SUMMARY Homology-directed repair deficiency (HRd) encompasses mutations in multiple genes yet is treated clinically as a single entity. Here, through parallel analyses of isogenic knockouts of multiple HR pathway genes, integrating multi-omic analyses with genome-wide CRISPR-Cas9-dependency and resistance screens, we show that HRd is not a single entity but exists along a molecular and functional continuum. BRCA1, BRCA2, PALB2, RAD51C, and RAD51D mutants shared many HRd-associated mutational signatures, while RAD51B, BRIP1 , CDK12 exhibited distinct genomic patterns. Functional heterogeneity was equally apparent: synthetic lethal interactions including CIP2A and a novel dependency on PRDX1 were penetrant across most HRd genotypes, whereas FANCM dependency was linked to HRd subtypes characterized by tandem duplications. PARPi resistance screens in distinct HRd contexts uncovered BRIP1 and RECQL5 as new BRCA2 -specific resistance genes. HRd is thus a complex continuum, underscoring why modernizing the molecular taxonomy utilizing all genomic features available per patient is crucial to informing precision interventions.

Ductal Carcinoma In Situ (DCIS) is a non-obligate precursor of invasive breast cancer. Due to a lack of reliable prognostic markers, nearly all women with DCIS undergo intensive treatment-often unnecessarily. The LORD trial addresses this by offering active surveillance to women with screen-detected, ER-positive, HER2-negative, grade 1 or 2 DCIS, aiming to reduce overtreatment. To support this, we developed a deep learning pipeline based on foundation models to predict grade, ER, and HER2 status directly from H&E-stained digitized pathology slides. Models were trained and tested on a Dutch multicenter dataset (n = 887) and externally validated on a UK dataset (n = 259). On the Dutch data, the models achieved mean AUROCs of 0.90 (ER), 0.84 (HER2), and 0.86 (grade); external validation yielded 0.80, 0.74, and 0.75, respectively. Using these outputs, we stratified patients according to active surveillance criteria, reaching balanced accuracies of 0.81 (Dutch) and 0.64 (UK), with corresponding NPVs of 0.86 and 0.76. Our models generalize across cohorts and reliably predict key biomarkers, supporting the identification of DCIS patients eligible for less aggressive management.

Pain and fatigue are common but poorly understood features of post-COVID Syndrome (PCS). To probe the mechanistic basis of these symptoms, we investigated sensory functions in patients with widespread pain attributed to PCS. Quantitative sensory testing revealed increased mechanical pain sensitivity and altered thermal sensitivities and microneurography demonstrated that patients with pain displayed abnormal spontaneous C-fibre activity. Administration of IgG from PCS patients with pain and fatigue (PCS-PF) to mice, conferred mechanical and cold hypersensitivities and reduced intra-epidermal nerve fibre density (IENFd). IgG from patients with fatigue but without pain (PCS-F) did not induce hypersensitivities but similarly reduced IENFd. In line with behavioural responses, sensory nerves from PCS-PF IgG treated mice showed increased responsiveness to mechanical and cold stimulation. PCS-PF IgG bound to isolated sensory neurons with staining intensities that correlated with the level of pain experienced by patients with PCS. These results indicate a causal role for autoantibodies in the pathogenesis of pain and sensory disturbances associated with PCS.

Objective: To identify associations between the occurrence of sacrocaudal fusion and the potential morphology of certain hind limb bones in actively racing greyhounds. Methods: The calcaneus, talus and patella from each hind limb were collected from 94 male and 77 female mature greyhound cadavers and grouped into four groups; right or left bones from greyhounds with a standard or fused sacrum. The measurements were recorded for the following parameters: body mass of the greyhound, mass, length, and width of the right and left calcanei, tali and patellae. Results: A fused sacrum (4 sacral vertebrae) was present in 41% of specimens. The right and left calcanei, tali and patellae in greyhounds with a standard or fused sacrum were anatomically similar. Overall, left to right asymmetry was found, in the width of calcaneus (P < 0.01) and the talus (P < 0.05) and the length of calcaneus (P < 0.001) all these being larger in bones from the left hind limbs. Comparing bones from dogs with a fused or unfused sacrum showed that the right calcaneus length (P < 0.05) was significantly less than the left in those greyhounds with standard sacrum; the right calcaneus width was significantly less (P < 0.01) than the left in those with a fused sacrum. There were no significant differences in the means of measurements of bones between greyhounds with a standard and those with a fused sacrum except for the mass of the right (95% CI 0.22 to 1.10, P < 0.01) and left (95% CI 0.18 to 1.04, P < 0.01) calcaneus which were heavier in greyhounds with a fused sacrum than those with a standard sacrum. Conclusion: In a population of greyhounds that race on anticlockwise tracks, the left calcaneus was wider and longer than the right and the left talus was wider. This asymmetry was more significant in dogs with sacrocaudal fusion and those dogs had more massive calcanei than dogs with standard sacrums, suggesting a difference in the way these bones were loaded in dogs with sacrocaudal fusion compared to dogs with the standard sacral anatomy.

Background CYLD cutaneous syndrome (CCS) is caused by germline heterozygous pathogenic variants in CYLD and results in the progressive formation of cylindromas, spiradenomas or trichoepitheliomas. Malignant cylindroma is a rare skin adnexal tumour occurring in CCS, which can metastasize with lethal outcomes and has limited genomic characterization. BRCA2 loss in CCS is not described and may modulate the cutaneous cancer risk of CCS. Objectives To establish whether BRCA deficiency drives metastatic malignant cylindroma and to report the phenotype of three siblings with digenic inheritance of CYLD and BRCA2 pathogenic variants (PVs), one of whom developed metastatic cylindroma at 28 years old. Methods A kindred study reporting seven members of a family with CCS was conducted in a tertiary hospital setting within the United Kingdom from April 2021 to February 2023. Clinical phenotype, pathological, radiological and genetic findings and treatment data were collected. Whole-genome sequencing of the primary malignant cylindroma occurring in one patient was performed to identify targetable driver mutations and signatures. Results Malignant cylindroma arose in one (proband) of the two male siblings with digenic inheritance of BRCA2 (c.5158insT) and CYLD (c.2689-2A>G) pathogenic variants. A further female sibling with digenic inheritance of the same BRCA2 and CYLD PVs developed early breast cancer. Whole-genome sequencing of the primary malignant cylindroma in the affected patient showed loss of heterozygosity of both BRCA2 and CYLD. Bioinformatic analysis confirmed homologous repair deficiency (HRD). These data supported the use of the PARP [poly(ADP-ribose) polymerase] inhibitor rucaparib to target HRD in a non-canonical BRCA-deficient skin cancer. Conclusions Digenic inheritance of pathogenic variants in cancer-predisposing genes should prompt clinicians to be vigilant for atypical malignant presentations. We demonstrate that rapid whole-genome sequencing can inform the treatment of metastatic malignant cylindroma and identify novel systemic therapies.

Background Changes in the third metacarpal (MC3) mid-diaphyseal dimensions were measured radiographically in young Thoroughbred racehorses through race training and found to be associated with a higher risk of shin soreness. However, it is still unclear whether such negative MC3 morphologies are set congenitally or through development. Objective To quantify MC3 bone dimensional changes through growth. Study Design Longitudinal. Method Twenty privately owned Thoroughbred foals were radiographed bimonthly from birth to 14 months of age. Four linear MC3 parameters (proximodistal MC3 length, mid-diaphyseal dorsal and palmar cortical bone thickness, and dorsopalmar medullary width) were measured using ImageJ on lateromedial radiographs. Associations with age (growth), MC3 length, differences between left and right limbs, and between sexes were analysed separately using a linear mixed effects model. Results The dorsal cortical bone thickness showed a positive linear relationship with growth (0.4 +/- 0.02 mm per month, p < 0.001, 95% CI [0.37, 0.44]) while the medullary width did not change. The relative thickness of the dorsal cortical bone thickness compared with the medullary cavity width and palmar cortical bone thickness also increased through growth (0.2 +/- 0.01 mm per month, p < 0.001, 95% CI [0.14, 0.19]). The dorsal cortical bone thickness was positively associated with MC3 length (0.07 +/- 0.0001 mm per mm increase in MC3 length, p < 0.001, 95% CI [0.05, 0.08]) and the relative thickness of the dorsal cortical bone thickness (RI) to the rest of the diaphyseal parameters (0.02 +/- 0.01 per mm increase in MC3 length, p = 0.01, 95% CI [0.01, 0.03]). Main Limitations A limited number of foals were included with similar breeding. Conclusion The mid-diaphyseal dorsal cortical bone thickness and proportions could be measured to monitor musculoskeletal development and identify foals at increased risk of shin soreness or those that are immature or otherwise need more time before the commencement of fast training.

ABSTRACT The morphology of the superficial fascia in the canine hindlimb is still poorly understood and incompletely described. The present study aimed to elucidate the macroscopic and microscopic structures of the superficial fascia, thereby clarifying its functional role. Cadavers were investigated for anatomic description ( N = 38), ultrasonic scanning ( N = 2), and histological analyses ( N = 10) of this tissue in the hindlimb. The superficial fascia was identified as a shiny, white fibroelastic layer that adhered to the skin through fibrous septa. It was organized into sublayers enveloping the cutaneous muscle and large blood vessels. In certain areas, superficial fascia fused with the deep fascia or skin, creating fascial bursae. These bursae included the ischiatic bursa, an iliac bursa, a prepatellar subfascial bursa, a prepatellar subcutaneous bursa, and the tarsal fascial bursa. Microscopically, the superficial fascia presented as a layer of dense connective tissue characterized by irregularly arranged collagen and elastic fibers. The superficial fascia was firmly attached to the skin and deep fascia by numerous fibrous tissue strands. Within both, the superficial fascia and fascial bursae, several mechanoreceptors and nerve endings were identified, including Ruffini's corpuscles, Pacinian corpuscles, and Golgi‐Mazzoni corpuscles. The organization of the superficial fascia and its attachments suggest a mechanical role in supporting structures and resisting loads during movement. The fibrous septa anchors fascia to the skin, providing stability and resistance against external forces, as well as protecting the nerves and blood vessels that pass towards the skin. Existing fascial bursae probably assist in decreasing pressure and facilitating freedom of movement adjacent to bony prominences. Elasticity and connectivity of the superficial fascia may explain the various responses to multidirectional loading. Furthermore, the presence of free nerve endings and mechanoreceptors within the fascia suggests that it may contribute to proprioception of the hindlimb, enhancing the awareness of body movement.

BackgroundThe risk of carpal injury in racehorses may be related to the morphology, yet whether carpal morphologies are set from birth or change through growth remains unclear.ObjectiveTo quantify carpal bone changes through growth.MethodTwenty privately owned Thoroughbred foals born between January 2022 and May 2023 were radiographed bimonthly from birth to 10 months of age. Imprint training was used to take radiographs safely without chemical restraints. Fifteen individual and 11 relative angular carpal parameters were measured using ImageJ on dorsopalmar radiographs of the carpus at zero degrees of vertical and horizontal rotation. Associations with age (growth), sex and the differences between left and right limbs were analysed separately using a linear mixed effects model.ResultsSix individual carpal parameters changed with age (radial carpal joint [RCJ], Prx.dor. radial carpal [Cr], Prx.Cu, Dis.dor. third carpal [C3], Dis.pal.C3 and Dis.pal. intermediate carpal), and one was influenced by side, that is higher in the left carpus (Dis.pal.Cr). Seven relative parameters changed with age, and one relative parameter was influenced by side, that is higher in the left (Ra.met-RCJ). The proximo-dorsal bone surface angle of Cr and disto-dorsal bone surface angle of C3 became flatter over time, which may be associated with the re-direction of the load towards the sagittal carpal plane. Sex did not influence any of the carpal parameters, nor did the combined effect of age, side of the limb and sex.ConclusionSpecific individual and relative angular carpal parameters changed significantly over time and some differed between the left and right limb, whereas other parameters did not change. The steeper carpal bone angles achieved proximally with the parameters that did change may improve stability by redirecting the load more medially through the carpus and the proximal and distal bones. This report describes the change in specific radiographic carpal parameters in Thoroughbred foals through the first 10 months of growth. Several carpal parameters changed significantly over time, whereas variations in two parameters were explained by side (left or right). The steeper carpal bone angles achieved proximally together with the parameters that remained constant may act to improve stability by redirecting the load more medially through the carpus and to the proximal and distal bones. image

Outcomes following SARS-CoV-2 infection are variable; whilst the majority of patients recover without serious complications, a subset of patients develop prolonged illness termed Long COVID or post-acute sequelae of SARS-CoV-2 infection (PASC). The pathophysiology underlying Long COVID remains unclear but appears to involve multiple mechanisms including persistent inflammation, coagulopathy, autoimmunity, and organ damage. Studies suggest that microclots, also known as fibrinaloids, play a role in Long COVID. In this context, we developed a method to quantify microclots and investigated the relationship between microclot counts and Long COVID. We show that as a cohort, platelet-poor plasma from Long COVID samples had a higher microclot count compared to control groups but retained a wide distribution of counts. Recent COVID-19 infections were also seen to be associated with microclot counts higher than the control groups and equivalent to the Long COVID cohort, with a subsequent time-dependent reduction of counts. Our findings suggest that microclots could be a potential biomarker of disease and/or a treatment target in some Long COVID patients. ### Competing Interest Statement AB is an employee of Agilent; and supported the design of the Cytation protocols using blinded data. The other authors have no competing interests to declare.   ### Funding Statement Funding was provided by the Patient-Led Research Fund, a project of Balvi Filantropic Fund (Balvi) and Patient-Led Research Collaborative (PLRC) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval for the study was obtained from the ethics committees of Sheffield Hallam University (reference number E39973246), and the North West Research Ethics Committee - Preston (reference number 10/H1016/25). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England's 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an "FFPEImpact" score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many. Formalin fixation is commonly used in tissue storage; however, this process has traditionally limited downstream whole genome sequencing usage. Here, the authors identify artefactual signatures in FFPE-derived sequencing data and demonstrate the preservation of clinical utility, thus enabling FFPE whole genome sequencing when required.

يعد الطب الإشعاعي أداة تشخيصية مهمة ذات تدخل جراحي بسيط تستخدم لتحديد الحالات المرضية وعلاجها. تم تطوير المعلمات الشعاعية للمفصل السنعي السلامي (MCPJ) لتقييم تشكيل MCPJ للخيول، ولكن لم يتم الإبلاغ عن تأثير زاوية MCPJ على مقاييس التصوير الشعاعي هذه. هدفت هذه التجربة إلى دراسة تأثير زاوية MCPJ على 27 معلمة (12 زاوية و 15 نسبة) لـ MCPJ وأيضاً تحديد المدى المقبول الذي حدث عنده الحد الأدنى من التغييرات في تلك المعلمات. تم جمع ستة أطراف أمامية من ستة خيول مختلفة مع عدم وجود تشوهات MCPJ أو أمراض مرئية. تم وضع كل MCPJ عموديًا وتصويرًا إشعاعيًا رقميًا ظهرانيًا عند سبع زوايا MCPJ مختلفة ضمن النطاق الطبيعي وبفواصل زمنية قدرها 5.5 درجة. تم تحقيق زوايا MCPJ من خلال تطبيق أحمال مختلفة باستخدام خلية تحميل. تم قياس جميع المعلمات على الصور الشعاعية الـ 42 باستخدام برنامج EponaTech Metron. تم تقدير الاختلافات لكل تغيير قدره 5.5 درجة في زاوية MCPJ لجميع المعلمات. كانت معلمات النسبة بشكل عام أقل تأثراً من المعلمات الزاوية. كان مقدار التغيير صغيرًا في غالبية المعلمات الزاوية وصغيرًا جدًا في جميع معلمات النسبة. تعتبر زوايا MCPJ التي تتراوح بين 146 درجة و157 درجة نطاقات مقبولة لجميع هذه المعلمات.

Orthostatic intolerance (OI), including postural orthostatic tachycardia syndrome (PoTS) and orthostatic hypotension (OH), are often reported in long covid, but published studies are small with inconsistent results. We sought to estimate the prevalence of objective OI in patients attending long covid clinics and healthy volunteers and associations with OI symptoms and comorbidities. Participants with a diagnosis of long covid were recruited from eight UK long covid clinics, and healthy volunteers from general population. All undertook standardized National Aeronautics and Space Administration Lean Test (NLT). Participants' history of typical OI symptoms (e.g., dizziness, palpitations) before and during the NLT were recorded. Two hundred seventy-seven long covid patients and 50 frequency-matched healthy volunteers were tested. Healthy volunteers had no history of OI symptoms or symptoms during NLT or PoTS, 10% had asymptomatic OH. One hundred thirty (47%) long covid patients had previous history of OI symptoms and 144 (52%) developed symptoms during the NLT. Forty-one (15%) had an abnormal NLT, 20 (7%) met criteria for PoTS, and 21 (8%) had OH. Of patients with an abnormal NLT, 45% had no prior symptoms of OI. Relaxing the diagnostic thresholds for PoTS from two consecutive abnormal readings to one abnormal reading during the NLT, resulted in 11% of long covid participants (an additional 4%) meeting criteria for PoTS, but not in healthy volunteers. More than half of long covid patients experienced OI symptoms during NLT and more than one in 10 patients met the criteria for either PoTS or OH, half of whom did not report previous typical OI symptoms. We therefore recommend all patients attending long covid clinics are offered an NLT and appropriate management commenced.

Xeroderma pigmentosum (XP) is caused by defective nucleotide excision repair of DNA damage. This results in hypersensitivity to ultraviolet light and increased skin cancer risk, as sunlight -induced photoproducts remain unrepaired. However, many XP patients also display early -onset neurodegeneration, which leads to premature death. The mechanism of neurodegeneration is unknown. Here, we investigate XP neurodegeneration using pluripotent stem cells derived from XP patients and healthy relatives, performing functional multi-omics on samples during neuronal differentiation. We show substantially increased levels of 5 ' ,8-cyclopurine and 8-oxopurine in XP neuronal DNA secondary to marked oxidative stress. Furthermore, we find that the endoplasmic reticulum stress response is upregulated and reversal of the mutant genotype is associated with phenotypic rescue. Critically, XP neurons exhibit inappropriate downregulation of the protein clearance ubiquitin-proteasome system (UPS). Chemical enhancement of UPS activity in XP neuronal models improves phenotypes, albeit inadequately. Although more work is required, this study presents insights with intervention potential.

BackgroundLong COVID encompasses a heterogeneous set of ongoing symptoms that affect many individuals after recovery from infection with SARS-CoV-2. The underlying biological mechanisms nonetheless remain obscure, precluding accurate diagnosis and effective intervention. Complement dysregulation is a hallmark of acute COVID-19 but has not been investigated as a potential determinant of long COVID.MethodsWe quantified a series of complement proteins, including markers of activation and regulation, in plasma samples from healthy convalescent individuals with a confirmed history of infection with SARS-CoV-2 and age/ethnicity/sex/infection/vaccine-matched patients with long COVID.FindingsMarkers of classical (C1s-C1INH complex), alternative (Ba, iC3b), and terminal pathway (C5a, TCC) activation were significantly elevated in patients with long COVID. These markers in combination had a receiver operating characteristic predictive power of 0.794. Other complement proteins and regulators were also quantitatively different between healthy convalescent individuals and patients with long COVID. Generalized linear modeling further revealed that a clinically tractable combination of just four of these markers, namely the activation fragments iC3b, TCC, Ba, and C5a, had a predictive power of 0.785.ConclusionsThese findings suggest that complement biomarkers could facilitate the diagnosis of long COVID and further suggest that currently available inhibitors of complement activation could be used to treat long COVID.FundingThis work was funded by the National Institute for Health Research (COV-LT2-0041), the PolyBio Research Foundation, and the UK Dementia Research Institute.

Supplementary Glioma Gene Mutations from A Hypermutation Phenotype and Somatic MSH6 Mutations in Recurrent Human Malignant Gliomas after Alkylator Chemotherapy

Passive dynamics is an aspect of locomotion which is entirely dependent on the mechanical configuration and linkages of adjacent body segments. Tension distribution along mechanical linkages enables the execution of movement patterns with reduced need for complex neurological pathways and may play a role in reestablishing postural stability following external disturbances. Here we demonstrate a uni-directional mechanical relationship between the equine forelimb, head and neck, which may have implications for balance and forelimb loading in the horse. These observations suggest that forelimb, head and neck movement coordination (observed in the horse during unrestrained locomotion) is significantly influenced by the mechanical linkages between body segments, rather than being entirely dependent on neurological input as previously thought. This highlights the potential significance of research directed at investigating passively induced movements in understanding common locomotory patterns. Additionally, it suggests a mode of postural control which may provide instantaneous adjustments to postural disturbances, thus promoting rapid and efficient locomotion.

<p>Substitution signature exposures estimated in 640 breast cancer WGS and the ovarian sample (AOCS-166).</p>