CRaTER enrichment for on-target gene editing enables generation of variant libraries in hiPSCs.

Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of variants in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different variants. We differentiated these hiPSCs to cardiomyocytes and show MHC-ß fusion proteins can localize as expected. Additionally, single-cell contractility analyses revealed cardiomyocytes with a pathogenic, hypertrophic cardiomyopathy-associated MYH7 variant exhibit salient HCM physiology relative to isogenic controls. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of functional transgenic cell lines at unprecedented scale.

ELF3 mediates IL-1α induced differentiation of mesenchymal stem cells to inflammatory iCAFs.

Stromal cells in the tumor microenvironment regulate the immune landscape and tumor progression. Yet, the ontogeny and heterogeneity of reactive stromal cells within tumors is not well understood. Carcinoma-associated fibroblasts exhibiting an inflammatory phenotype (iCAFs) have been identified within multiple cancers; however, mechanisms that lead to their recruitment and differentiation also remain undefined. Targeting these mechanisms therapeutically may be important in managing cancer progression. Here, we identify the ELF3 transcription factor as the canonical mediator of IL-1α-induced differentiation of prostate mesenchymal stem cells to an iCAF phenotype, typical of the tumor microenvironment. Furthermore, IL-1α-induced iCAFs were subsequently refractive to TGF-ß1 induced trans-differentiation to a myofibroblast phenotype (myCAF), another key carcinoma-associated fibroblast subtype typical of reactive stroma in cancer. Restricted trans-differentiation was associated with phosphorylation of the YAP protein, indicating that interplay between ELF3 action and activation of the Hippo pathway are critical for restricting trans-differentiation of iCAFs. Together, these data show that the IL-1α/ELF3/YAP pathways are coordinate for regulating inflammatory carcinoma-associated fibroblast differentiation.

Short-term Effects of Canceled Elective Procedures Due to COVID-19: Evidence From the Veterans Affairs Healthcare System.

OBJECTIVE: To determine whether delayed or canceled elective procedures due to COVID-19 resulted in higher rates of ED utilization and/or increased mortality. SUMMARY OF BACKGROUND DATA: On March 15, 2020, the VA issued a nationwide order to temporarily pause elective cases due to COVID-19. The effects of this disruption on patient outcomes are not yet known. METHODS: This retrospective cohort study used data from the VA Corporate Data Warehouse. Surgical procedures canceled due to COVID-19 in 2020 (n = 3326) were matched to similar completed procedures in 2018 (n = 151,863) and 2019 (n = 146,582). Outcome measures included 30- and 90-day VA ED use and mortality in the period following the completed or canceled procedure. We used exact matching on surgical procedure category and nearest neighbor matching on patient characteristics, procedure year, and facility. RESULTS: Patients with elective surgical procedures canceled due to COVID-19 were no more likely to have an ED visit in the 30- [Difference: -4.3% pts; 95% confidence interval (CI): -0.078, -0.007] and 90 days (-0.9% pts; 95% CI: -0.068, 0.05) following the expected case date. Patients with cancellations had no difference in 30- (Difference: 0.1% pts; 95% CI: -0.008, 0.01) and 90-day (Difference: -0.4% pts; 95% CI: -0.016, 0.009) mortality rates when compared to similar patients with similar procedures that were completed in previous years. CONCLUSIONS: The pause in elective surgical cases was not associated with short-term adverse outcomes in VA hospitals, suggesting appropriate surgical case triage and management. Further study will be essential to determine if the delayed cases were associated with longer-term effects.

Emergency Medicaid Acquisition Through the Affordable Care Act: The Association Between Hospital Enrollment in California and Hospital Revenue.

BACKGROUND: Hospital Presumptive Eligibility (HPE) is a national policy stemming from the Affordable Care Act that allows qualified hospitals, working with state officials, to enroll eligible patients for temporary Medicaid coverage. Although all states are required to operate an HPE program, hospital participation is elective and variable. It is unclear which hospitals choose to participate in HPE and how participation affects hospital utilization and revenue. OBJECTIVE: We examined hospital factors associated with HPE participation in the state of California and assessed pre and post changes in hospital revenue and utilization for HPE and non-HPE hospitals. RESEARCH DESIGN: We performed a logistic regression to identify hospital attributes associated with HPE participation. We then used a difference in differences methodology with a hospital fixed effect to test whether HPE enrollment was associated with changes in annual revenues by payer source, uncompensated care costs, outpatient visits, and/or discharges. RESULTS: Three quarters (76%) of qualified hospitals elected to participate in HPE by the end of 2018. Hospitals with 100 or more beds had over 10 times greater odds of participating in HPE compared with smaller hospitals. Hospitals that did not provide outpatient care were significantly less likely to participate. Among hospitals included in trend analyses, enrollment in HPE was associated with increased annual net patient Medicaid revenue and decreased uncompensated care charges. We predicted that HPE enrollment was associated with an average of 9.7% (95% confidence interval: 3.4%-16.4%) increase in annual net patient Medicaid revenue. As of 2018, ∼33,000 adults and children were enrolled in California's HPE program per month. CONCLUSION: Hospital enrollment in the HPE program shifted costs from uncompensated care to Medicaid.

Determinants of Resistance to Checkpoint Inhibitors.

The development of immune checkpoint inhibitors (ICIs) has drastically altered the landscape of cancer treatment. Since approval of the first ICI for the treatment of advanced melanoma in 2011, several therapeutic agents have been Food and Drug Administration (FDA)-approved for multiple cancers, and hundreds of clinical trials are currently ongoing. These antibodies disrupt T-cell inhibitory pathways established by tumor cells and thus re-activate the host's antitumor immune response. While successful in many cancers, several types remain relatively refractory to treatment or patients develop early recurrence. Hence, there is a great need to further elucidate mechanisms of resistant disease and determine novel, effective, and tolerable combination therapies to enhance efficacy of ICIs.

Replication of Zika Virus in Human Prostate Cells: A Potential Source of Sexually Transmitted Virus.

Background: While Zika virus (ZIKV) is mainly transmitted by mosquitoes, numerous cases of sexual transmission have been reported during recent outbreaks. Little is known about which host cell types or entry factors aid in mediating this sexual transmission. Methods: In this study, we investigated ZIKV cell tropism by infecting 2 types of human prostate cells with 3 contemporary ZIKV isolates from persons infected in the Americas. We used real-time quantitative polymerase chain reaction and immunofluorescence analyses to measure infection and flow cytometry to detect entry factor expression. Results: Here we show that ZIKV infects, replicates, and produces infectious virus in prostate stromal mesenchymal stem cells, epithelial cells, and organoids made with a combination of these cells. We also show that prostate cells express several well-characterized flavivirus attachment factors. In contrast, dengue virus does not infect or does not replicate in these prostate cells, although it is known to use similar receptors. Conclusions: Our results indicate that ZIKV favors infection of stromal cells more so than epithelial cells in organoids, possibly indicating a preference for stem cells in general. Overall, these results suggest that ZIKV replication occurs in the human prostate and can account for ZIKV secretion in semen, thus leading to sexual transmission.

Clinical characteristics and quality of life of older adults with cognitive impairment in Macao.

BACKGROUND: Little is known about the characteristics of older adults with cognitive impairment in Macao. This study aimed to determine the prevalence of cognitive impairment and the quality of life (QOL) of older adults living in the community and nursing homes. METHODS: A consecutive sample of 413 subjects (199 from the community; 214 from nursing homes) was recruited and interviewed using standardized instruments. Cognition was measured with the Repeatable Battery for the Assessment of Neuropsychological Status and QOL with the brief version of the World Health Organization Quality of Life instrument. RESULTS: Altogether 87 subjects (21.0%) had cognitive impairment. On multivariate analyses, advanced age (P < 0.001, OR = 1.06, 95%CI: 1.03-1.1) and depressive symptoms (P = 0.03, OR = 1.07, 95%CI: 0.005-1.1) were positively associated with cognitive impairment. Married marital status (P = 0.01, OR = 0.3, 95%CI: 0.1-0.7) and higher education level (P < 0.001, OR = 0.1, 95%CI: 0.06-0.3) were negatively associated with cognitive impairment. After the confounders were controlled for, cognitive impairment was significantly associated with the lower psychological (F (11,412) = 6.3, P = 0.01) and social relationship domains of QOL (F (11,412) = 4.0, P = 0.04). CONCLUSION: Cognitive impairment was found to be common in community-dwelling and nursing home resident older adults in Macao. Given cognitive impairment's negative impact on QOL, appropriate strategies should be implemented to improve access to treatment in this population.

RUNX1 is essential for mesenchymal stem cell proliferation and myofibroblast differentiation.

Myofibroblasts are a key cell type in wound repair, cardiovascular disease, and fibrosis and in the tumor-promoting microenvironment. The high accumulation of myofibroblasts in reactive stroma is predictive of the rate of cancer progression in many different tumors, yet the cell types of origin and the mechanisms that regulate proliferation and differentiation are unknown. We report here, for the first time to our knowledge, the characterization of normal human prostate-derived mesenchymal stem cells (MSCs) and the TGF-ß1-regulated pathways that modulate MSC proliferation and myofibroblast differentiation. Human prostate MSCs combined with prostate cancer cells expressing TGF-ß1 resulted in commitment to myofibroblasts. TGF-ß1-regulated runt-related transcription factor 1 (RUNX1) was required for cell cycle progression and proliferation of progenitors. RUNX1 also inhibited, yet did not block, differentiation. Knockdown of RUNX1 in prostate or bone marrow-derived MSCs resulted in cell cycle arrest, attenuated proliferation, and constitutive differentiation to myofibroblasts. These data show that RUNX1 is a key transcription factor for MSC proliferation and cell fate commitment in myofibroblast differentiation. This work also shows that the normal human prostate gland contains tissue-derived MSCs that exhibit multilineage differentiation similar to bone marrow-derived MSCs. Targeting RUNX1 pathways may represent a therapeutic approach to affect myofibroblast proliferation and biology in multiple disease states.

Quality of life and clinical correlates in older adults living in the community and in nursing homes in Macao.

AIM: There have been no previous studies of quality of life (QOL) in older adults in Macao. This study aimed to examine QOL in relation to the sociodemographic and clinical characteristics of adults aged ≥50 years in Macao. METHODS: A sample of 451 subjects (203 living in the community, 248 living in nursing homes) was interviewed using standardized instruments. Basic sociodemographic and clinical data including QOL were collected. RESULT: There were no significant differences between the community and nursing home groups in any of the QOL domains. Multiple linear regression analyses revealed that poor physical QOL was significantly predicted by severe depressive symptoms, insomnia, major medical conditions, unmarried status, and lower education ( F 11,438 = 26.2, P < 0.001), which accounted for 38.2% of the variance. Poor psychological QOL was significantly predicted by severe depressive symptoms and lower educational level ( F 11,438 = 24.3, P < 0.001), which accounted for 36.4% of the variance. Poor social QOL was significantly predicted by severe depressive symptoms, male gender, and unmarried status ( F 11,438 = 5.6, P < 0.001), which accounted for 12.5% of the variance. Poor environment QOL was significantly predicted by lower educational level, severe depressive symptoms, and younger age ( F 11,438 = 6.6, P < 0.001), which accounted for 12.1% of the variance. CONCLUSION: Older Macanese adults had poorer scores on physical and social QOL domains than the general Hong Kong Chinese population. Their QOL was more strongly related to severe depressive symptoms, major medical conditions, and insomnia.

Pharmacologic treatment of type 2 diabetes: oral medications.

OBJECTIVE: To review the oral and injectable pharmacologic treatment options for type 2 diabetes. DATA SOURCES: A literature search was conducted using PubMed electronic database for studies published in English between 1993 and September 2014. Search terms included diabetes mellitus, type 2 diabetes, and the individual name for each antidiabetic medication reviewed. In addition, manual searches were performed for cross-references from publications. Package inserts, United States Food and Drug Administration (FDA) Web site, Institute for Safe Medication Practices Web site, American Diabetes Association Web site and scientific session poster presentations, and individual drug company Web pages were also reviewed. STUDY SELECTION AND DATA EXTRACTION: This review focused on information elucidated over the past 10 years to assist prescribers in choosing optimal therapy based on individual patient characteristics. Studies leading to the approval of or raising safety concerns for the antidiabetic medications reviewed in this article were included. DATA SYNTHESIS: In the past 10 years, there have been 4 novel oral antidiabetic medication classes and 9 new injectable agents and insulin products approved by the FDA for the treatment of type 2 diabetes as well as new information regarding the safety and use of several older antidiabetic medication classes. The distinctions were reviewed for each individual agent, and a comparison was completed if there was more than one agent in a particular therapeutic class. Using current information available, select investigational agents in phase III trials or those with a pending new drug application were highlighted. CONCLUSION: There are now 9 distinct oral pharmacologic classes and a variety of insulin and noninsulin injectable medications available for the treatment of type 2 diabetes. Metformin remains the first-line treatment option for most patients. When considering options for alternative or additional treatment, prescribers must weigh the benefits and risks using individual patient characteristics.

Pharmacologic treatment of type 2 diabetes: injectable medications.

OBJECTIVE: To review the oral and injectable pharmacologic treatment options for type 2 diabetes. DATA SOURCES: A literature search was conducted using PubMed electronic database for studies published in English between 1993 and September 2014. Search terms included diabetes mellitus, type 2 diabetes, and the individual name for each antidiabetic medication reviewed. In addition, manual searches were performed for cross-references from publications. Package inserts, United States Food and Drug Administration (FDA) Web site, Institute for Safe Medication Practices Web site, American Diabetes Association Web site and scientific session poster presentations, and individual drug company Web pages were also reviewed. STUDY SELECTION AND DATA EXTRACTION: This review focused on information elucidated over the past 10 years to assist prescribers in choosing optimal therapy based on individual patient characteristics. Studies leading to the approval of or raising safety concerns for the antidiabetic medications reviewed in this article were included. DATA SYNTHESIS: In the past 10 years, there have been 4 novel oral antidiabetic medication classes and 10 new injectable agents and insulin products approved by the FDA for the treatment of type 2 diabetes as well as new information regarding the safety and use of several older antidiabetic medication classes. The distinctions were reviewed for each individual agent, and a comparison was completed if there was more than one agent in a particular therapeutic class. Using current information available, select investigational agents in phase III trials or with a pending new drug application were highlighted. CONCLUSION: There are now 9 distinct oral pharmacologic classes and a variety of insulin and noninsulin injectable medications available for the treatment of type 2 diabetes. Metformin remains the first-line treatment option for most patients. When considering options for alternative or additional treatment, prescribers must weigh the benefits and risks using individual patient characteristics.

Selective alpha(1)-adrenoceptor blockade prevents fructose-induced hypertension.

The purpose of this study was to investigate the effect of chronic treatment with prazosin, a selective α1-adrenoceptor antagonist, on the development of hypertension in fructose-fed rats (FFR). High-fructose feeding and treatment with prazosin (1 mg/kg/day via drinking water) were initiated simultaneously in male Wistar rats. Systolic blood pressure, fasted plasma parameters, insulin sensitivity, plasma norepinephrine (NE), uric acid, and angiotensin II (Ang II) were determined following 9 weeks of treatment. FFR exhibited insulin resistance, hyperinsulinemia, hypertriglyceridemia, and hypertension, as well as elevations in plasma NE and Ang II levels. Treatment with prazosin prevented the rise in blood pressure without affecting insulin levels, insulin sensitivity, uric acid, or Ang II levels, while normalizing plasma NE levels in FFR. These data suggest that over-activation of the sympathetic nervous system, specifically α1-adrenoceptors, contributes to the development of fructose-induced hypertension, however, this over-activation does not appear to an initial, precipitating event in FFR.

Renal remodeling after abdominal radiation therapy: parenchymal and functional changes.

OBJECTIVE: The purpose of this study was to quantify changes in renal length, volume, and function over time after upper abdominal radiation therapy. MATERIALS AND METHODS: Imaging and clinical data were retrospectively reviewed for 27 adults with abdominal radiation therapy between 2001 and 2012. All had two kidneys, radiation exposure to one kidney, and survival of at least 1 year after therapy. Mean prescribed dose was 52 ± 9 Gy to extrarenal targets. Length and volume of exposed and unexposed kidneys were measured on CT scans before treatment (baseline) and at intervals 0-3, 3-6, 6-12, 12-24, 24-36, and more than 36 months after completion of radiotherapy. Serum creatinine was correlated at each interval. Mixed-models ANOVA was used to test renal length and volume, serum creatinine, and time against multiple models to assess for temporal effects; specific time intervals were compared in pairwise manner. RESULTS: Mean follow-up duration was 35 months (range, 5-94 months). Exposed kidney length and volume progressively decreased from baseline throughout follow-up, with mean loss of 23% (p < 0.001) and 47% (p < 0.001), respectively. Slight increase in unexposed kidney length was not significant. Mean serum creatinine increased from 0.86 ± 0.18 mg/dL at baseline to 1.12 ± 0.27 mg/dL at 12-24 months (p < 0.001), then stabilized. CONCLUSION: Kidneys exposed to radiation during therapy of adjacent malignancies exhibited continuous progressive atrophy for the entire follow-up period, nearly 8 years. Volume changes were twice as great as length changes. Renal function also declined. To accurately interpret follow-up studies in cancer survivors, radiologists should be aware of the potential for progressive renal atrophy, even many years after radiation therapy.

Community Emergency Care Use by Veterans in an Era of Expanding Choice.

Importance: Recently passed legislation aimed at improving access to care has considerably expanded options for veterans to receive emergency care in community, or non-Veterans Affairs (VA) settings. However, national trends in community emergency department (ED) use by veterans are unknown. Objective: To examine national, temporal trends in the frequencies and types of ED visits provided in community settings and explore the association between facilities' purchase of community care with facility and regional characteristics. Design, Setting, and Participants: Retrospective, observational cross-sectional study of ED visits over fiscal years (FY) 2016 to 2022. VA and community ED encounter data were obtained from the VA Corporate Data Warehouse and the Office of Integrated Veteran Care. Participants were veterans receiving ED care at VA facilities or paid for by the VA in the community. Data were analyzed from June to September 2023. Main Outcomes and Measures: The primary outcome measures included community ED visit volume, disposition, and payments over time. Also, the most common and costly ED visits were assessed. Negative binomial regression analysis examined associations between facility and regional characteristics and the rate of ED visits purchased in community settings relative to all ED visits. Results: There were 19 787 056 ED visits, predominantly at VA facilities (14 532 261 visits [73.4%]), made by 3 972 503 unique veterans from FY 2016 to 2022. The majority of ED users were male (3 576 120 individuals [90.0%]), and the median (IQR) age was 63 (48-73) years. The proportion of community ED visits increased in absolute terms from 18% in FY 2016 to 37% in FY 2022. Total community ED payments, adjusted to 2021 dollars, were $1.18 billion in FY 2016 and over $6.14 billion in FY 2022. The most common reasons for ED visits in the community were for nonspecific chest pain (305 082 visits [6%]), abdominal pain (174 836 visits [3%]), and septicemia (149 968 visits [3%]). The average proportion of ED visits purchased by a VA facility increased from 14% in FY 2016 to 32% by FY 2022. In multivariable analyses, facilities with greater ED volume and low-complexity facilities had higher expected rates of community emergency care than lower volume and high-complexity facilities, respectively. Conclusions and Relevance: As veterans increasingly use community EDs for acute, unscheduled needs, attention to factors associated with veterans' use of acute care services in different settings are important to identify access barriers and to ensure veterans' health care needs are met.

Do hospital-based emergency Medicaid programs benefit trauma centers? A mixed-methods analysis.

INTRODUCTION: Hospital Presumptive Eligibility (HPE) is a temporary Medicaid insurance at hospitalization, which can offset patient costs of care, increase access to postdischarge resources, and provide a path to sustain coverage through Medicaid. Less is known about the implications of HPE programs on trauma centers (TCs). We aimed to describe the association with HPE and hospital Medicaid reimbursement and characterize incentives for HPE participation among hospitals and TCs. We hypothesized that there would be financial, operational, and mission-based incentives. METHODS: We performed a convergent mixed methods study of HPE hospitals in California (including all verified TCs). We analyzed Annual Financial Disclosure Reports from California's Department of Health Care Access and Information (2005-2021). Our primary outcome was Medicaid net revenue. We also conducted thematic analysis of semistructured interviews with hospital stakeholders to understand incentives for HPE participation (n = 8). RESULTS: Among 367 California hospitals analyzed, 285 (77.7%) participate in HPE, 77 (21%) of which are TCs. As of early 2015, 100% of TCs had elected to enroll in HPE. There is a significant positive association between HPE participation and net Medicaid revenue. The highest Medicaid revenues are in HPE level I and level II TCs. Controlling for changes associated with the Affordable Care Act, HPE enrollment is associated with increased net patient Medicaid revenue ( b = 6.74, p < 0.001) and decreased uncompensated care costs ( b = -2.22, p < 0.05). Stakeholder interviewees' explanatory incentives for HPE participation included reduction of hospital bad debt, improved patient satisfaction, and community benefit in access to care. CONCLUSION: Hospital Presumptive Eligibility programs not only are a promising pathway for long-term insurance coverage for trauma patients but also play a role in TC viability. Future interventions will target streamlining the HPE Medicaid enrollment process to reduce resource burden on participating hospitals and ensure ongoing patient engagement in the program. LEVEL OF EVIDENCE: Economic And Value Based Evaluations; Level II.

Characterizing the flavodoxin landscape in Clostridioides difficile.

Clostridioides difficile infections have become a major challenge in medical facilities. The bacterium is capable of spore formation allowing the survival of antibiotic treatment. Therefore, research on the physiology of C. difficile is important for the development of alternative treatment strategies. In this study, we investigated eight putative flavodoxins of C. difficile 630. Flavodoxins are small electron transfer proteins of specifically low potential. The unusually high number of flavodoxins in C. difficile suggests that they are expressed under different conditions. We determined high transcription levels for several flavodoxins during the exponential growth phase, especially for floX. Since flavodoxins are capable of replacing ferredoxins under iron deficiency conditions in other bacteria, we also examined their expression in C. difficile under low iron and no iron levels. In particular, the amount of fldX increased with decreasing iron concentration and thus could possibly replace ferredoxins. Moreover, we demonstrated that fldX is increasingly expressed under different oxidative stress conditions and thus may play an important role in the oxidative stress response. While increased fldX expression was detectable at both RNA and protein level, CD2825 showed increased expression only at mRNA level under H2O2 stress with sufficient iron availability and may indicate hydroxyl radical-dependent transcription. Although the exact function of the individual flavodoxins in C. difficile needs to be further investigated, the present study shows that flavodoxins could play an important role in several physiological processes and under infection-relevant conditions. IMPORTANCE: The gram-positive, anaerobic, and spore-forming bacterium Clostridioides difficile has become a vast problem in human health care facilities. The antibiotic-associated infection with this intestinal pathogen causes serious and recurrent inflammation of the intestinal epithelium, in many cases with a severe course. To come up with novel targeted therapies against C. difficile infections, a more detailed knowledge on the pathogen's physiology is mandatory. Eight putative flavodoxins, an extraordinarily high copy number of this type of small electron transfer proteins, are annotated for C. difficile. Flavodoxins are known to be essential electron carriers in other bacteria, for instance, during infection-relevant conditions such as iron limitation and oxidative stress. This work is a first and comprehensive overview on characteristics and expression profiles of the putative flavodoxins in the pathogen C. difficile.

Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes.

BACKGROUND: Pathogenic autosomal-dominant missense variants in MYH7 (myosin heavy chain 7), which encodes the sarcomeric protein (ß-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically actionable. However, ≈75% of MYH7 missense variants are of unknown significance. While human-induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes to enable the interrogation of MYH7 variant effect in a disease-relevant context, deep mutational scanning has not been executed using diploid hiPSC derivates due to low hiPSC gene-editing efficiency. Moreover, multiplexable phenotypes enabling deep mutational scanning of MYH7 variant hiPSC-derived cardiomyocytes are unknown. METHODS: To overcome these obstacles, we used CRISPRa On-Target Editing Retrieval enrichment to generate an hiPSC library containing 113 MYH7 codon variants suitable for deep mutational scanning. We first established that ß-MHC protein loss occurs in a hypertrophic cardiomyopathy human heart with a pathogenic MYH7 variant. We then differentiated the MYH7 missense variant hiPSC library to cardiomyocytes for multiplexed assessment of ß-MHC variant abundance by massively parallel sequencing and hiPSC-derived cardiomyocyte survival. RESULTS: Both the multiplexed assessment of ß-MHC abundance and hiPSC-derived cardiomyocyte survival accurately segregated all known pathogenic variants from synonymous variants. Functional data were generated for 4 variants of unknown significance and 58 additional MYH7 missense variants not yet detected in patients. CONCLUSIONS: This study leveraged hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants for the first time. Phenotyping strategies used here enable the application of deep mutational scanning to clinically actionable genes, which should reduce the burden of variants of unknown significance on patients and clinicians.

Medical advice lines offering on-demand access to providers reduced emergency department visits.

Instant access to clinicians through virtual care is designed to allow patients to receive care they need while avoiding high-cost visits in acute-care settings. This study investigates the effect of offering patients the option to instantly connect with emergency care providers instead of being referred to the emergency department (ED) following calls to a medical advice line. We used a staggered rollout design to assess the effects of implementing this program on key outcomes among Veterans Affairs enrollees. Analyzing over 1 million calls from 2019 to 2022, we found that access to a provider reduced the proportion of patients who subsequently visited the ED compared with those with access to the standard medical advice line (38% vs 36%). There was no significant difference observed in subsequent inpatient admissions or 30-day mortality. We found that a majority of callers (65%) achieved issue resolution or were directed to lower acuity settings for further evaluation. Although substantial direct cost savings were not evident, our findings demonstrate that on-demand access to a virtual provider can effectively decrease ED visits.

Emergency Medicaid programs may be an effective means of providing sustained insurance among trauma patients: A statewide longitudinal analysis.

BACKGROUND: Hospital Presumptive Eligibility (HPE) is a temporary Medicaid insurance at hospitalization that offsets costs of care, increases access to postdischarge resources, and provides patients with a path to sustain coverage through Medicaid. Because HPE only lasts up to 60 days, we aimed to determine Medicaid insurance status 6 months after injury among HPE-approved trauma patients and identify factors associated with successful sustainment. METHODS: Using a customized longitudinal claims data set for HPE-approved patients from the California Department of Health Care Services, we analyzed adults with a primary trauma diagnosis (International Classification of Diseases version 10) who were HPE approved in 2016 and 2017. Our primary outcome was Medicaid sustainment at 6 months. Univariate and multivariate analyses were performed. RESULTS: A total of 9,749 trauma patients with HPE were analyzed; 6,795 (69.7%) sustained Medicaid at 6 months. Compared with patients who did not sustain, those who sustained had higher Injury Severity Score (ISS > 15: 73.5% vs. 68.7%, p < 0.001), more frequent surgical intervention (74.8% vs. 64.5%, p < 0.001), and were more likely to be discharged to postacute services (23.9% vs. 10.4%, p < 0.001). Medicaid sustainment was high among patients who identified as White (86.7%), Hispanic (86.7%), Black (84.3%), and Asian (83.7%). Medicaid sustainment was low among the 2,505 patients (25.7%) who declined to report race, ethnicity, or preferred language (14.8% sustainment). In adjusted analyses, major injuries (ISS > 16) (vs. ISS < 15: adjusted odds ratio [aOR], 1.51; p = 0.02) and surgery (aOR, 1.85; p < 0.001) were associated with increased likelihood of Medicaid sustainment. Declining to disclose race, ethnicity, or language (aOR, 0.05; p < 0.001) decreased the likelihood of Medicaid sustainment. CONCLUSION: Hospital Presumptive Eligibility programs are a promising pathway for securing long-term insurance coverage for trauma patients, particularly among the severely injured who likely require ongoing access to health care services. Patient and provider interviews would help to elucidate barriers for patients who do not sustain. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.