Its Own Architect: Flipping Cardiolipin Synthase

Current dogma assumes that lipid asymmetry in biological membranes is actively maintained and dispensable for cell viability. The inner (cytoplasmic) membrane (IM) of Escherichia coli is asymmetric. However, the molecular mechanism that maintains this uneven distribution is unknown. We engineered a conditionally lethal phosphatidylethanolamine (PE)-deficient mutant in which the presence of cardiolipin (CL) on the periplasmic leaflet of the IM is essential for viability, revealing a mechanism that provides CL on the desired leaflet of the IM. CL synthase (ClsA) flips its catalytic cytoplasmic domain upon depletion of PE to supply nonbilayer-prone CL in the periplasmic leaflet of the IM for cell viability. In the presence of a physiological amount of PE, osmotic down-shock induces a topological inversion of ClsA, establishing the biological relevance of membrane protein reorientations in wild-type cells. These findings support a flippase-less mechanism for maintaining membrane lipid asymmetry in biogenic membranes by self-organization of a lipid-synthesizing enzyme

Clonal Dynamics and Somatic Evolution of Haematopoiesis in Mouse

Haematopoietic stem cells maintain blood production throughout life1. Although extensively characterized using the laboratory mouse, little is known about clonal selection and population dynamics of the haematopoietic stem cell pool during murine ageing. We isolated stem cells and progenitors from young and old mice, identifying 221,890 somatic mutations genome-wide in 1,845 single-cell-derived colonies. Mouse stem cells and progenitors accrue approximately 45 somatic mutations per year, a rate only approximately threefold greater than human progenitors despite the vastly different organismal sizes and lifespans. Phylogenetic patterns show that stem and multipotent progenitor cell pools are established during embryogenesis, after which they independently self-renew in parallel over life, evenly contributing to differentiated progenitors and peripheral blood. The stem cell pool grows steadily over the mouse lifespan to about 70,000 cells, self-renewing about every 6 weeks. Aged mice did not display the profound loss of clonal diversity characteristic of human haematopoietic ageing. However, targeted sequencing showed small, expanded clones in the context of murine ageing, which were larger and more numerous following haematological perturbations, exhibiting a selection landscape similar to humans. Our data illustrate both conserved features of population dynamics of blood and distinct patterns of age-associated somatic evolution in the short-lived mouse

Prevalence and Patterns of Opioid Use in Chronic Pancreatitis

Introduction: Opioids are used to treat pain in chronic pancreatitis (CP), but little is known about current use patterns. The aim of this study was to characterize the utilization of opioids and associations with clinical characteristics in adult patients with CP. Methods: This cross-sectional analysis used baseline data from participants with definite CP enrolled in a cohort study in the United States (PROspective Evaluation of CP for EpidEmiologic and Translational StuDies). Data on demographics, pain medication use, healthcare utilization, disability, and pain patterns were systematically collected in case report forms while quality of life was assessed with patient-reported outcome instruments. Opioid use was classified according to strength (weak or strong) and frequency (scheduled or as-needed). Results: A total of 681 participants (n = 364, 53% male) were included: 299 (44%) were current opioid users (22% only weak opioids and 22% at least 1 strong opioid). Increasing frequency and severity of pain was associated with increase of weak, strong, as-needed, or scheduled opioids. Neuromodulators were used by ∼40% of participants; increasing use was associated with increasing frequency and severity of pain. On multivariate analysis, independent predictors associated with strength and frequency of current opioid use were pain patterns (odds ratios [ORs] 1.84-8.32 and ORs 1.92-8.52, respectively, P \u3c 0.001) and prior celiac plexus block (OR 3.54, 95% confidence intervals 1.82-6.87 and OR 3.42, 95% confidence intervals 1.76-6.64, respectively). Participants using opioids had higher prevalence of disability, healthcare utilization, and poorer quality of life. Discussion: Opioid use in CP is common and associated with increased pain severity and constancy. These data provide foundational estimates for future trials that can elucidate the complex interactions between patient factors, pain, and interventions

Impact of an Adapted Diabetes Prevention Program in a Spanish Primary Care Setting: Protocol for a Type II Hybrid Effectiveness-Implementation Cluster-Randomized Trial (ALADIM)

Background: Type 2 diabetes (T2D) is a global health concern affecting 10.5% of the adult population and is projected to rise significantly in the coming decades. Lifestyle modification programs, such as the Diabetes Prevention Program (DPP), can effectively reduce T2D risk among individuals with prediabetes. However, their implementation in real-world healthcare settings remains poor, particularly in Spain, where T2D prevalence is the highest in Europe. The ALADIM study aims to evaluate the effectiveness and implementation of an adapted DPP in Spanish Primary Care Centers (PCCs). The primary effectiveness outcome is weight, the co-primary implementation outcome is implementation fidelity. We will also assess the effect of DPP implementation on overall prediabetes management within the PCCs (spillover) by measuring the percentage of people with prediabetes receiving lifestyle advice. Methods: The ALADIM trial is a hybrid type II effectiveness-implementation cluster-randomized controlled trial involving 10 PCCs of Mallorca (Balearic Islands, Spain). PCCs will be randomized to the intervention (5 PCCs) or control (5 PCCs) group in a 1:1 ratio. The intervention group will receive training and materials to implement and deliver the adapted DPP over 12 months. The control group will continue providing usual care. The DPP will be culturally adapted using the Intervention Mapping-ADAPT (IM-ADAPT) approach. The implementation strategy will be designed using Implementation Mapping. Measures of effectiveness will be assessed at the participant level at baseline, 6 and 12 months during the intervention period, and 18 months after baseline. Implementation outcomes will be assessed at the PCC level at multiple time-points throughout the study period. Spillover will be assessed at PCC level at months -1, 6 and 18. An intention-to-treat analysis will assess effectiveness and spillover effect using generalized estimating equations. Implementation outcomes will be evaluated using a mixed-methods approach. Discussion: The ALADIM study has the potential to address the gap between research and practice by employing implementation science for evaluation, adaptation and implementation of an evidence-based diabetes intervention. The findings will contribute to the development of a sustainable and scalable implementation strategy for T2D prevention, with potential implications for policy and practice at regional and national levels. Trial registration: ClinicalTrials.gov, NCT06871059. Registered 10 March 2025, https://clinicaltrials.gov/study/NCT0687105

β-Catenin Drives the FOXC2-Mediated Epithelial-Mesenchymal Transition and Acquisition of Stem Cell Properties

Background: Aggressive forms of breast cancer, such as triple-negative breast cancer (TNBC), are associated with an increase in cancer cells that exhibit stem cell properties. The activation of the epithelial-mesenchymal transition (EMT) program, mediated by the transcription factor FOXC2, generates these stem-like cells. FOXC2 is linked to poor prognoses across various cancer types and is notably upregulated in TNBC, where it establishes and sustains these stem-like cells within the tumor population. Methods: Here, we decode the pathways regulating FOXC2 activation using EMT-enriched cell line models. Stemness was assessed using mammosphere assays and mesenchymal markers by western blot. Expression correlations with clinical data was examined using the EMTome. Results: We demonstrate that β-catenin serves as a critical mediator of mesenchymal and stemness characteristics through FOXC2 upregulation. By disrupting β-catenin, we find that FOXC2 expression, mesenchymal properties, and stemness are reduced; however, the introduction of exogenous FOXC2 expression in β-catenin deficient cells is enough to restore the mesenchymal and stemness phenotype. These findings support the idea that FOXC2 acts as the downstream regulator of β-catenin and influences both mesenchymal and stemness properties. Moreover, there is a positive correlation between the expression of β-catenin and FOXC2 in various cancer subtypes observed in clinical patient samples. Conclusions: Our study clarifies the role of the β-catenin/FOXC2 signaling axis in maintaining stemness properties, suggesting potential targets for TNBC and other cancers driven by EMT-related mesenchymal and stemness characteristics

Forward Genetic Screen in Zebrafish Identifies New Fungal Regulators That Limit Host-Protective Candida-Innate Immune Interaction

Candida is one of the most frequent causes of bloodstream infections, and our first line of defense against these invasive infections is the innate immune system. The early immune response is critical in controlling Candida albicans infection, but C. albicans has several strategies to evade host immune attack. Phagocytosis of C. albicans blocks hyphal growth, limiting host damage and virulence, but how C. albicans limits early recruitment and phagocytosis in vertebrate infection is poorly understood. To study innate immune evasion by intravital imaging, we utilized the transparent larval zebrafish infection model to screen 131 C. albicans mutants for altered virulence and phagocyte response. Infections with each of the seven hypovirulent mutants led to altered phagocyte recruitment and/or phagocytosis, falling into four categories. Of particular interest among these is NMD5, a predicted β-importin and newly identified virulence factor. The nmd5∆/∆ mutant fails to limit phagocytosis, and its virulence defects are eliminated when phagocyte activity is compromised, suggesting that its role in virulence is limited to immune evasion. These quantitative intravital imaging experiments are the first to document altered Candida-phagocyte interactions for several additional mutants and clearly distinguish recruitment from phagocytic uptake, suggesting that Candida modulates both events. This initial large-scale screen of individual C. albicans mutants in a vertebrate, coupled with high-resolution imaging of Candida-phagocyte interactions, provides a more nuanced view of how diverse mutations can lead to more effective phagocytosis, a key immune process that blocks germination and drives anti-fungal immunity. Importance: Candida albicans is part of the human microbial community and is a dangerous opportunistic pathogen, able to prevent its elimination by the host immune system. Although Candida avoids immune attack through several strategies, we still understand little about how it regulates when immune phagocytes get recruited to the infection site and when they engulf fungal cells. We tested over 130 selected Candida mutants for their ability to cause lethal infection and found several hypovirulent mutants, which provoked altered innate immune responses, resulting in lower overall inflammation and greater host survival. Of particular interest is NMD5, which acts to limit fungal phagocytosis and is predicted to regulate the activity of stress-associated transcription factors. Our high-content screening was enabled by modeling Candida infection in transparent vertebrate zebrafish larva. Our findings help us understand how Candida survives immune attack during commensal and pathogenic growth, and may eventually inform new strategies for controlling disease

Changes of Eeg Beta Band Power and Functional Connectivity During Spaceflight: A Retrospective Study

Spaceflight exposes astronauts to unique conditions like microgravity, which may affect brain function, though it remains underexplored compared to other physiological systems. Astronauts often report temporary neurological symptoms, such as disorientation, visual disturbances, and motor issues, potentially linked to structural and electrophysiological brain changes. To investigate this, electroencephalography (EEG) is a reliable tool to study brain activity in space, measuring oscillatory activity and functional connectivity (FC). This study analyzed EEG data from five male astronauts during three stages: pre-flight, during low Earth orbit (LEO), and post-flight in a 2-min task-free eyes-closed (EC) condition followed by another 2-min of eyes-open (EO) condition. The focus was on beta band (12-30 Hz) activity, which is associated with motor control and proprioception. Results showed increased beta power during spaceflight when compared to pre-flight (EC: p \u3c 0.01) and post-flight (EC: p \u3c 0.01; EO: p \u3c 0.05) conditions. FC strength also increased during spaceflight when compared to pre-flight (EO: p \u3c 0.05) and post-flight (EC: p \u3c 0.01; EO: p \u3c 0.01) conditions. These differences were found primarily in the sensorimotor cortex (SMC) and frontotemporal regions, suggesting the brain\u27s adaptation to altered vestibular and proprioceptive inputs during microgravity. As these results reflect astronaut\u27s movement adaptation to microgravity, this study highlights the importance of understanding central nervous system (CNS) changes during spaceflights to ensure optimal performance and protect astronaut\u27s health during long-duration missions

Synthetic Versus Biologic Mesh for Complex Open Ventral Hernia Repair: 3-Year Follow-Up of a Pilot Randomized Controlled Trial

Background: Biologic mesh is often used in complex hernia repair, but there has been limited clinical evidence to date to support this practice. The aim of this study was to compare clinical and patient-reported outcomes of biologic versus synthetic mesh for complex open ventral hernia repair (OVHR) at 3 years. Methods: Patients from a single center, randomized, controlled, pilot trial comparing biologic versus synthetic mesh in complex OVHR were followed for 3 years. The primary outcome focused on major complications, namely mesh infections, hernia recurrences, reoperations, and deaths. Secondary outcomes included surgical site infections, surgical site occurrences, and patient-reported outcomes. Outcomes were assessed using frequentist generalized linear models. Results: A total of 87 patients (44 biologic mesh, 43 synthetic mesh) were randomized, and 61 patients (70%; 28 biologic and 33 synthetic) completed 3-year follow-up. Baseline demographics were similar in both groups. No significant differences were seen in major complications (50% vs 30%, P = .123), mesh infection (14% vs 3%, P = .144), recurrence (39% vs 24%, P = .214), reoperation (14% vs 9%, P = .531), or mortality (4% vs 0%, P = .459) between the 2 arms. A single death occurred as a result of bacteremia in a patient with hepatocellular carcinoma. Similarly, no significant differences were seen in secondary or patient-reported outcomes. Both groups demonstrated clinically important improvements in quality of life and pain scores at 3 years. Conclusion: This study failed to find benefits with biologic mesh as opposed to synthetic mesh in complex OVHR at 3 years when comparing both clinical and patient-reported outcomes

Development and Pilot Testing of an Addiction Clinic-Based Pre-Exposure Prophylaxis Uptake and Adherence Intervention for Women with Substance Use Disorders: Protocol for a Pilot Randomized Trial

Background: Black and Hispanic women in the United States continue to bear disproportionate incidence of HIV related to sexual transmission and injection drug use. Specifically, women with substance use disorders (SUDs) are more likely to engage in vaginal or anal condomless sex associated with HIV transmission. Pre-exposure prophylaxis (PrEP) is a highly effective HIV prevention tool but is not widely used by racial or ethnic minority women. Effective interventions for engaging women with SUDs in HIV prevention interventions that are culturally appropriate and, therefore, more appealing to racial or ethnic minority women with SUDs are critically needed. Objective: This 3-phased study, including a pilot randomized controlled trial (RCT), will assess the initial efficacy, feasibility, and acceptability of an addiction clinic-based behavioral and PrEP services intervention to increase the uptake and adherence to PrEP among racial or ethnic minority women. Methods: A 3-phased mixed methods research design will involve formative qualitative methods using thematic analysis to design the intervention (phase 1), theatre testing to adapt and refine the intervention (phase 2), and RCT methods to pilot test the intervention for efficacy, feasibility, and acceptability (phase 3). The pilot RCT will enroll and randomize 60 women to either the standard SUD treatment program or SUD treatment integrated with PrEP services. The addiction clinic-based behavioral intervention will include 4 motivational counseling sessions guided by the Information-Motivation-Behavioral Skills Model to increase the uptake of PrEP. A mobile health app will be used to engage participants with the intention of motivating PrEP initiation and supporting adherence to PrEP. Following phase 3, generalized linear modeling will be used to model effects of the proportion of participants who fill their prescription and take at least 1 dose as a function of the intervention group. Results: Findings from individual qualitative interviews informed the development of the addiction clinic-based behavioral intervention. Study recruitment for the randomized pilot (phase 3) launched in May 2024. Additional statistical analyses will be performed upon completion of the study. Conclusions: This addiction clinic-based behavioral intervention aims to increase PrEP uptake and adherence among racial or ethnic minority women who engage in sexual and substance use behaviors associated with increased susceptibility to HIV transmission. The addiction clinic-based behavioral intervention has the potential to reduce HIV-related disparities among Black and Hispanic women with SUDs. Findings from this study will provide a foundation for future HIV prevention interventions for racial or ethnic minority women with SUDs. Trial registration: ClinicalTrials.gov NCT06158607; https://clinicaltrials.gov/study/NCT06158607?term=NCT06158607&rank=1. International registered report identifier (irrid): DERR1-10.2196/64961

Ensemble Learning With Explainable AI for Improved Heart Disease Prediction Based on Multiple Datasets

Heart disease is one of the leading causes of death worldwide. Predicting and detecting heart disease early is crucial, as it allows medical professionals to take appropriate and necessary actions at earlier stages. Healthcare professionals can diagnose cardiac conditions more accurately by applying machine learning technology. This study aimed to enhance heart disease prediction using stacking and voting ensemble methods. Fifteen base models were trained on two different heart disease datasets. After evaluating various combinations, six base models were pipelined to develop ensemble models employing a meta-model (stacking) and a majority vote (voting). The performance of the stacking and voting models was compared to that of the individual base models. To ensure the robustness of the performance evaluation, we conducted a statistical analysis using the Friedman aligned ranks test and Holm post-hoc pairwise comparisons. The results indicated that the developed ensemble models, particularly stacking, consistently outperformed the other models, achieving higher accuracy and improved predictive outcomes. This rigorous statistical validation emphasised the reliability of the proposed methods. Furthermore, we incorporated explainable AI (XAI) through SHAP analysis to interpret the model predictions, providing transparency and insight into how individual features influence heart disease prediction. These findings suggest that combining the predictions of multiple models through stacking or voting may enhance the performance of heart disease prediction and serve as a valuable tool in clinical decision-making