Genome-Wide CRISPR Screen Identifies KEAP1 Perturbation as a Vulnerability of ARID1A-Deficient Cells.

ARID1A is the core DNA-binding subunit of the BAF chromatin remodeling complex and is mutated in about 8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at > 50% of cases. Despite a growing understanding of the consequences of ARID1A loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of ARID1A-deficient cells.

Valproic acid-induced teratogenicity is driven by senescence and prevented by Rapamycin in human spinal cord and animal models.

Valproic acid (VPA) is an effective and widely used anti-seizure medication but is teratogenic when used during pregnancy, affecting brain and spinal cord development for reasons that remain largely unclear. Here we designed a genetic recombinase-based SOX10 reporter system in human pluripotent stem cells that enables tracking and lineage tracing of Neural Crest cells (NCCs) in a human organoid model of the developing neural tube. We found that VPA induces extensive cellular senescence and promotes mesenchymal differentiation of human NCCs. We next show that the clinically approved drug Rapamycin inhibits senescence and restores aberrant NCC differentiation trajectory after VPA exposure in human organoids and in developing zebrafish, highlighting the therapeutic promise of this approach. Finally, we identify the pioneer factor AP1 as a key element of this process. Collectively our data reveal cellular senescence as a central driver of VPA-associated neurodevelopmental teratogenicity and identifies a new pharmacological strategy for prevention. These results exemplify the power of genetically modified human stem cell-derived organoid models for drug discovery.

Impact of Vietnam-era G.I. Bill eligibility on later-life blood pressure distribution: evidence from the Vietnam draft lottery natural experiment.

We quantified the impact of Vietnam-era G.I Bill eligibility, which subsidized college education for eligible Veterans, on the later-life blood pressure distribution by exploiting the Vietnam draft lottery natural experiment. We restricted Health and Retirement Study data (2006-2018) to men born between 1947-1953 (N=1,970). We estimated intention-to-treat effects at the mean and 1st-99th quantiles of blood pressure using linear and quantile regressions. Our outcomes were systolic blood pressure (SBP), diastolic blood pressure (DBP), hypertension, and self-reported stroke. We proxied G.I. Bill eligibility using lottery-defined draft eligibility. We also conducted analyses stratified by childhood socioeconomic status (cSES) defined based on a previously validated measure. Draft eligibility reduced mean blood pressure outcomes (e.g., effect on SBP: -1.33 [95% confidence interval (CI) -2.85, 0.19]). Draft eligibility also had larger protective effects at higher quantiles of the SBP and DBP distributions relative to lower quantiles (effects on SBP at the 10th and 90th quantiles: -0.33mmHg [95% CI -2.35,1.68]; -3.00mmHg [95% CI -5.68,-0.32]). Draft eligibility had protective effects on blood pressure among low and medium cSES men but opposite effects among high cSES men. G.I. Bill eligibility reshaped the blood pressure distribution to one of lower morbidity risk, particularly among low and medium cSES men.

Acceptability of heart rate-based remote monitoring of smoking status.

Introduction: Digital interventions present a scalable solution to overcome barriers to smoking cessation treatment, and changes in resting heart rate (HR) may offer a viable option for monitoring smoking status remotely. The goal of this study was to explore the acceptability of using smartphone cameras and activity trackers to measure heart rate for use in a smoking cessation intervention. Methods: Participants (N=410), most of whom identified as female (75.8 %) with mean age 38.3 years (SD 11.4), were recruited via the Smoke Free app. They rated the perceived comfort, convenience, and likelihood of using smartphone cameras and wrist-worn devices for HR monitoring as an objective measure of smoking abstinence. Wilcoxon signed-rank tests and Kruskal-Wallis tests assessed differences in acceptability across device types and whether the participant owned an activity tracker/smartwatch or smartphone. Results: Participants reported high levels of acceptability for both HR monitoring methods, with activity trackers/smartwatches rated more favorably in terms of comfort, convenience, and likelihood of use compared to smartphone cameras. Participants indicated a statistically significantly greater likelihood of using the activity tracker/smartwatch over the smartphone camera. Participants viewed the activity tracker/smartwatch as more acceptable than the smartphone camera (87.0% vs 50.0%). Conclusions: HR monitoring via smartphone cameras and wrist-worn devices was deemed acceptable among people interested in quitting smoking. Wrist-worn devices, in particular, were preferred, suggesting their potential as a scalable, user-friendly method for remotely monitoring smoking status. These findings support the need for further exploration and implementation of HR monitoring technology in smoking cessation research and interventions.

Advances in Difference-in-differences Methods for Policy Evaluation Research.

Difference-in-differences (DiD) is a powerful, quasi-experimental research design widely used in longitudinal policy evaluations with health outcomes. However, DiD designs face several challenges to ensuring reliable causal inference, such as when policy settings are more complex. Recent economics literature has revealed that DiD estimators may exhibit bias when heterogeneous treatment effects, a common consequence of staggered policy implementation, are present. To deepen our understanding of these advancements in epidemiology, in this methodologic primer, we start by presenting an overview of DiD methods. We then summarize fundamental problems associated with DiD designs with heterogeneous treatment effects and provide guidance on recently proposed heterogeneity-robust DiD estimators, which are increasingly being implemented by epidemiologists. We also extend the discussion to violations of the parallel trends assumption, which has received less attention. Last, we present results from a simulation study that compares the performance of several DiD estimators under different scenarios to enhance understanding and application of these methods.

Driving Osteocytogenesis from Mesenchymal Stem Cells in Osteon-like Biomimetic Nanofibrous Scaffolds.

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.

Mediating pathways between neighborhood disadvantage and cardiovascular risk: Quasi-experimental evidence from a Danish refugee dispersal policy.

Research has documented that neighborhood disadvantage is associated with increased cardiovascular disease risk, but it is unclear which mechanistic pathways mediate this association across the life course. Leveraging a natural experiment in which refugees to Denmark were quasi-randomly assigned to neighborhoods across the country during 1986-1998 and using 30 years of follow-up data from population and health registers, we assessed whether and how individual-level poverty, unstable employment, and poor mental health mediate the relation between neighborhood disadvantage and the risk of hypertension, hyperlipidemia, and type 2 diabetes among Danish refugees (N= 40,811). Linear probability models using the discrete time-survival framework showed that neighborhood disadvantage was associated with increased risk of hypertension (0.05 percentage points [pp] per year [95%CI -0.00, 0.10]); hyperlipidemia (0.03 pp per year [95%CI -0.01, 0.07]), and diabetes (0.01 pp per year (95%CI -0.02, 0.03)). The Baron-Kenny product-of-coefficients method for counterfactual mediation analysis indicated that cumulative income mediated 6%-28% of the disadvantage effect on these outcomes. We find limited evidence of mediation by unstable employment and poor mental health. This study informs our theoretical understanding of the pathways linking neighborhood disadvantage with cardiovascular disease risk and identifies income security as a promising point of intervention in future research.

Individual and regional differences in the effects of school racial segregation on Black students' health.

Background: School racial segregation in the US has risen steadily since the 1990s, propelled by Supreme Court decisions rolling back the legacy of Brown v. Board. Quasi-experimental research has shown this resegregation harms Black students' health. However, whether individual or family characteristics (e.g., higher family incomes) are protective against segregation's health harms-or whether segregation is more damaging in regions of the US with fewer public sector investments-remains unclear. We leverage the quasi-random timing of school districts being released from Brown-era integration plans to examine heterogeneity in the association between resegregation and Black students' health. Methods & findings: We took an instrumental variables approach, using the timing of integration order releases as an instrument for school segregation and analyzing a pre-specified list of theoretically-motivated modifiers in the Panel Study of Income Dynamics. In sensitivity analyses, we fit OLS models that directly adjusted for relevant covariates. Results suggest resegregation may have been particularly harmful in the South, where districts resegregated more quickly after order releases. We find little evidence that the effects of school segregation differed across family income, gender, or age. Conclusion: The end of court-ordered integration threatens the health of Black communities-especially in the US South. Modestly higher incomes do not appear protective against school segregation's harms. Research using larger samples and alternative measures of school segregation-e.g., between districts, instead of within districts-may further our understanding of segregation's health effects, especially in Northern states.

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2.

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.

Superior Induced Pluripotent Stem Cell Generation through Phactr3-Driven Mechanomodulation of Both Early and Late Phases of Cell Reprogramming.

Human cell reprogramming traditionally involves time-intensive, multistage, costly tissue culture polystyrene-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft 2- and 3-dimensional hydrogel substrates/matrices (of stiffnesses ≤ 1 kPa) can drive ~2× improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multistage protocols lack intrinsic scalability, and, furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximize reprogramming outcomes. In screening the largest range of polyacrylamide (pAAm) hydrogels of varying stiffness to date (1 kPa to 1.3 MPa), we have found that a medium stiffness gel (~100 kPa) increased the overall number of reprogrammed cells by up to 10-fold (10×), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced induced pluripotent stem cells (iPSCs) having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalization of these pAAm hydrogels with poly-l-dopamine enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the tissue culture polystyrene practice is a 2-stage process). Comparative RNA sequencing analyses coupled with experimental validation revealed that a novel reprogramming regulator, protein phosphatase and actin regulator 3, up-regulated under the gel condition at a very early time point, was responsible for the observed enhanced reprogramming outcomes. This study provides a novel culture protocol and substrate for continuous hydrogel-based cell reprogramming and previously unattained clarity of the underlying mechanisms via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes.

The end of court-ordered desegregation and U.S. children's health: Quasi-experimental evidence.

School racial segregation significantly impacts racial disparities in U.S. children's health. Recently, school segregation has been increasing, partially due to Supreme Court decisions since 1991 that have made it easier for school districts to be released from court-ordered desegregation. We investigated the association of the end of court-ordered desegregation with child health using the 1997-2018 waves of the National Health Interview Survey (N=8,182 Black, 16,930 White children). We exploited quasi-random variation in the timing of school districts' releases from court orders to estimate effects on general health, body weight, mental health, and asthma, using difference-in-differences and event-study methods (including traditional and heterogeneity-robust estimators). Heterogeneity-robust difference-in-differences analyses show that release was associated with increased school segregation, improved mental health among Black children, and better self-reported health among White children. For heterogeneity-robust event-study analyses, school segregation increased steadily over time after release, with worse self-reported health and higher risk of asthma episodes among Black children 18+ years after release. Black children's mental health temporarily improved in the short term. In contrast, White children had improved self-reported health, mental health, and risk of asthma episodes in some years. Interventions to address the harms of school segregation are important for reducing racial health inequities.

A Unique Delusion with Depression in Early Stage of Neurocognitive Disorder Due to Vascular Disease: Considerations for Clinicians.

As global aging becomes more prominent, neurocognitive disorders (NCD) incidence has increased. Patients with NCD usually have an impairment in one or more cognitive domains, such as attention, planning, inhibition, learning, memory, language, visual perception, and spatial or social skills. Studies indicate that 50-80% of these adults will develop neuropsychiatric symptoms (NPS), such as apathy, depression, anxiety, disinhibition, delusions, hallucinations, and aberrant motor behavior. The progression of NCD and subsequent NPS requires tremendous care from trained medical professionals and family members. The behavioral symptoms are often more distressing than cognitive changes, causing caregiver distress/depression, more emergency room visits and hospitalizations, and even earlier institutionalization. This signifies the need for early identification of individuals at higher risk of NPS, understanding the trajectory of their NCD, and exploring treatment modalities. In this case report and review, we present an 82-year-old male admitted to our facility for new-onset symptoms of depression, anxiety, and persecutory delusions. He has no significant past psychiatric history, and his medical history is significant for extensive ischemic vascular disease requiring multiple surgeries and two episodes of cerebrovascular accident (CVA). On further evaluation, the patient was diagnosed with major NCD, vascular subtype. We discuss differential diagnoses and development of NPS from NCD in order to explain the significance of more thorough evaluation by clinicians for early detection and understanding of NCD prognosis.

Oocyte cryopreservation and reciprocal in vitro fertilization in a transgender man on long term testosterone gender-affirming hormone therapy: a case report.

Objective: To report a successful case of oocyte cryopreservation and subsequent in vitro fertilization (IVF) in a transgender male receiving continued testosterone gender-affirming hormone therapy, followed by reciprocal embryo transfer (ET). Design: A case report of a rare case of fertility preservation in a transgender man with concomitant use of testosterone therapy for 4 years before and during ovarian stimulation. Setting: Private fertility clinic with university affiliation. Patients: A 26-year-old transgender man undergoing oocyte cryopreservation before gender-affirming surgery. Interventions: Fertility preservation using oocyte cryopreservation and IVF with reciprocal fresh ET into a cisfemale partner. Main Outcome Measures: Successful oocyte cryopreservation, oocyte thawing, and reciprocal IVF cycle. Results: Oocyte cryopreservation of 29 mature oocytes. Sixteen mature oocytes survived the thaw, and 12 were fertilized with intracytoplasmic sperm injection. A fresh ET of an advanced blastocyst resulted in a clinical pregnancy and live birth. Conclusions: Fertility preservation with oocyte cryopreservation or IVF with embryo cryopreservation is feasible for patients on continued long-term testosterone gender-affirming therapy. Future studies on egg quality and reproductive outcomes are required. Our case report demonstrates a promising outcome in this patient population.

Medicaid Expansion and Perinatal Health Outcomes: A Quasi-Experimental Study.

OBJECTIVE: There has been little evidence of the impact of preventive services during pregnancy covered under the Affordable Care Act (ACA) on birthing parent and infant outcomes. To address this gap, this study examines the association between Medicaid expansion under the ACA and birthing parent and infant outcomes of low-income pregnant people. METHODS: This study used individual-level data from the 2004-2017 annual waves of the Pregnancy Risk Assessment Monitoring System (PRAMS). PRAMS is a surveillance project of the Centers for Disease Control and Prevention and health departments that annually includes a representative sample of 1,300 to 3,400 births per state, selected from birth certificates. Birthing parents' outcomes of interest included timing of prenatal care, gestational diabetes, hypertensive disorders of pregnancy, cigarette smoking during pregnancy, and postpartum care. Infant outcomes included initiation and duration of breastfeeding, preterm birth, and birth weight. The association between ACA Medicaid expansion and the birthing parent and infant outcomes were examined using difference-in-differences estimation. RESULTS: There was no association between Medicaid expansion and the outcomes examined after correcting for multiple testing. This finding was robust to several sensitivity analyses. CONCLUSIONS FOR PRACTICE: Study findings suggest that expanded access to more complete insurance benefits with limited cost-sharing for pregnant people, a group that already had high rates of insurance coverage, did not impact the birthing parents' and infant health outcomes examined.

Evaluation of Changes in Prices and Purchases Following Implementation of Sugar-Sweetened Beverage Taxes Across the US.

Importance: Sugar-sweetened beverage (SSB) taxes are promoted as key policies to reduce cardiometabolic diseases and other conditions, but comprehensive analyses of SSB taxes in the US have been difficult because of the absence of sufficiently large data samples and methods limitations. Objective: To estimate changes in SSB prices and purchases following SSB taxes in 5 large US cities. Design, Setting, and Participants: In this cross-sectional study with an augmented synthetic control analysis, changes in prices and purchases of SSBs were estimated following SSB tax implementation in Boulder, Colorado; Philadelphia, Pennsylvania; Oakland, California; Seattle, Washington; and San Francisco, California. Changes in SSB prices (in US dollars) and purchases (volume in ounces) in these cities in the 2 years following tax implementation were estimated and compared with control groups constructed from other cities. Changes in adjacent, untaxed areas were assessed to detect any increase in cross-border purchases. Data used for this analysis spanned from January 1, 2012, to February 29, 2020, and were analyzed between June 1, 2022, and September 29, 2023. Main Outcomes and Measures: The main outcomes were the changes in SSB prices and volume purchased. Results: Using nutritional information, 5500 unique universal product codes were classified as SSBs, according to tax designations. The sample included 26 338 stores-496 located in treated localities, 1340 in bordering localities, and 24 502 in the donor pool. Prices of SSBs increased by an average of 33.1% (95% CI, 14.0% to 52.2%; P < .001) during the 2 years following tax implementation, corresponding to an average price increase of 1.3¢ per oz and a 92% tax pass-through rate from distributors to consumers. SSB purchases declined in total volume by an average of 33.0% (95% CI, -2.2% to -63.8%; P = .04) following tax implementation, corresponding to a -1.00 price elasticity of demand. The observed price increase and corresponding volume decrease immediately followed tax implementation, and both outcomes were sustained in the months thereafter. No evidence of increased cross-border purchases following tax implementation was found. Conclusions and Relevance: In this cross-sectional study, SSB taxes led to substantial, consistent declines in SSB purchases across 5 taxed cities following price increases associated with those taxes. Scaling SSB taxes nationally could yield substantial public health benefits.

Exploring the cell interactome: deciphering relative impacts of cell-cell communication in cell co-culture using a novel microfluidic device.

The human body is made up of approximately 40 trillion cells in close contact, with the cellular density of individual tissues varying from 1 million to 1 billion cells per cubic centimetre. Interactions between different cell types (termed heterotypic) are thus common in vivo. Communication between cells can take the form of direct cell-cell contact mediated by plasma membrane proteins or through paracrine signalling mediated through the release, diffusion, and receipt of soluble factors. There is currently no systematic method to investigate the relative contributions of these mechanisms to cell behaviour. In this paper, we detail the conception, development and validation of a microfluidic device that allows cell-cell contact and paracrine signalling in defined areas and over a variety of biologically relevant length scales, referred to as the interactome-device or 'I-device'. Importantly, by intrinsic device design features, cells in different regions in the device are exposed to four different interaction types, including a) no heterotypic cell interaction, b) only paracrine signalling, c) only cell-cell direct contact, or d) both forms of interaction (paracrine and cell-cell direct contact) together. The device design was validated by both mathematical modelling and experiments. Perfused stem cell culture over the medium term and the formation of direct contact between cells in the culture chambers was confirmed. The I-device offers significant flexibility, being able to be applied to any combination of adherent cells to determine the relative contributions of different communication mechanisms to cellular outcomes.

Transforming undergraduate laboratory courses with interlinked real-world challenges.

Undergraduate laboratory course components often provide training in various techniques without connections to an interlinked real-world scenario. This article emphasizes the benefits of longitudinal integration of research techniques to enhance learning and emphasize societal relevance. An example of a biomedical engineering challenge involving a new pandemic is described.

Deconstructing heterogeneity of replicative senescence in human mesenchymal stem cells at single cell resolution.

Following prolonged cell division, mesenchymal stem cells enter replicative senescence, a state of permanent cell cycle arrest that constrains the use of this cell type in regenerative medicine applications and that in vivo substantially contributes to organismal ageing. Multiple cellular processes such as telomere dysfunction, DNA damage and oncogene activation are implicated in promoting replicative senescence, but whether mesenchymal stem cells enter different pre-senescent and senescent states has remained unclear. To address this knowledge gap, we subjected serially passaged human ESC-derived mesenchymal stem cells (esMSCs) to single cell profiling and single cell RNA-sequencing during their progressive entry into replicative senescence. We found that esMSC transitioned through newly identified pre-senescent cell states before entering into three different senescent cell states. By deconstructing this heterogeneity and temporally ordering these pre-senescent and senescent esMSC subpopulations into developmental trajectories, we identified markers and predicted drivers of these cell states. Regulatory networks that capture connections between genes at each timepoint demonstrated a loss of connectivity, and specific genes altered their gene expression distributions as cells entered senescence. Collectively, this data reconciles previous observations that identified different senescence programs within an individual cell type and should enable the design of novel senotherapeutic regimes that can overcome in vitro MSC expansion constraints or that can perhaps slow organismal ageing.