Digital home monitoring for capturing daily fluctuation of symptoms; a longitudinal repeated measures study: Long Covid Multi-disciplinary Consortium to Optimise Treatments and Services across the NHS (a LOCOMOTION study).

INTRODUCTION: A substantial proportion of COVID-19 survivors continue to have symptoms more than 3 months after infection, especially of those who required medical intervention. Lasting symptoms are wide-ranging, and presentation varies between individuals and fluctuates within an individual. Improved understanding of undulation in symptoms and triggers may improve efficacy of healthcare providers and enable individuals to better self-manage their Long Covid. We present a protocol where we aim to develop and examine the feasibility and usability of digital home monitoring for capturing daily fluctuation of symptoms in individuals with Long Covid and provide data to facilitate a personalised approach to the classification and management of Long Covid symptoms. METHODS AND ANALYSIS: This study is a longitudinal prospective cohort study of adults with Long Covid accessing 10 National Health Service (NHS) rehabilitation services in the UK. We aim to recruit 400 people from participating NHS sites. At referral to study, 6 weeks and 12 weeks, participants will complete demographic data (referral to study) and clinical outcome measures, including ecological momentary assessment (EMA) using personal mobile devices. EMA items are adapted from the COVID-19 Yorkshire Rehabilitation Scale items and include self-reported activities, symptoms and psychological factors. Passive activity data will be collected through wrist-worn sensors. We will use latent class growth models to identify trajectories of experience, potential phenotypes defined by co-occurrence of symptoms and inter-relationships between stressors, symptoms and participation in daily activities. We anticipate that n=300 participants provide 80% power to detect a 20% improvement in fatigue over 12 weeks in one class of patients relative to another. ETHICS AND DISSEMINATION: The study was approved by the Yorkshire & The Humber-Bradford Leeds Research Ethics Committee (ref: 21/YH/0276). Findings will be disseminated in peer-reviewed publications and presented at conferences. TRIAL REGISTRATION NUMBER: ISRCTN15022307.

Hospital-based preventative interventions for people experiencing homelessness in high-income countries: A systematic review.

Background: People experiencing homelessness have significant unmet needs and high rates of unplanned care. We aimed to describe preventative interventions, defined in their broadest sense, for people experiencing homelessness in a hospital context. Secondary aims included mapping outcomes and assessing intervention effectiveness. Methods: We searched online databases (MEDLINE, Embase, PsycINFO, HMIC, CINAHL, Web of Science, Cochrane Library) from 1999-2019 and conducted backward and forward citation searches to 31 December 2020 (PROSPERO CRD42019154036). We included quantitative studies in emergency and inpatient settings measuring health or social outcomes for adults experiencing homelessness in high income countries. We assessed rigour using the "Quality Assessment Tool for Quantitative Studies" and summarised findings using descriptive quantitative methods, a binomial test, a Harvest Plot, and narrative synthesis. We used PRISMA and SWiM reporting guidelines. Findings: Twenty-eight studies identified eight intervention types: care coordination (n=18); advocacy, support, and outreach (n=13); social welfare assistance (n=13); discharge planning (n=12); homelessness identification (n=6); psychological therapy and treatment (n=6); infectious disease prevention (n=5); and screening, treatment, and referrals (n=5). The evidence strength was weak (n=16) to moderate (n=10), with two high quality randomised controlled trials. We identified six outcome categories with potential benefits observed for psychosocial outcomes, including housing (11/13 studies, 95%CI=54.6-98.1%, p=0.023), healthcare use (14/17, 56.6-96.2%, p=0.013), and healthcare costs (8/8, 63.1-100%, p=0.008). Benefits were less likely for health outcomes (4/5, 28.3-99.5%, p=0.375), integration with onward care (2/4, 6.8-93.2%, p=1.000), and feasibility/acceptability (5/6, 35.9-99.6%, p=0.219), but confidence intervals were very wide. We observed no harms. Most studies showing potential benefits were multi-component interventions. Interpretation: Hospital-based preventative interventions for people experiencing homelessness are potentially beneficial, but more rigorous research is needed. In the context of high needs and extreme inequities, policymakers and healthcare providers may consider implementing multi-component preventative interventions. Funding: SL is supported by an NIHR Clinical Doctoral Research Fellowship (ICA-CDRF-2016-02-042). JD is supported by an NIHR School of Public Health Research Pre-doctoral Fellowship (NU-004252). RWA is supported by a Wellcome Clinical Research Career Development Fellowship (206602).

Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas.

Cancer genome sequencing has identified dozens of mutations with a putative role in lymphomagenesis and leukemogenesis. Validation of driver mutations responsible for B cell neoplasms is complicated by the volume of mutations worthy of investigation and by the complex ways that multiple mutations arising from different stages of B cell development can cooperate. Forward and reverse genetic strategies in mice can provide complementary validation of human driver genes and in some cases comparative genomics of these models with human tumors has directed the identification of new drivers in human malignancies. We review a collection of forward genetic screens performed using insertional mutagenesis, chemical mutagenesis and exome sequencing and discuss how the high coverage of subclonal mutations in insertional mutagenesis screens can identify cooperating mutations at rates not possible using human tumor genomes. We also compare a set of independently conducted screens from Pax5 mutant mice that converge upon a common set of mutations observed in human acute lymphoblastic leukemia (ALL). We also discuss reverse genetic models and screens that use CRISPR-Cas, ORFs and shRNAs to provide high throughput in vivo proof of oncogenic function, with an emphasis on models using adoptive transfer of ex vivo cultured cells. Finally, we summarize mouse models that offer temporal regulation of candidate genes in an in vivo setting to demonstrate the potential of their encoded proteins as therapeutic targets.

Dual-task walking and automaticity after Stroke: Insights from a secondary analysis and imaging sub-study of a randomised controlled trial.

OBJECTIVE: To test the extent to which initial walking speed influences dual-task performance after walking intervention, hypothesising that slow walking speed affects automatic gait control, limiting executive resource availability. DESIGN: A secondary analysis of a trial of dual-task (DT) and single-task (ST) walking interventions comparing those with good (walking speed ⩾0.8 m s-1, n = 21) and limited (walking speed <0.79 m s-1, n = 24) capacity at baseline. SETTING: Community. SUBJECTS: Adults six-months post stroke with walking impairment. INTERVENTIONS: Twenty sessions of 30 minutes treadmill walking over 10 weeks with (DT) or without (ST) cognitive distraction. Good and limited groups were formed regardless of intervention received. MAIN MEASURES: A two-minute walk with (DT) and without (ST) a cognitive distraction assessed walking. fNIRS measured prefrontal cortex activation during treadmill walking with (DT) and without (ST) Stroop and planning tasks and an fMRI sub-study used ankle-dorsiflexion to simulate walking. RESULTS: ST walking improved in both groups (∆baseline: Good = 8.9 ± 13.4 m, limited = 5.3±8.9 m, Group × time = P < 0.151) but only the good walkers improved DT walking (∆baseline: Good = 10.4 ± 13.9 m, limited = 1.3 ± 7.7 m, Group × time = P < 0.025). fNIRS indicated increased ispilesional prefrontal cortex activation during DT walking following intervention (P = 0.021). fMRI revealed greater DT cost activation for limited walkers, and increased resting state connectivity of contralesional M1 with cortical areas associated with conscious gait control at baseline. After the intervention, resting state connectivity between ipsilesional M1 and bilateral superior parietal lobe, involved in integrating sensory and motor signals, increased in the good walkers compared with limited walkers. CONCLUSION: In individual who walk slowly it may be difficult to improve dual-task walking ability.Registration: ISRCTN50586966.

LUMI-PCR: an Illumina platform ligation-mediated PCR protocol for integration site cloning, provides molecular quantitation of integration sites.

BACKGROUND: Ligation-mediated PCR protocols have diverse uses including the identification of integration sites of insertional mutagens, integrating vectors and naturally occurring mobile genetic elements. For approaches that employ NGS sequencing, the relative abundance of integrations within a complex mixture is typically determined through the use of read counts or unique fragment lengths from a ligation of sheared DNA; however, these estimates may be skewed by PCR amplification biases and saturation of sequencing coverage. RESULTS: Here we describe a modification of our previous splinkerette based ligation-mediated PCR using a novel Illumina-compatible adapter design that prevents amplification of non-target DNA and incorporates unique molecular identifiers. This design reduces the number of PCR cycles required and improves relative quantitation of integration abundance for saturating sequencing coverage. By inverting the forked adapter strands from a standard orientation, the integration-genome junction can be sequenced without affecting the sequence diversity required for cluster generation on the flow cell. Replicate libraries of murine leukemia virus-infected spleen samples yielded highly reproducible quantitation of clonal integrations as well as a deep coverage of subclonal integrations. A dilution series of DNAs bearing integrations of MuLV or piggyBac transposon shows linearity of the quantitation over a range of concentrations. CONCLUSIONS: Merging ligation and library generation steps can reduce total PCR amplification cycles without sacrificing coverage or fidelity. The protocol is robust enough for use in a 96 well format using an automated liquid handler and we include programs for use of a Beckman Biomek liquid handling workstation. We also include an informatics pipeline that maps reads, builds integration contigs and quantitates integration abundance using both fragment lengths and unique molecular identifiers. Suggestions for optimizing the protocol to other target DNA sequences are included. The reproducible distinction of clonal and subclonal integration sites from each other allows for analysis of populations of cells undergoing selection, such as those found in insertional mutagenesis screens.

Comparing the Effectiveness of Blood Flow Restriction and Traditional Heavy Load Resistance Training in the Post-Surgery Rehabilitation of Anterior Cruciate Ligament Reconstruction Patients: A UK National Health Service Randomised Controlled Trial.

BACKGROUND: We implemented a blood flow restriction resistance training (BFR-RT) intervention during an 8-week rehabilitation programme in anterior cruciate ligament reconstruction (ACLR) patients within a National Health Service setting. OBJECTIVE: To compare the effectiveness of BFR-RT and standard-care traditional heavy-load resistance training (HL-RT) at improving skeletal muscle hypertrophy and strength, physical function, pain and effusion in ACLR patients following surgery. METHODS: 28 patients scheduled for unilateral ACLR surgery with hamstring autograft were recruited for this parallel-group, two-arm, single-assessor blinded, randomised clinical trial following appropriate power analysis. Following surgery, a criteria-driven approach to rehabilitation was utilised and participants were block randomised to either HL-RT at 70% repetition maximum (1RM) (n = 14) or BFR-RT (n = 14) at 30% 1RM. Participants completed 8 weeks of biweekly unilateral leg press training on both limbs, totalling 16 sessions, alongside standard hospital rehabilitation. Resistance exercise protocols were designed consistent with standard recommended protocols for each type of exercise. Scaled maximal isotonic strength (10RM), muscle morphology of the vastus lateralis of the injured limb, self-reported function, Y-balance test performance and knee joint pain, effusion and range of motion (ROM) were assessed at pre-surgery, post-surgery, mid-training and post-training. Knee joint laxity and scaled maximal isokinetic knee extension and flexion strength at 60°/s, 150°/s and 300°/s were measured at pre-surgery and post-training. RESULTS: Four participants were lost, with 24 participants completing the study (12 per group). There were no adverse events or differences between groups for any baseline anthropometric variable or pre- to post-surgery change in any outcome measure. Scaled 10RM strength significantly increased in the injured limb (104 ± 30% and 106 ± 43%) and non-injured limb (33 ± 13% and 39 ± 17%) with BFR-RT and HL-RT, respectively, with no group differences. Significant increases in knee extension and flexion peak torque were observed at all speeds in the non-injured limb with no group differences. Significantly greater attenuation of knee extensor peak torque loss at 150°/s and 300°/s and knee flexor torque loss at all speeds was observed with BFR-RT. No group differences in knee extensor peak torque loss were found at 60°/s. Significant and comparable increases in muscle thickness (5.8 ± 0.2% and 6.7 ± 0.3%) and pennation angle (4.1 ± 0.3% and 3.4 ± 0.1%) were observed with BFR-RT and HL-RT, respectively, with no group differences. No significant changes in fascicle length were observed. Significantly greater and clinically important increases in several measures of self-reported function (50-218 ± 48% vs. 35-152 ± 56%), Y-balance performance (18-59 ± 22% vs. 18-33 ± 19%), ROM (78 ± 22% vs. 48 ± 13%) and reductions in knee joint pain (67 ± 15% vs. 39 ± 12%) and effusion (6 ± 2% vs. 2 ± 2%) were observed with BFR-RT compared to HL-RT, respectively. CONCLUSION: BFR-RT can improve skeletal muscle hypertrophy and strength to a similar extent to HL-RT with a greater reduction in knee joint pain and effusion, leading to greater overall improvements in physical function. Therefore, BFR-RT may be more appropriate for early rehabilitation in ACLR patient populations within the National Health Service.

Examination of the comfort and pain experienced with blood flow restriction training during post-surgery rehabilitation of anterior cruciate ligament reconstruction patients: A UK National Health Service trial.

OBJECTIVE: Examine the comfort and pain experienced with blow flow restriction resistance training (BFR-RT) compared to standard care heavy load resistance training (HL-RT) during anterior cruciate ligament reconstruction (ACLR) patient rehabilitation. DESIGN: Randomised controlled trial. SETTING: United Kingdom National Health Service. PARTICIPANTS: Twenty eight patients undergoing unilateral ACLR surgery with hamstring autograft were recruited. Following surgery participants were block randomised to either HL-RT at 70% repetition maximum (1RM) (n = 14) or BFR-RT (n = 14) at 30% 1RM and completed 8 weeks of twice weekly unilateral leg press training on both limbs. MAIN OUTCOME MEASURES: Perceived knee pain, muscle pain and rating of perceived exertion (RPE) were assessed using Borg's (1998) RPE and pain scales during training. Knee pain was also assessed 24 h post-training. RESULTS: There were no adverse events. Knee pain was lower with BFR-RT during (p < 0.05) and at 24 h post-training (p < 0.05) with BFR-RT for all sessions. Muscle pain was higher (p < 0.05) with BFR-RT compared to HL-RT during all sessions. RPE remained unchanged (p > 0.05) for both BFR-RT and HL-RT. CONCLUSION: ACLR patients experienced less knee joint pain and reported similar ratings of perceived exertion during and following leg press exercise with BFR-RT compared to traditional HL-RT. BFR-RT may be more advantageous during the early phases of post-surgery ACLR rehabilitation.

Author Correction: Subclonal mutation selection in mouse lymphomagenesis identifies known cancer loci and suggests novel candidates.

The original version of this Article contained an error in the hyperlink for the online repository http://mulvdb.org which was incorrectly given as http://mulv.lms.mrc.ac.uk. This has been corrected in both the PDF and HTML versions of the Article.

Subclonal mutation selection in mouse lymphomagenesis identifies known cancer loci and suggests novel candidates.

Determining whether recurrent but rare cancer mutations are bona fide driver mutations remains a bottleneck in cancer research. Here we present the most comprehensive analysis of murine leukemia virus-driven lymphomagenesis produced to date, sequencing 700,000 mutations from >500 malignancies collected at time points throughout tumor development. This scale of data allows novel statistical approaches for identifying selected mutations and yields a high-resolution, genome-wide map of the selective forces surrounding cancer gene loci. We also demonstrate negative selection of mutations that may be deleterious to tumor development indicating novel avenues for therapy. Screening of two BCL2 transgenic models confirmed known drivers of human non-Hodgkin lymphoma, and implicates novel candidates including modifiers of immunosurveillance and MHC loci. Correlating mutations with genotypic and phenotypic features independently of local variance in mutation density also provides support for weakly evidenced cancer genes. An online resource http://mulv.lms.mrc.ac.uk allows customized queries of the entire dataset.