious adverse events. There was no evidence of needing higher doses with time to indicate tolerance. The longest follow-up period was 6 months. There were no long-term consequences (including neurocognitive) reported within this time frame. CONCLUSIONS: These results suggest that, despite methodological limitations of the studies, ketamine is well tolerated and safe for use in children, even when given repeatedly in regimens analogous to those used for the treatment of depression in adults. This finding supports the expansion of research into the use of ketamine as a novel antidepressant in children.

Management of Agitation in Behaviours That Challenge in Dementia Care: Multidisciplinary Perspectives on Non-Pharmacological Strategies.

Objective: NICE guidelines recommend non-pharmacological interventions as the first-line approach for the management of behaviours that challenge. Recent work, however, highlights dissatisfaction with the lack of detailed guidance in the national guidelines regarding non-drug interventions. This study examines the views of practitioners regarding non-pharmacological treatments. It further explores perspectives on non-pharmacological strategies used in the management of agitation occurring within episodes of behaviours that challenge. Methods: Forty-two experienced practitioners attended a workshop where behaviours that challenge were described as occurring in three phases of agitation, using a framework adapted from the Positive Behaviour Support framework (pre-agitation, triggering and escalating, high level). The participants were asked to populate a template derived from the adapted framework. The completed templates recorded the clinical strategies the participants found useful to (i) prevent the occurrence of agitation, (ii) de-escalate distress and (iii) deal with perceived high levels of agitation. Results: The Positive Behaviour Support conceptual framework was perceived by participants as helpful in organising their clinical work. A number of interventions were suggested as preventative strategies: music therapy, doll therapy, physical activity and generic person-centred communication skills to enhance wellbeing. In contrast, de-escalation strategies identified by the participants focused on reducing emotional distress. The approaches for dealing with continued high levels of agitation involved a number of "control and restraint" techniques as well as medication. Conclusion: The template allowed specialist multidisciplinary professionals to identify skills for the management of distress and agitated behaviour linked to the respective phase of arousal. The template has scope to guide practitioners to identify the detail needed for the management of behaviours that challenge. The findings have the potential to influence the contents of forthcoming guidelines on alternatives to psychotropics in dementia care.

A fit and function analysis of the UK OSPREY body armour system for female users.

INTRODUCTION: OSPREY personal armour has been issued to UK forces since 2005. From 2015, the VIRTUS personal armour and load carriage system have been progressively replacing OSPREY. In 2016, the ban on women in ground close combat roles throughout the UK's Armed Forces was lifted. In anticipation of this, work has been ongoing to prepare future ballistic protection programmes for a potential increase in the number of female users. METHOD: A human factors questionnaire was provided to 150 female users of OSPREY body armour to complete while on combat operations in Afghanistan. The questionnaire asked the users to rate the comfort of their OSPREY body armour along with their ability to carry out basic tasks. Other background data such as size of body armour worn and bra size were also collected for the analysis. RESULTS: The female participants reported various types of discomfort when wearing their OSPREY body armour, with 135 instances of discomfort experienced in the hip region, for example. Challenges were reported in the ability to carry out basic movements, with the tasks rated on a Likert scale as difficult or very difficult by between 29% and 59% of participants. In addition, a restriction in ability to access personal equipment worn on the person (including pouches, trouser pockets) was commented on by 39% of participants. CONCLUSIONS: Female users reported challenges relating to the fit and function of OSPREY body armour. The VIRTUS body armour system for UK Armed Forces Personnel has already addressed many of the reported issues with OSPREY. Further optimisation for VIRTUS with regard to female users is planned and includes sizing of ballistic hard plates.

The Association between the Histological Subtypes of Mesothelioma and Asbestos Exposure Characteristics.

Asbestos mining operations have left South Africa with a legacy of asbestos contamination and asbestos-related diseases continue to be a problem. The large-scale mining of three types of asbestos presents a unique opportunity to study malignant mesothelioma of the pleura (mesothelioma) in South Africa. This study aimed to describe the demographics of deceased individuals diagnosed with mesothelioma and explore any associations between the histological morphology of mesothelioma and asbestos characteristics. We reviewed the records of all deceased miners and ex-miners from the Pathology Automation System (PATHAUT) database of the National Institute of Occupational Health (NIOH) that were histologically diagnosed with mesothelioma in the period from January 2006-December 2016 (11 years). The study population does not include all cases of mesothelioma in South Africa but rather those that reached the compensation system. Crocidolite asbestos fibres were identified in the majority of mesothelioma cases (n = 140; 53.4%). The epithelioid subtype was most commonly present in both occupational and environmental cases. Cases with the sarcomatous subtype were older at death and fewer female cases were diagnosed with this subtype. No relationship between mesothelioma subtype and asbestos type or asbestos burden or fibre size was established.

Intronic NEFH variant is associated with reduced risk for sporadic ALS and later age of disease onset.

Neurofilament heavy (NEFH) is one of the critical proteins required for the formation of the neuronal cytoskeleton and polymorphisms in NEFH are reported as a rare cause of sporadic ALS (sALS). In the current study, a candidate tetranucleotide (TTTA) repeat variant in NEFH was selected using an in-silico short structural variant (SSV) evaluation algorithm and investigated in two cohorts of North American sALS patients, both separately and combined (Duke cohort n = 138, Coriell cohort n = 333; combined cohort n = 471), compared to a group of healthy controls from the Coriell Institute biobank (n = 496). Stratification according to site of disease onset revealed that the 9 TTTA allele was associated with reduced disease risk, specifically confined to spinal-onset sALS patients in the Duke cohort (p = 0.001). Furthermore, carriage of the 10 TTTA allele was associated with a 2.7 year later age of disease onset in the larger combined sALS cohort (p = 0.02). These results suggest that the 9 and 10 TTTA motif length may have a protective advantage for potentially lowering the risk of sALS and delaying the age of disease onset, however, these results need to be replicated in larger multicenter and multi-ethnic cohorts.

Rational engineering of a synthetic insect-bacterial mutualism.

Many insects maintain mutualistic associations with bacterial endosymbionts, but little is known about how they originate in nature. In this study, we describe the establishment and manipulation of a synthetic insect-bacterial symbiosis in a weevil host. Following egg injection, the nascent symbiont colonized many tissues, including prototypical somatic and germinal bacteriomes, yielding maternal transmission over many generations. We then engineered the nascent symbiont to overproduce the aromatic amino acids tyrosine and phenylalanine, which facilitate weevil cuticle strengthening and accelerated larval development, replicating the function of mutualistic symbionts that are widely distributed among weevils and other beetles in nature. Our work provides empirical support for the notion that mutualistic symbioses can be initiated in insects by the acquisition of environmental bacteria. It also shows that certain bacterial genera, including the Sodalis spp. used in our study, are predisposed to develop these associations due to their ability to maintain benign infections and undergo vertical transmission in diverse insect hosts, facilitating the partner-fidelity feedback that is critical for the evolution of obligate mutualism. These experimental advances provide a new platform for laboratory studies focusing on the molecular mechanisms and evolutionary processes underlying insect-bacterial symbiosis.

Reflections on a personalized cognitive rehabilitation intervention: Experiences of people living with dementia and their carers participating in the GREAT trial.

Cognitive rehabilitation for people living with early-stage dementia improves functional ability in areas targeted in the therapy, but little is known about how participants experience this intervention. This qualitative paper investigates participants' views about a cognitive rehabilitation intervention in a randomized controlled trial (the GREAT trial) and aims to help explain and interpret the findings and to inform further intervention development. Using in-depth thematic analysis, 43 semi-structured interviews (35 individual and 8 dyadic) were conducted with 25 people living with dementia and 26 family carers from three sites. The person-centred, individualized approach was valued. Some participants' views about dementia were questioned as a consequence of taking part in the therapy; they considered the effectiveness of the intervention in the context of the progressive nature of the condition. Certain participants continued to be doubtful, focussing on the inevitability of decline, rather than the possibility of reablement. Such views may have influenced engagement. The therapeutic relationship played a vital role as it was how personalized care was provided and participants' views had changed positively. Therapists engendered greater confidence and reduced anxiety and social isolation. Positive responses support personalized rehabilitative care to address the specific needs of people living with dementia.

Two-Part Phase 1 Multiple-Ascending-Dose Study to Evaluate the Safety, Tolerability, Pharmacodynamics, and Pharmacokinetics of TRV734 in Healthy Adults.

TRV734, an oral G-protein biased ligand at the µ-opioid receptor has demonstrated differentiated pharmacology in preclinical studies compared to unbiased ligands. First-time-in-human data suggested that TRV734 was safe and well tolerated and caused effective pain relief after single doses of 150 to 250 mg. In this study, safety and tolerability of multiple ascending doses of TRV734, and single doses of TRV734 125 mg following various administration paradigms, in healthy subjects were evaluated. In both parts of the study, TRV734 was generally well tolerated with no serious adverse events. Pharmacokinetics of TRV734 were similar when TRV734 125 mg was administered following a high-fat or standard meal. Compared to either of the fed conditions, maximum concentration and area under the plasma concentration-time curve did not change, and time to maximum concentration was 1.5 hours later when TRV734 125 mg was administered as 3 split portions over 120 minutes under fasted conditions. Split doses of TRV734 delayed time to peak decrease in pupil diameter. Following multiple-dose administration of TRV734 60 to 175 mg every 6 hours, there was a trend of slightly less-than-dose proportional increase of maximum concentration, and area under the plasma concentration-time curve and accumulation was modest. Time to maximum concentration was ≈1 to 2 hours and elimination half-life ≈1.9 to 2.5 hours. The analgesic effect of TRV734 on the cold pain test was generally dose proportional and similar to that of oxycodone 10 mg immediate release, after both the first and last doses. There was a dose-related decrease in pupil diameter following administration of TRV734 up to TRV734 125 mg every 6 hours. A favorable trend in bowel function index for TRV734 warrants continued study.

Professional perspectives on applying the NICE and British Psychological Society Guidelines for the management of Behaviours that Challenge in dementia care: an e-survey.

OBJECTIVES: Behaviours that challenge (BtC) reflect the most costly and burdensome aspects of dementia where non-pharmacological interventions rather than antipsychotic medication have been recommended as first-line approaches for over a decade (NICE 2006). This paper outlines professionals' views about their application of the Dementia NICE Guideline 97 (2018) and a British Psychological Society, Division of Clinical Psychology (BPS-DCP) Briefing paper (2013) on alternatives to antipsychotics. METHODS: A mixed-methods 34-item e-survey, with five items about the use of the NICE Guideline 97 (2018) and the BPS-DCP Briefing paper (2013) for the management of BtC, was conducted. Participants were recruited through multidisciplinary professional dementia networks across the United Kingdom. Quantitative data were descriptively summarized and thematic analysis of open-ended questions undertaken. RESULTS: Two hundred and forty-seven participants completed the questions relating to guidelines. Mean ratings of 'moderately useful' for both the NICE and BPS-DCP guidance were obtained across professions and geographical locations, with the exception of psychiatrists who rated the NICE guidance as 'slightly useful'. The qualitative themes identified were a mix of positive and cautionary perspectives, relating to 'evidence base', the 'accessibility of the guides', 'problems with implementation', and 'lack of detail and clarity'. CONCLUSION: Professionals were cautiously positive regarding the guidance for BtC management, but highlighted a need for improved clarity about the use of non-pharmacological approaches, and more specificity about how these can be implemented in clinical settings. Tailored 'setting-specific' toolkits are required to update and refine the BPS-DCP (2013) if the aspirations of the NICE Dementia Guideline 97 (2018) are to inform professional practice. PRACTITIONER POINTS: Owing to major concerns about the problematic side effects of using psychotropics in the treatment of behaviours that challenge (BtC), there is a need for national guidance on the use on non-drug alternatives. The NICE (2018) guidance was seen by participants as accessible and clear but lacking in detail in the use of non-pharmacological interventions, which are the first-line treatments for BtC. The BPS Guidelines on 'Alternatives to antipsychotics' (2013) were seen as having good structured advice for allocating non-pharmacological resources but were lacking in flexibility for meeting individual needs or what might be an acceptable fit for clinical services. The findings suggest that we need to develop UK-wide bespoke specific advice for practitioners and services for both the use and the delivery of non-pharmacological evidence-based interventions for BtC.

Pharmacodynamics and molecular correlates of response to glofitamab in relapsed/refractory non-Hodgkin lymphoma.

Glofitamab, a novel CD20xCD3, T-cell-engaging bispecific antibody, exhibited single-agent activity in Study NP30179, a first-in-human, phase 1 trial in relapsed/refractory B-cell non-Hodgkin lymphoma. Preclinical studies showed that glofitamab leads to T-cell activation, proliferation, and tumor cell killing upon binding to CD20 on malignant cells. Here, we provide evidence of glofitamab's clinical activity, including pharmacodynamic profile, mode of action, and factors associated with clinical response, by evaluating biomarkers in patient samples from the dose-escalation part of this trial. Patients enrolled in Study NP30179 received single-dose obinutuzumab pretreatment (1000 mg) 7 days before IV glofitamab (5 µg-25 mg). Glofitamab treatment lasted ≤12 cycles once every 2 or 3 weeks. Blood samples were collected at predefined time points per the clinical protocol; T-cell populations were evaluated centrally by flow cytometry, and cytokine profiles were analyzed. Immunohistochemical and genomic biomarker analyses were performed on tumor biopsy samples. Pharmacodynamic modulation was observed with glofitamab treatment, including dose-dependent induction of cytokines, and T-cell margination, proliferation, and activation in peripheral blood. Gene expression analysis of pretreatment tumor biopsy samples indicated that tumor cell intrinsic factors such as TP53 signaling are associated with resistance to glofitamab, but they may also be interlinked with a diminished effector T-cell profile in resistant tumors and thus represent a poor prognostic factor per se. This integrative biomarker data analysis provides clinical evidence regarding glofitamab's mode of action, supports optimal biological dose selection, and will further guide clinical development. This trial was registered at www.clinicaltrials.gov as #NCT03075696.

The experiences of care home staff during the COVID-19 pandemic: A systematic review.

Objectives: To examine experiences of care home staff to better understand how to support them during the ongoing pandemic and in the future.Method: A systematic review examining experiences of care staff over the last year (March 2020-2021).Results: Fourteen papers related to experiences of staff and one was an intervention study. Quantitatively there was evidence of anxiety, PTSD and depression amongst the staff. Qualitatively, seven themes were identified: Poor working conditions; Lack of skills and knowledge; Psychological/Mental health concerns; Feeling undervalued and abandoned; Fears of contagion; Support and the positive impacts of COVID. The intervention study recommended employing needs-based approaches, including educational and wellbeing components.Conclusion: Recommendations are made in terms of how to work with staff, both practically and clinically. There are also suggestions about how to deal with similar situations if they were to reoccur. It is evident that lessons need to be learned because errors were made. Indeed, from a UK perspective, discharging thousands back to care homes, without testing, cost lives. This may have been done to protect the NHS, but it unwittingly 'lockdown' the virus within the care sector.

Generation of a Novel SARS-CoV-2 Sub-genomic RNA Due to the R203K/G204R Variant in Nucleocapsid: Homologous Recombination has Potential to Change SARS-CoV-2 at Both Protein and RNA Level.

BACKGROUND: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host's anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. METHODS: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. CONCLUSIONS: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

Critical evaluation of stents in coronary angioplasty: a systematic review.

BACKGROUND: Coronary stents are routinely placed in the treatment and prophylaxis of coronary artery disease (CAD). Current coronary stent designs are prone to developing blockages: in-stent thrombosis (IST) and in-stent re-stenosis (ISR). This is a systematic review of the design of current coronary stent models, their structural properties and their modes of application, with a focus on their associated risks of IST and ISR. The primary aim of this review is to identify the best stent design features for reducing the risk of IST and ISR. To review the three major types of stents used in clinical settings today, determining best and relevant clinical practice by exploring which types and features of offer improved patient outcomes regarding coronary angioplasty. This information can potentially be used to increase the success rate of coronary angioplasty and stent technology in the future taking into account costs and benefits. METHODS: Scientific databases were searched to find studies concerning stents. After the exclusion criteria were applied, 19 of the 3192 searched literature were included in this review. Studies investigating three major types of stent design were found: bare-metal stents (BMS), drug-eluting stents (DES) and bioresorbable stents (BRS). The number of participants varied between 14 and 1264. On average 77.4% were male, with a mean age of 64 years. RESULTS: From the findings of these studies, it is clear that DES are superior in reducing the risk of ISR when compared to BMS. Conflicting results do not clarify whether BRS are superior to DES at reducing IST occurrence, although studies into newer BRS technologies show reducing events of IST to 0, creating a promising future for BRS showing them to be non-inferior. Thinner stents were shown to reduce IST rates, due to better re-endothelialisation. Scaffold material has also been shown to play a role with cobalt alloy stents reducing the risk of IST. This study found that thinner stents that release drugs were better at preventing re-blockages. Some dissolvable stents might be better at stopping blood clots blocking the arteries when compared to metal stents. The method and procedure of implanting the stent during coronary angioplasty influences success rate of these stents, meaning stent design is not the only significant factor to consider. CONCLUSIONS: Positive developments in coronary angioplasty could be made by designing new stents that encompass all the most desirable properties of existing stent technology. Further work is needed to investigate the benefits of BRS in reducing the risk of IST compared to DES, as well as to investigate the effects of different scaffold materials on IST and ISR outcomes.

How to improve healthcare for autistic people: A qualitative study of the views of autistic people and clinicians.

LAY ABSTRACT: Research has shown that on average, autistic people are more likely to die earlier than non-autistic people, and barriers can stop autistic people accessing healthcare. We carried out a study where we interviewed healthcare professionals (including doctors and nurses), and held discussion groups of autistic people. Our results highlighted several key points: seeing the same professional is important for autistic people and clinicians; both clinicians and autistic people think making adjustments to healthcare is important (and often possible); autistic people process information in a different way and so may need extra support in appointments; and that clinicians are often constrained by time pressures or targets.

Novel STMN2 Variant Linked to Amyotrophic Lateral Sclerosis Risk and Clinical Phenotype.

OBJECTIVE: There is a critical need to establish genetic markers that explain the complex phenotypes and pathogenicity of ALS. This study identified a polymorphism in the Stathmin-2 gene and investigated its association with sporadic ALS (sALS) disease risk, age-of onset and survival duration. METHODS: The candidate CA repeat was systematically analyzed using PCR, Sanger sequencing and high throughput capillary separation for genotyping. Stathmin-2 expression was investigated using RT-PCR in patient olfactory neurosphere-derived (ONS) cells and RNA sequencing in laser-captured spinal motor neurons. RESULTS: In a case-control analysis of a combined North American sALS cohort (n = 321) and population control group (n = 332), long/long CA genotypes were significantly associated with disease risk (p = 0.042), and most strongly when one allele was a 24 CA repeat (p = 0.0023). In addition, longer CA allele length was associated with earlier age-of-onset (p = 0.039), and shorter survival duration in bulbar-onset cases (p = 0.006). In an Australian longitudinal sALS cohort (n = 67), ALS functional rating scale scores were significantly lower in carriers of the long/long genotype (p = 0.034). Stathmin-2 mRNA expression was reduced in sporadic patient ONS cells. Additionally, sALS patients and controls exhibited variable expression of Stathmin-2 mRNA according to CA genotype in laser-captured spinal motor neurons. CONCLUSIONS: We report a novel non-coding CA repeat in Stathmin-2 which is associated with sALS disease risk and has disease modifying effects. The potential value of this variant as a disease marker and tool for cohort enrichment in clinical trials warrants further investigation.

Generation of a novel SARS-CoV-2 sub-genomic RNA due to the R203K/G204R variant in nucleocapsid: homologous recombination has potential to change SARS-CoV-2 at both protein and RNA level.

BACKGROUND: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over-time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. METHODS: Deep sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. RESULTS: Sequence analysis suggests that the three adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence (CS) of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. CONCLUSIONS: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans resulting in both coding changes and novel sub-genomic RNA transcripts suggests this as a mechanism for diversification and adaptation within its new host.

Estradiol-Responsive miR-365a-3p Interacts with Tissue Factor 3'UTR to Modulate Tissue Factor-Initiated Thrombin Generation.

BACKGROUND: High estradiol (E2) levels are linked to an increased risk of venous thromboembolism; however, the underlying molecular mechanism(s) remain poorly understood. We previously identified an E2-responsive microRNA (miR), miR-494-3p, that downregulates protein S expression, and posited additional coagulation factors, such as tissue factor, may be regulated in a similar manner via miRs. OBJECTIVES: To evaluate the coagulation capacity of cohorts with high physiological E2, and to further characterize novel E2-responsive miR and miR regulation on tissue factor in E2-related hypercoagulability. METHODS: Ceveron Alpha thrombin generation assay (TGA) was used to assess plasma coagulation profile of three cohorts. The effect of physiological levels of E2, 10 nM, on miR expression in HuH-7 cells was compared using NanoString nCounter and validated with independent assays. The effect of tissue factor-interacting miR was confirmed by dual-luciferase reporter assays, immunoblotting, flow cytometry, biochemistry assays, and TGA. RESULTS: Plasma samples from pregnant women and women on the contraceptive pill were confirmed to be hypercoagulable (compared with sex-matched controls). At equivalent and high physiological levels of E2, miR-365a-3p displayed concordant E2 downregulation in two independent miR quantification platforms, and tissue factor protein was upregulated by E2 treatment. Direct interaction between miR-365a-3p and F3-3'UTR was confirmed and overexpression of miR-365a-3p led to a decrease of (1) tissue factor mRNA transcripts, (2) protein levels, (3) activity, and (4) tissue factor-initiated thrombin generation. CONCLUSION: miR-365a-3p is a novel tissue factor regulator. High E2 concentrations induce a hypercoagulable state via a miR network specific for coagulation factors.