Replication-coupled gene editing using locked nucleic acid-modified single-stranded DNA oligonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, oligonucleotide-directed mutation screening (ODMS) was developed to determine whether variants of uncertain clinical significance of DNA mismatch repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine-resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas mouse embryonic stem cells (mESCs) hemizygous for MMR genes were used previously, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6-Thioguanine resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; and second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggests that the efficiency of LMO-mediated gene targeting at a nonselectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.

A 7-month-old boy presented to our clinic with developmental delay, Magnetic Resonance Imaging (MRI) features of delayed myelination and diffusion restriction, and a homozygous variant of uncertain significance (c.4T>G, p.Phe2Val) in HIKESHI, a gene associated with autosomal-recessive hypomyelinating leukodystrophy 13. We hypothesized that the variant is disease-causing and aimed to rescue the cellular phenotype with vector-mediated gene replacement. HIKESHI mediates heat-induced nuclear accumulation of heat-shock proteins, including HSP70, to protect cells from stress. We generated skin fibroblasts from the proband and proband\'s mother (heterozygous) to compare protein expression and subcellular localization of HSP70 under heat stress conditions, and the effect of vector-mediated overexpression of HIKESHI in the proband\'s cells under the same heat stress conditions. Western blot analysis revealed absent HIKESHI protein from proband fibroblasts, contrasted with ample expression in parental cells. Under heat stress conditions, while the mother\'s cells displayed appropriate nuclear localization of HSP70, the proband\'s cells displayed impaired nuclear translocalization. When patient fibroblasts were provided exogenous HIKESHI, the transfected proband\'s cells showed restored heat-induced nuclear translocalization of HSP70 under conditions of heat stress. These functional data establish that the patient\'s variant is a pathogenic loss-of-function mutation, thus confirming a diagnosis of hypomyelinating leukodystrophy 13 and that vector-mediated gene replacement may be an effective treatment approach for patients with this disorder.

UNASSIGNED: While controversy remains as to the relative benefit of operative (OM) versus non-operative management (NOM) of Achilles tendon (AT) ruptures (ATR), few studies have examined the effect on high impact maneuvers such as jumping and hopping.
UNASSIGNED: The purpose of this study was to determine if functional performance including strength, jumping, and hopping outcomes differed between OM and NOM of acute ATR. The secondary objective was to assess the degree of association between AT morphology and performance outcomes.
UNASSIGNED: Retrospective cohort with a single prospective evaluation.
UNASSIGNED: All patients were treated at an institutional secondary care center. Eligible participants (n=12 OM; 12 NOM) who were treated with OM or NOM of ATR within three weeks of injury were evaluated a minimum 20 months following ATR. AT length, thickness and gastrocnemius muscle thickness were assessed with B-mode ultrasound. Isokinetic plantar flexor strength, hop tests and countermovement and drop jumps were completed. Two-way ANOVAS were completed on all tests with unilateral values, independent t-tests were used for bilateral outcomes, and linear regressions were completed to assess the relationship between normalized AT length and performance.
UNASSIGNED: Affected limb AT was elongated and thickened (p\\<0.01), gastrocnemius was atrophied (p\\< 0.01) and angle-specific plantar flexor torque was reduced at 120°/s when measured at 20° plantar flexion (p = 0.028). Single leg drop vertical jump was higher in OM (p = 0.015) with no difference for hop and jump tests. AT length was related to plantar flexor torque but had no relationship with hopping performance.
UNASSIGNED: Hop test performance was maintained despite plantarflexion weakness, gastrocnemius atrophy, and AT elongation. This may be the result of compensatory movement patterns. AT length holds limited explanatory power in plantar flexor strength, although this relationship should be evaluated further.
UNASSIGNED: Level III.

This article provides a summary of the clinical spectrum of no obstructive coronary arteries. We describe the pathologies, invasive and noninvasive assessment, and management strategies.

OBJECTIVE: Assess inter- and intra-rater reliability of the Qualitative Analysis of Single Leg Squat (QASLS) during a single-leg triple hop landing in subjects following anterior cruciate ligament reconstruction (ACLR). Explore if differences in reliability existed between novice and experienced clinicians. Determine if QASLS scores differed between the surgical and nonsurgical limbs.
METHODS: Repeated Measures.
METHODS: 20 subjects ≥6 months post-ACLR.
METHODS: Subjects were recorded performing a single-leg triple hop bilaterally. Videos were independently rated by five raters (2 physical therapists and 3 physical therapy students). Intraclass correlation coefficient (ICC) was calculated to measure reliability of the QASLS on the surgical limb. Wilcoxon signed-rank test was utilized to assess if differences in QASLS scores existed between limbs.
RESULTS: The cumulative inter-rater reliability was moderate (ICC (2,1): 0.703) and the cumulative intra-rater reliability was good (ICC (3,1): 0.857). Little difference was found between experienced and novice raters for inter- and intra-rater reliability. There was no statistically significant difference in QASLS scores between limbs (P = 0.64).
CONCLUSIONS: The QASLS tool offers moderate inter- and good intra-rater reliability for evaluating movement quality during a single-leg triple hop landing, irrespective of rater experience. Additionally, there was no observed difference in QASLS scores between surgical and nonsurgical limbs.

Background: The effectiveness of knee orthoses as part of conservative treatment for patients with medial knee osteoarthritis has not been fully explored. The purpose of this study was to evaluate the effects of a novel semi-rigid knee orthosis on pain, physical activity, and functional capacity. Methods: Pain levels, physical activity, and functional capacity were assessed in 24 participants experiencing symptomatic medial knee osteoarthritis one week before (i.e., pretest) initiating a six-week orthosis intervention and again during the final week of the intervention (i.e., post-test). Results: Night pain, pain during walking, pain during stair climbing, and pain during sitting consistently decreased by 41% to 48% while wearing the knee orthosis. Device-based measured physical activity showed a 20.2-min increase in vigorous physical activity during the post-test, while light and moderate physical activity did not show significant changes. After six weeks of orthosis application, there was a 5% increased distance for the six-minute walk test, and participants reported fewer limitations both in everyday and athletic activities, as well as an enhanced quality of life. Conclusions: These findings highlight the potential effectiveness of a semi-rigid knee orthosis to enhancing functional capacity and quality of life. More extensive and longer clinical trials are needed to improve confidence in these findings and understand their impact on disease progression.

UNASSIGNED: Functional testing has recently become more and more popular to assess athletes, both for injury prevention, as well as in an objective of performance. However, the relationship between the results of these tests and performances (or injuries) or their interpretation remains unclear.
UNASSIGNED: The aim of this study is to explore the usefulness, the characteristics, and the interpretation of the most frequently used upper-limb functional test.
UNASSIGNED: Twenty-two experts with an excellent knowledge of upper limb functional tests and an expertise in sports medicine and/or sports training of at least 5 years were recruited. They answered to qualitative and quantitative questions about functional testing trough structured questionnaires (online).
UNASSIGNED: Four rounds were needed to reach a consensus about the usefulness as well as the characteristics of each test. Different sports-specific batteries of tests were also suggested by the experts and reached consensus. However, concerning the interpretation of the test, a consensus was only found for half of the tests considered.
UNASSIGNED: The current study summarizes the characteristics and the usefulness of the most popular upper-limb functional tests. However, the interpretation of some tests will have to be further explored since no consensus was found for them.

Extraskeletal osteosarcoma (ESOS) is a rare malignant mesenchymal tumor that originates in the soft tissue. ESOS accounts for less than 1% of all soft tissue sarcomas and exhibits an aggressive behavior with a high propensity for local recurrence and distant metastasis. Despite advances in treatment, the prognosis for ESOS remains poor, with a five-year survival rate of less than 50% and 27% for metastatic patients. Ex vivo models derived from patient samples are critical tools for studying rare diseases with poor prognoses, such as ESOS, and identifying potential new treatment strategies. In this work, we established a novel ESOS ex vivo sarco-sphere model from a metastatic lesion to the dermis for research and functional testing purposes. The ex vivo cell model accurately recapitulated the native tumor, as evidenced by histomorphology and molecular profiles. Through a functional screening approach, we were able to identify novel individual anti-cancer drug sensitivities for different drugs such as romidepsin, miverbresib and to multiple kinase inhibitors. Overall, our new ESOS ex vivo cell model represents a valuable tool for investigating disease mechanisms and answering basic and translational research questions.

Basic and clinical cancer research requires tumor models that consistently recapitulate the characteristics of prima tumors. As ex vivo 3D cultures of patient tumor cells, patient-derived tumor organoids possess the biological properties of primary tumors and are therefore excellent preclinical models for cancer research. Patient-derived organoids can be established using primary tumor tissues, peripheral blood, pleural fluid, ascites, and other samples containing tumor cells. Circulating tumor cells acquired by non-invasive sampling feature dynamic circulation and high heterogeneity. Circulating tumor cell-derived organoids are prospective tools for the dynamic monitoring of tumor mutation evolution profiles because they reflect the heterogeneity of the original tumors to a certain extent. This review discusses the advantages and applications of patient-derived organoids. Meanwhile, this work highlights the biological functions of circulating tumor cells, the latest advancement in research of circulating tumor cell-derived organoids, and potential application and challenges of this technology.

The growing global burden of visual impairment necessitates better population eye screening for early detection of eye diseases. However, accessibility to testing is often limited and centralized at in-hospital settings. Furthermore, many eye screening programs were disrupted by the COVID-19 pandemic, presenting an urgent need for out-of-hospital solutions.
This study investigates the performance of a novel remote perimetry application designed in a virtual reality metaverse environment to enable functional testing in community-based and primary care settings.
This was a prospective observational study investigating the performance of a novel remote perimetry solution in comparison with the gold standard Humphrey visual field (HVF) perimeter. Subjects received a comprehensive ophthalmologic assessment, HVF perimetry, and remote perimetry testing. The primary outcome measure was the agreement in the classification of overall perimetry result normality by the HVF (Swedish interactive threshold algorithm-fast) and testing with the novel algorithm. Secondary outcome measures included concordance of individual testing points and perimetry topographic maps.
We recruited 10 subjects with an average age of 59.6 (range 28-81) years. Of these, 7 (70%) were male and 3 (30%) were female. The agreement in the classification of overall perimetry results was high (9/10, 90%). The pointwise concordance in the automated classification of individual test points was 83.3% (8.2%; range 75%-100%). In addition, there was good perimetry topographic concordance with the HVF in all subjects.
Remote perimetry in a metaverse environment had good concordance with gold standard perimetry using the HVF and could potentially avail functional eye screening in out-of-hospital settings.