Synthesis, In Silico and Kinetics Evaluation of N-(ß-d-glucopyranosyl)-2-arylimidazole-4(5)-carboxamides and N-(ß-d-glucopyranosyl)-4(5)-arylimidazole-2-carboxamides as Glycogen Phosphorylase Inhibitors.

Recently studied N-(ß-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N-(ß-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per-O-acetylated ß-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GPb revealed low micromolar inhibitors, with the best inhibition constants (Kis) of ~3-4 µM obtained for 1- and 2-naphthyl-substituted N-(ß-d-glucopyranosyl)-imidazolecarboxamides, 2b-c. The predicted protein-ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed.

Antibiotic Resistance and Disinfectant Resistance Among Escherichia coli Isolated During Red Meat Production.

Escherichia coli commonly found in the gastrointestinal tracts of food animals include Shiga toxin-producing E. coli (STEC, stx+, eae-), Enterohemorrhagic E. coli (EHEC, stx+, eae+), Enteropathogenic E. coli (EPEC, stx-, eae+), and "nondiarrheagenic" E. coli (NDEC, stx-, eae-). EHEC, EPEC, and STEC are associated with foodborne disease outbreaks. During meat processing, disinfectants are employed to control various bacteria, including human pathogens. Concerns exist that E. coli resistant to antibiotics are less susceptible to disinfectants used during meat processing. Since EHEC, EPEC, and STEC with reduced susceptibility to disinfectants are potential public health risks, the goal of this study was to evaluate the association of antibiotic resistant (ABR) E. coli with increased tolerance to 4% lactic acid (LA) and 150 ppm quaternary ammonium compounds (QACs). A pool of 3,367 E. coli isolated from beef cattle, veal calves, swine, and sheep at various processing stages was screened to identify ABR E. coli. Resistance to ≥1 of the six antibiotics examined was identified in 27.9%, 36.1%, 54.5%, and 28.7% among the NDEC (n = 579), EHEC (n = 693), EPEC (n = 787), and STEC (n = 1308) isolates evaluated, respectively. Disinfectant tolerance did not differ (P > 0.05) between ABR and antibiotic susceptible EHEC isolates. Comparable frequencies (P > 0.05) of biofilm formation or congo red binding were observed between ABR and antibiotic susceptible strains of E. coli. Understanding the frequencies of ABR and disinfectant tolerance among E. coli present in food-animal is a critically important component of meat safety.

Development of the AMPDECIDE Decision Aid to Facilitate Shared Decision Making in Patients Facing Amputation Secondary to Chronic Limb Threatening Ischemia.

INTRODUCTION: We developed a patient decision aid to enhance patient participation in amputation level decision making when there is a choice between a transmetatarsal or transtibial amputation. METHODS: In accordance with International Patient Decision Aid Standards, we developed an amputation level patient decision aid for patients who are being considered for either a transmetatarsal or transtibial amputation, incorporating qualitative literature data, quantitative literature data, qualitative provider and patient interviews, expert panel input and iterative patient feedback. RESULTS: The rapid qualitative literature review and qualitative interviews identified five domains outcome priority domains important to patients facing amputation secondary to chronic limb threatening ischemia: 1) the ability to walk, 2) healing and risk for reamputation, 3) rehabilitation program intensity, 4) ease of prosthetic use, and 5) limb length after amputation. The rapid quantitative review identified only two domains with adequate evidence comparing differences in outcomes between the two amputation levels: mobility and reamputation. Patient, surgeon, rehabilitation and decision aid expert feedback allowed us to integrate critical facets of the decision including addressing the emotional context of loss of limb, fear and anxiety as an obstacle to decision making, shaping the decision in the context of remaining life years, and how to facilitate patient knowledge of value tradeoffs. CONCLUSIONS: Amputation level choice is associated with significant outcome trade-offs. The AMPDECIDE patient decision aid can facilitate acknowledgment of patient fears, enhance knowledge of amputation level outcomes, assist patients in determining their personal outcome priorities, and facilitate shared amputation level decision making.

Midwife-led obstetric triage to increase providers' knowledge and improve timeliness of care: A pre and posttest design.

BACKGROUND: Research in low- and middle-income countries has shown that maternal mortality is directly related to inadequate or absent obstetric (OB) triage systems. Standard triage systems and knowledge on triaging for obstetric emergencies are often absent or lacking in most healthcare systems in Liberia. OBJECTIVE: The objective of this research was to address the third delay defined as receiving adequate, quality care when a facility is reached by increasing knowledge through the establishment of a midwife-led, hospital-based OB triage system to stratify care based on risk and imminence of birth and to improve timely assessment at two district referral hospitals. METHODS: A quasi-experimental study design using a pre/post survey was employed for a midwife-led OB triage training course. Using a train-the-trainer model, five midwives were trained as champions, who in turn trained an additional 62 providers. Test results were analyzed with the R statistical software using paired sample t-test and descriptive statistics. RESULTS: Pretest results revealed a knowledge and practice gap among OB providers on key components of the standard triage package. However, post-test mean scores were significantly higher (M = 79.6, SD = 2.32) than pre-test mean scores (M = 59.0, SD = 2.30) for participants following a 2-day training (p = <0.001). DISCUSSION: Following a structured OB triage training, participants showed significant improvement in post-test OB triage scores. CONCLUSION: Standard OB triage protocols incorporated into the policies and procedures of obstetric wards have the potential to improve knowledge and practice, addressing the third delay and reducing preventable, obstetrics-related deaths.

Unlocking the adaptive advantage: correlation and machine learning classification to identify optimal online adaptive stereotactic partial breast candidates.

OBJECTIVE: Online adaptive radiotherapy (OART) is a promising technique for delivering stereotactic accelerated partial breast irradiation (APBI), as lumpectomy cavities vary in location and size between simulation and treatment. However, OART is resource-intensive, increasing planning and treatment times and decreasing machine throughput compared to the standard of care (SOC). Thus, it is pertinent to identify high-yield OART candidates to best allocate resources. Approach. Reference plans (plans based on simulation anatomy), SOC plans (reference plans recalculated onto daily anatomy), and daily adaptive plans were analyzed for 31 sequential APBI targets, resulting in the analysis of 333 treatment plans. Spearman correlations between 22 reference plan metrics and 10 adaptive benefits, defined as the difference between mean SOC and delivered metrics, were analyzed to select a univariate predictor of OART benefit. A multivariate logistic regression model was then trained to stratify high- and low-benefit candidates. Main Results. Adaptively delivered plans showed dosimetric benefit as compared to SOC plans for most plan metrics, although the degree of adaptive benefit varied per patient. The univariate model showed high likelihood for dosimetric adaptive benefit when the reference plan ipsilateral breast V15Gy exceeds 23.5%. Recursive feature elimination identified 5 metrics that predict high-dosimetric-benefit adaptive patients. Using leave-one-out cross validation, the univariate and multivariate models classified targets with 74.2% and 83.9% accuracy, resulting in improvement in per-fraction adaptive benefit between targets identified as high- and low-yield for 7/10 and 8/10 plan metrics, respectively. Significance. This retrospective, exploratory study demonstrated that dosimetric benefit can be predicted using only ipsilateral breast V15 Gy on the reference treatment plan, allowing for a simple, interpretable model. Using multivariate logistic regression for adaptive benefit prediction led to increased accuracy at the cost of a more complicated model. This work presents a methodology for clinics wishing to triage OART resource allocation. .

Tumor metabolism in pheochromocytomas: clinical and therapeutic implications.

Pheochromocytomas and paragangliomas (PPGLs) have emerged as one of the most common endocrine tumors. It epitomizes fascinating crossroads of genetic, metabolic, and endocrine oncology, providing a canvas to explore the molecular intricacies of tumor biology. Predominantly rooted in the aberration of metabolic pathways, particularly the Krebs cycle and related enzymatic functionalities, PPGLs manifest an intriguing metabolic profile, highlighting elevated levels of oncometabolites like succinate and fumarate, and furthering cellular malignancy and genomic instability. This comprehensive review aims to delineate the multifaceted aspects of tumor metabolism in PPGLs, encapsulating genetic factors, oncometabolites, and potential therapeutic avenues, thereby providing a cohesive understanding of metabolic disturbances and their ramifications in tumorigenesis and disease progression. Initial investigations into PPGLs metabolomics unveiled a stark correlation between specific genetic mutations, notably in the succinate dehydrogenase complex (SDHx) genes, and the accumulation of oncometabolites, establishing a pivotal role in epigenetic alterations and hypoxia-inducible pathways. By scrutinizing voluminous metabolic studies and exploiting technologies, novel insights into the metabolic and genetic aspects of PPGLs are perpetually being gathered elucidating complex interactions and molecular machinations. Additionally, the exploration of therapeutic strategies targeting metabolic abnormalities has burgeoned harboring potential for innovative and efficacious treatment modalities. This review encapsulates the profound metabolic complexities of PPGLs, aiming to foster an enriched understanding and pave the way for future investigations and therapeutic innovations in managing these metabolically unique tumors.

Do Rib-Based Anchors Impair Chest Wall Motion in Early Onset Scoliosis (EOS)?

Purpose: There is a concern in pediatric surgery practice that rib-based fixation may limit chest wall motion in early onset scoliosis (EOS). The purpose of this study is to address the above concern by assessing the contribution of chest wall excursion to respiration before and after surgery. Methods: Quantitative dynamic magnetic resonance imaging (QdMRI) is performed on EOS patients (before and after surgery) and normal children in this retrospective study. QdMRI is purely an image-based approach and allows free breathing image acquisition. Tidal volume parameters for chest walls (CWtv) and hemi-diaphragms (Dtv) were analyzed on concave and convex sides of the spinal curve. EOS patients (1-14 years) and normal children (5-18 years) were enrolled, with an average interval of two years for dMRI acquisition before and after surgery. Results: CWtv significantly increased after surgery in the global comparison including all EOS patients (p < 0.05). For main thoracic curve (MTC) EOS patients, CWtv significantly improved by 50.24% (concave side) and 35.17% (convex side) after age correction (p < 0.05) after surgery. The average ratio of Dtv to CWtv on the convex side in MTC EOS patients was not significantly different from that in normal children (p=0.78), although the concave side showed the difference to be significant. Conclusion: Chest wall component tidal volumes in EOS patients measured via QdMRI did not decrease after rib-based surgery, suggesting that rib-based fixation does not impair chest wall motion in pediatric patients with EOS.

Virtual Growing Child (VGC): A general normative comparative system via quantitative dynamic MRI for quantifying pediatric regional respiratory anomalies with application in thoracic insufficiency syndrome (TIS).

Background: A normative database of regional respiratory structure and function in healthy children does not exist. Methods: VGC provides a database with four categories of regional respiratory measurement parameters including morphological, architectural, dynamic, and developmental. The database has 3,820 3D segmentations (around 100,000 2D slices with segmentations). Age and gender group analysis and comparisons for healthy children were performed using those parameters via two-sided t-testing to compare mean measurements, for left and right sides at end-inspiration (EI) and end-expiration (EE), for different age and gender specific groups. We also apply VGC measurements for comparison with TIS patients via an extrapolation approach to estimate the association between measurement and age via a linear model and to predict measurements for TIS patients. Furthermore, we check the Mahalanobis distance between TIS patients and healthy children of corresponding age. Findings: The difference between male and female groups (10-12 years) behave differently from that in other age groups which is consistent with physiology/natural growth behavior related to adolescence with higher right lung and right diaphragm tidal volumes for females(p<0.05). The comparison of TIS patients before and after surgery show that the right and left components are not symmetrical, and the left side diaphragm height and tidal volume has been significantly improved after surgery (p <0.05). The left lung volume at EE, and left diaphragm height at EI of TIS patients after surgery are closer to the normal children with a significant smaller Mahalanobis distance (MD) after surgery (p<0.05). Interpretation: The VGC system can serve as a reference standard to quantify regional respiratory abnormalities on dMRI in young patients with various respiratory conditions and facilitate treatment planning and response assessment. Funding: The grant R01HL150147 from the National Institutes of Health (PI Udupa).

Interactive Segmentation of Lung Tissue and Lung Excursion in Thoracic Dynamic MRI Based on Shape-guided Convolutional Neural Networks.

Purpose: Lung tissue and lung excursion segmentation in thoracic dynamic magnetic resonance imaging (dMRI) is a critical step for quantitative analysis of thoracic structure and function in patients with respiratory disorders such as Thoracic Insufficiency Syndrome (TIS). However, the complex variability of intensity and shape of anatomical structures and the low contrast between the lung and surrounding tissue in MR images seriously hamper the accuracy and robustness of automatic segmentation methods. In this paper, we develop an interactive deep-learning based segmentation system to solve this problem. Material & Methods: Considering the significant difference in lung morphological characteristics between normal subjects and TIS subjects, we utilized two independent data sets of normal subjects and TIS subjects to train and test our model. 202 dMRI scans from 101 normal pediatric subjects and 92 dMRI scans from 46 TIS pediatric subjects were acquired for this study and were randomly divided into training, validation, and test sets by an approximate ratio of 5:1:4. First, we designed an interactive region of interest (ROI) strategy to detect the lung ROI in dMRI for accelerating the training speed and reducing the negative influence of tissue located far away from the lung on lung segmentation. Second, we utilized a modified 2D U-Net to segment the lung tissue in lung ROIs, in which the adjacent slices are utilized as the input data to take advantage of the spatial information of the lungs. Third, we extracted the lung shell from the lung segmentation results as the shape feature and inputted the lung ROIs with shape feature into another modified 2D U-Net to segment the lung excursion in dMRI. To evaluate the performance of our approach, we computed the Dice coefficient (DC) and max-mean Hausdorff distance (MM-HD) between manual and automatic segmentations. In addition, we utilized Coefficient of Variation (CV) to assess the variability of our method on repeated dMRI scans and the differences of lung tidal volumes computed from the manual and automatic segmentation results. Results: The proposed system yielded mean Dice coefficients of 0.96±0.02 and 0.89±0.05 for lung segmentation in dMRI of normal subjects and TIS subjects, respectively, demonstrating excellent agreement with manual delineation results. The Coefficient of Variation and p-values show that the estimated lung tidal volumes of our approach are statistically indistinguishable from those derived by manual segmentations. Conclusions: The proposed approach can be applied to lung tissue and lung excursion segmentation from dynamic MR images with high accuracy and efficiency. The proposed approach has the potential to be utilized in the assessment of patients with TIS via dMRI routinely.

Extruded alginate tubes with myogenic potential.

BACKGROUND: There are currently no proven methods to reverse muscle loss in humans, which is caused by trauma (e.g., volumetric muscle loss, VML), genetic neuromuscular diseases (e.g., muscular dystrophies, MDs), and accelerated senescence (e.g., sarcopenia). Since muscle tissue is capable of regeneration through muscle satellite cells (MuSCs), the implantation of autologous (or other) donor MuSCs and MuSC-derived myoblasts into host muscles can promote donor-cell-derived myogenesis. Direct injection or implantation of MuSCs or MuSC-derived myoblasts into host muscles only promotes minimal donor-cell-derived myogenesis, whereas implantation of MuSCs/myoblasts along with associated muscle tissue (muscle fibers, extracellular matrix, neurovascular pathways, etc.) gives better results. METHODS: We aim to leverage the benefits of constraining donor myogenic cells within a template that resembles muscle tissue. In this paper, we present a workflow for basic and translational studies aimed at promoting donor-cell-derived myogenesis to increase functional muscle mass in mice. Our workflow involves preparing a slurry of 10% sodium alginate mixed with myogenic cells in cell culture media, extruding the cell-containing slurry into 10% calcium lactate to form tubes, and implanting the cellularized alginate tubes into host muscle. RESULTS: Our data suggest that, the extruded alginate tubes can tolerate a peak stress of 1892 ± 527 mN, that the elastic range is at ~75-125% strain beyond initial length, and that the Young's modulus (stiffness) is 14.17 ± 1.68 %/mm 2 . Importantly, these mechanical properties render the alginate tubes suitable for a published technique known as minimally-invasive muscle embedding (MIME) that was developed by us to implant myogenic material into host muscle. MIME involves threading donor myogenic tissue into a needle track created within a host muscle. Cellularized alginate tubes implanted into the tibialis anterior muscle of previously euthanized mice had numerous hematoxylin-stained structures similar to nuclear staining, supporting the idea that our alginate tubes can support cell seeding. Alginate tubes that were seeded with MuSCs, incubated in MuSC/myoblast growth (i.e., proliferation) media for two days, incubated in myotube differentiation media for six days, and then minced and reseeded in new dishes, were able to promote in vitro myoblast outgrowth over several days. DISCUSSION: This pilot study is limited in its translational scope because it was performed in vitro and with previously euthanized mice. Additional studies are needed to confirm that cellularized alginate tubes can promote the de novo development of donor-cell-derived muscle fibers, which can contribute to contractile force production. CONCLUSION: Alginate tubes with MuSC/myoblasts can be generated by a simple extrusion method. The alginate tubes have sufficient mechanical strength to tolerate insertion into a host muscle, in a minimally-invasive manner, through a needle track. The cellularized alginate tubes demonstrate myogenic potential since they are capable of being maintained in culture conditions for several days, after which they can still facilitate myoblast outgrowth in a dish.

Assessment of 3D hemi-diaphragmatic motion via free-breathing dynamic MRI in pediatric thoracic insufficiency syndrome.

Purpose: Thoracic insufficiency syndrome (TIS) affects ventilatory function due to spinal and thoracic deformities limiting lung space and diaphragmatic motion. Corrective orthopedic surgery can be used to help normalize skeletal anatomy, restoring lung space and diaphragmatic motion. This study employs free-breathing dynamic MRI (dMRI) and quantifies the 3D motion of each hemi-diaphragm surface in normal and TIS patients, and evaluates effects of surgical intervention. Materials and Methods: In a retrospective study of 149 pediatric patients with TIS and 190 healthy children, we constructed 4D images from free-breathing dMRI and manually delineated the diaphragm at end-expiration (EE) and end-inspiration (EI) time points. We automatically selected 25 points uniformly on each hemi-diaphragm surface, calculated their relative velocities between EE and EI, and derived mean velocities in 13 homologous regions for each hemi-diaphragm to provide measures of regional 3D hemi-diaphragm motion. T-testing was used to compare velocity changes before and after surgery, and to velocities in healthy controls. Results: The posterior-central region of the right hemi-diaphragm exhibited the highest average velocity post-operatively. Posterior regions showed greater velocity changes after surgery in both right and left hemi-diaphragms. Surgical reduction of thoracic Cobb angle displayed a stronger correlation with changes in diaphragm velocity than reduction in lumbar Cobb angle. Following surgery, the anterior regions of the left hemi-diaphragm tended to approach a more normal state. Conclusion: Quantification of regional motion of the 3D diaphragm surface in normal subjects and TIS patients via free-breathing dMRI is feasible. Derived measurements can be assessed in comparison to normal subjects to study TIS and the effects of surgery.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Comprehensive peripheral blood immunoprofiling reveals five immunotypes with immunotherapy response characteristics in patients with cancer.

The lack of comprehensive diagnostics and consensus analytical models for evaluating the status of a patient's immune system has hindered a wider adoption of immunoprofiling for treatment monitoring and response prediction in cancer patients. To address this unmet need, we developed an immunoprofiling platform that uses multiparameter flow cytometry to characterize immune cell heterogeneity in the peripheral blood of healthy donors and patients with advanced cancers. Using unsupervised clustering, we identified five immunotypes with unique distributions of different cell types and gene expression profiles. An independent analysis of 17,800 open-source transcriptomes with the same approach corroborated these findings. Continuous immunotype-based signature scores were developed to correlate systemic immunity with patient responses to different cancer treatments, including immunotherapy, prognostically and predictively. Our approach and findings illustrate the potential utility of a simple blood test as a flexible tool for stratifying cancer patients into therapy response groups based on systemic immunoprofiling.

Longitudinal analysis within one hospital in sub-Saharan Africa over 20 years reveals repeated replacements of dominant clones of Klebsiella pneumoniae and stresses the importance to include temporal patterns for vaccine design considerations.

BACKGROUND: Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. METHODS: We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. RESULTS: We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. CONCLUSIONS: Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one.

The translational gap for gene therapies in low- and middle-income countries.

Gene therapies are designed to address the root cause of disease. As scientific understanding of disease prevention, diagnosis, and treatment improves in tandem with technological innovation, gene therapies have the potential to become safe and effective treatment options for a wide range of genetic and nongenetic diseases. However, as the medical scope of gene therapies expands, consideration must be given to those who will benefit and what proactive steps must be taken to widen development and access potential, particularly in regions carrying a high disease burden.

Normothermic Preservation of the Intestinal Allograft.

Intestinal allotransplantation was first described in the 1960s and successfully performed in the 1980s. Since that time, less progress has been made in the preservation of the allograft before transplantation and static cold storage remains the current standard. Normothermic machine perfusion represents an opportunity to simultaneously preserve, assess, and recondition the organ for transplantation and improve the procurement radius for allografts. The substantial progress made in the field during the last 60 years, coupled with the success of the preclinical animal model of machine perfusion-preserved intestinal transplantation, suggest we are approaching the point of clinical application.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Limited Return to Preinjury Performance in NCAA Division I American Football Players With Hamstring Injuries.

Background: Hamstring strains are common among elite athletes, but their effect on return to the same level of play in American football has been incompletely characterized. Purpose: Data on National Collegiate Athletics Association Division I college football players with acute hamstring strains were gathered to identify the effects these injuries have on both return to play and athletic performance regarding velocity, workload, and acceleration. Study Design: Case Series; Level of evidence, 4. Methods: Injury data for a single Division I football team were prospectively recorded over a 4-year period. Players wore global navigation satellite system and local positioning system (GNSS/LPS) devices to record movement data in practices and games. The practice and game data were cross-referenced to evaluate players with isolated acute hamstring strains. Comparisons were made regarding players' pre- and postinjury ability to maintain high velocity (>12 mph [19.3 kph]), maximal velocity, triaxial acceleration, and inertial movement analysis (IMA). There were 58 hamstring injuries in 44 players, of which 25 injuries from 20 players had GNSS/LPS data. Results: Players were able to return to play from all 25 injury incidences at a mean of 9.2 days. At the final mean follow-up of 425 days, only 4 players had reached preinjury function in all measurements; 12 players were able to return in 2 of the 4 metrics; and only 8 players reached their preinjury ability to maintain high velocity. For those who did not achieve this metric, there was a significant difference between pre- and postinjury values (722 vs 442 m; P = .016). A total of 14 players were able to regain their IMA. Players who returned to prior velocity or acceleration metrics did so at a mean of 163 days across all metrics. Conclusion: While players may be able to return to play after hamstring strain, many players do not reach preinjury levels of acceleration or velocity, even after 13.5 months. Further studies are needed to confirm these findings, assess clinical relevance on imaging performance, and improve hamstring injury prevention and rehabilitation.

Healing Health Care Disparities: Development and Pilot Testing of a Virtual Reality Implicit Bias Training Module for Physicians in the Context of Black Maternal Health.

Grounded in communication models of cultural competence, this study reports on the development and testing of the first module in a larger virtual reality (VR) implicit bias training for physicians to help them better: (a) recognize implicit bias and its effects on communication, patients, and patient care; (b) identify their own implicit biases and exercise strategies for managing them; and (c) learn and practice communicating with BIPOC patients in a culture-centered manner that demonstrates respect and builds trust. Led by communication faculty, a large, interdisciplinary team of researchers, clinicians, and engineers developed the first module tested herein focused on training goal (a). Within the module, participants observe five scenes between patient Marilyn Hayes (a Black woman) and Dr. Richard Flynn (her obstetrician, a White man) during a postpartum visit. The interaction contains examples of implicit bias, and participants are asked to both identify and consider how implicit bias impacts communication, the patient, and patient care. The team recruited 30 medical students and resident physicians to participate in a lab-based study that included a pretest, a training experience of the module using a head-mounted VR display, and a posttest. Following the training, participants reported improved attitudes toward implicit bias instruction, greater importance of determining patients' beliefs and perspectives for history-taking, treatment, and providing quality health care; and greater communication efficacy. Participants' agreement with the importance of assessing patients' perspectives, opinions, and psychosocial and cultural contexts did not significantly change. Implications for medical education about cultural competency and implicit bias are discussed.