Human Reproduction

BackgroundX chromosome inactivation (XCI) is the mechanism which randomly silences one X chromosome to equalise gene expression between 46, XX females and 46, XY males. Though XCI is expected to result in a random pattern of mosaicism across tissues, some females display a significantly unbalanced ratio in immune cells, termed XCI-skew, in which [&ge;]75% of cells have the same X inactivated. XCI-skew is associated with adverse health outcomes and its prevalence increases with age - particularly after midlife - yet the specific risk factors have yet to be identified. The menopausal transition, which is driven by profound shifts in sex hormone levels, has significant impact on chronic disease risk yet the molecular and cellular effects are incompletely understood. We hypothesised that the menopausal transition may impact XCI-skew. MethodsUsing XCI data measured in blood-derived DNA from 1,395 females from the TwinsUK population cohort, along with questionnaires, genetic data, and sex hormone measures, we carried out a cross-sectional study to assess the impact of the menopausal transition and sex hormones on XCI-skew. ResultsWe demonstrate that early menopause (<45yrs) is associated with increased risk of XCI-skew. In subset analyses across those who had a surgically induced or natural menopause, we find the association restricted to those who underwent a surgical menopause. We next identify a low polygenic score (PGS) for testosterone levels is significantly associated with XCI-skew, which we replicate in an independent dataset (n=149), while a PGS for age at natural menopause is not associated. Finally, using longitudinal measures across two time points spanning [~]18 years we show XCI-skew is a stable cellular phenotype that typically increases over time. DiscussionThese data represent the first environmental and genetic risk factors of XCI-skew, both of which implicate endogenous sex hormone levels, particularly testosterone. We propose XCI-skew may have clinical relevance in postmenopausal females.

Research questionCan tissue clearing, combined with volumetric imaging, enable reliable, quantitative three-dimensional analysis of follicles and vasculature in intact human ovarian tissue? DesignA CUBIC-based clearing protocol was adapted for human ovarian medulla and cryopreserved cortex. Tissue from reproductive-aged donors was cleared, fluorescently labeled, and imaged using confocal and light sheet microscopy. Tissue expansion, imaging depth, and vascular morphometrics were quantified and follicle density was compared to conventional histology. ResultsClearing produced optically transparent tissue with a linear expansion factor of 1.2 across cortex and medulla. Imaging depth increased 6.5-11-fold in cortex and 6-8-fold in medulla. Follicle density measurements in immunolabeled cleared cortex were comparable to histology, supporting the validity of volumetric follicle quantification. Light sheet microscopy of lectin-labeled cortex revealed no significant donor-to-donor differences in vascular morphometrics, including mean vessel diameters of 12-14 {micro}m, branch point densities of 632-965 points/mm3, vessel length densities of 117-175 mm/mm3, and volume fractions of 1.9-2.3%. Volumetric imaging further illustrated heterogeneous spatial relationships between follicles and surrounding vessels. ConclusionTissue clearing and volumetric imaging complement routine histology and enable quantitative three-dimensional investigation of follicle-vascular interactions in intact human ovarian tissue, providing a framework for advancing fertility preservation and ovarian tissue transplantation research.

STRUCTURED ABSTRACTO_ST_ABSImportanceC_ST_ABSSexual dysfunction can occur after midurethral sling (MUS) and transvaginal prolapse surgery. It remains unclear whether these procedures impact the clitoris, despite its role in sexual function and proximity to the MUS and vagina. ObjectivesTo compare postoperative sexual function and clitoral features by MUS and vaginal surgery approach after transvaginal prolapse repair with/without concomitant MUS. DesignCross-sectional ancillary study of magnetic resonance imaging (MRI) and sexual function data from the Defining Mechanisms of Anterior Vaginal Wall Descent study. SettingEight clinical sites in the US Pelvic Floor Disorders Network. Participants: 88 women with uterovaginal prolapse who underwent vaginal mesh hysteropexy or vaginal hysterectomy with uterosacral ligament suspension with/without MUS between 2013-2015. Data were analyzed between September 2021-June 2023. ExposuresBetween June 2014-May 2018, participants underwent pelvic MRI 30-42 months after surgery, or earlier if reoperation was desired. Sexual activity and function at baseline and 24-48-month follow-up were evaluated using the Pelvic Organ Prolapse/Incontinence Sexual Questionnaire, IUGA-Revised (PISQ-IR). Clitoral features were obtained from postoperative MRI-based 3-dimensional models. Main Outcomes and MeasuresPISQ-IR scores and clitoral features (size, position). ResultsEighty-two women (median [range] age, 65 [47-79] years) were analyzed: 45 MUS (22 hysteropexy, 23 hysterectomy) and 37 No-MUS (19 hysteropexy, 18 hysterectomy). Postoperatively, 25 MUS, 12 No-MUS, 20 hysteropexy, and 17 hysterectomy patients were sexually active (SA). Overall, within the MUS and vaginal surgery groups, sexual function remained unchanged or improved (most PISQ-IR change from baseline scores were [&ge;]0) among SA and NSA women. Among SA women after surgery, the MUS group (vs No-MUS) had a poorer PISQ-IR arousal/orgasm (SA-AO) score (median, 3.5 vs 4.3; P=.02). The hysteropexy group (vs hysterectomy) had less improvement in PISQ-IR SA-AO score (median, 0.0 vs 0.3; P=.01). Women with MUS (vs without) had a smaller clitoral glans thickness (median, 9.0 mm vs 10.0 mm; P=.008) and clitoral body volume (median, 2783.5 mm3 vs 3587.4 mm3; P=.01). Conclusions and RelevanceSA women with MUS (vs without) or hysteropexy (vs hysterectomy) experienced poorer postoperative sexual function. MUS was linked to a smaller clitoris. Future studies should explore surgery-induced changes in clitoral anatomy and sexual function. KEY POINTSO_ST_ABSQuestionC_ST_ABSHow do sexual function and clitoral anatomy differ by midurethral sling placement and vaginal surgery approach? FindingsThis cross-sectional study compared patient-reported sexual function outcomes and 30-42-month postoperative magnetic resonance imaging-based 3-dimensional clitoral models of 82 women after vaginal prolapse surgery with or without concomitant midurethral sling. Midurethral sling (vs no sling) and vaginal mesh hysteropexy (vs vaginal hysterectomy) were associated with poorer postoperative sexual function outcomes. Additionally, midurethral sling was associated with a smaller clitoral glans and body. MeaningMidurethral sling and vaginal mesh hysteropexy were associated with, and may adversely alter, postoperative sexual function and/or clitoral anatomy. VISUAL ABSTRACT/PROMOTIONAL IMAGE O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/26351291v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@904497org.highwire.dtl.DTLVardef@187514aorg.highwire.dtl.DTLVardef@e9e799org.highwire.dtl.DTLVardef@640f1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

The menopausal transition represents a major neuroendocrine shift marked by declining estradiol and progesterone, rising follicle-stimulating hormone, and increased vulnerability to cognitive and affective symptoms. Despite extensive evidence of hormone-related neural changes, few biomarkers directly index hormone-sensitive neuronal adaptations in vivo. Neuron-enriched extracellular vesicles (nEVs) isolated from blood provide a minimally invasive window into central nervous system (CNS) biology by carrying microRNAs (miRNAs) linked to neuronal regulatory processes. This pilot study tested whether L1 cell adhesion molecule (L1CAM)-positive nEV miRNA profiles differ between early (STRAW stage - 2; n = 22) and late (STRAW stage - 1; n = 24) perimenopause. A pooled discovery screen of 179 miRNAs identified 10 candidates with substantial fold-change differences between groups; these were then quantified at the individual level using qPCR. Linear mixed-effects models showed a significant main effect of STRAW stage, with late perimenopause associated with higher {Delta}Cq values (lower overall expression) across the miRNA panel. The miRNA x STRAW stage interaction was not significant, indicating a coordinated shift across the measured miRNAs rather than miRNA-specific regulation. No evidence of an association between nEV miRNA expression and current estradiol levels or menopausal symptom severity was observed. Bioinformatic analysis of predicted mRNA targets identified significant enrichment of the gonadotropin-releasing hormone (GnRH) receptor pathway, along with related growth factor, immune, and intracellular signaling pathways, with preferential expression in brain-relevant tissues. These findings are consistent with stage-related differences in hormone-sensitive neuronal regulatory processes across the transition.

Objective. Menopause is a significant physiological transition with implications for health outcomes (e.g., cardiometabolic), yet gaps remain in understanding the menopause transition, including how menopause timing and type influence health outcomes. Large-scale cohort studies in midlife (age~40-60) females, including the All of Us Research Program (AoURP), provide opportunities to study menopause across diverse populations and data modalities. We characterized menopause-related data in AoURP, focusing on age distributions and concordance between EHR diagnosis codes and self-reported survey responses. Methods. We analyzed menopause-related survey, EHR diagnostic code, and genomic data among ~396,000 participants in AoURP with female sex. We summarized menopause data across modalities, overlap between survey, EHR, and genomic data, and age distributions overall and across sociodemographic characteristics. Results. Among ~396,000 females, surveys captured ~193,000 menopause observations, nearly seven times more than structured EHR diagnoses (~28,000), suggesting under- ascertainement in EHR data. Nearly all females (~99%) with an EHR menopause diagnosis also reported menopause in the survey. Approximately 22,000 participants had intersected EHR, survey, and genomic menopause-related data. Survey-based age patterns matched expectations, with participants <40 years predominantly reporting pre-menopausal status and those >60 years predominantly reporting post-menopausal status. A small subset (N{approx}1,700; 4%) (age>70 years) reported no menopause, suggesting response or recall bias. EHR menopause codes were concentrated after age>45 years, with a notable spike at age 65. Modest differences in survey-based menopause age distributions were observed by sociodemographic characteristics (e.g., race, ancestry). Conclusions. These findings inform sampling strategies, power calculations, phenotype definition, and study design for menopause research using AoURP.

Preconception weight loss by metabolic-bariatric surgery (MBS) improves maternal-fetal outcomes, but little is known about its impact on offspring growth and health. The preconception bariatric surgery and child health outcomes (POSIT) study aims to estimate the effects of maternal MBS-induced preconception weight loss on infant and childhood body size, growth, and related outcomes. This report presents the methods used to construct the POSIT cohort and its baseline characteristics. This retrospective cohort study sampled members from a United States healthcare system aged 18 and older with a singleton, live birth to create three study groups: 1) a treatment group including women who underwent preconception MBS and subsequently became pregnant (n=1,374); 2) a control group matched to the MBS pre-surgery body mass index (BMI) (pre-surgery controls, n=13,740); and 3) a second control group matched to the MBS post-surgical, pre-pregnancy BMI (pre-pregnancy controls, n=13,740). MBS and pre-surgery BMI controls showed slight imbalances in that pre-surgery BMI controls were on average ~6 months younger, had 0.6 lower BMI (44.5 kg/m2) at the time of their pregnancy and were more likely to have become pregnant in earlier years than the MBS group prior to surgery. MBS and pre-pregnancy controls had comparable age (mean {+/-} SD 33 {+/-} 5 years), pre-pregnancy BMI (33 {+/-} 6 kg/m2), and year of delivery. Following matching, the MBS group had similar socioeconomic and health disparities as the pre-surgery control group, and both were worse than pre-pregnancy control group. Pregestational maternal comorbidity index improved after MBS and matched the pre-pregnancy controls. Upon extraction of offspring growth patterns and mediation analyses of maternal weight loss and metabolic responses to MBS, study findings will investigate effects of preconception weight loss by MBS on short- and long-term child health outcomes. Results will guide future studies focusing on improving maternal preconception weight and maternal-fetal outcomes.

Objective The decline of the perinatal demise rate is slowing and demises are often unexplained. Significant research has been done regarding diagnostic yield and genetic causes of demise, but little is known about how Geneticist involvement impacts outcomes. The goal of the study was to evaluate post-mortem genetic testing practices and effects of the geneticists involvement. Methods Retrospective data from 111 perinatal demise cases was examined, including rates of prenatal genetic counseling, post-delivery genetics consult, genetic testing, and autopsy investigation. Results In this cohort 54% received genetic testing and 25% received a genetics consult. When compared to those without, cases with genetic specialist involvement were associated with significant increases in testing uptake (p=0.007), diagnostic yield (p<0.001), and patient education (p<0.001). Second trimester stillbirths and those with fewer ultrasound (US) abnormalities were less likely to receive genetic testing (both p values <0.001) and consults (p<0.001, p=0.020). Conclusion Though it was not possible to avoid ascertainment bias, this data demonstrates that geneticist involvement correlates with a higher rate of testing, greater diagnostic yield, and more thorough counseling. These findings underscore the importance of integrating genetics providers into perinatal postmortem healthcare teams.

Background: The etiology and nosological status of seasonal affective disorder (SAD) as a specifier of depressive episodes versus a transdiagnostic disorder are the subject of debate. In this study, we investigated the underlying etiology of SAD and dimensional seasonality by examining their association with latitude and genetic risk for a range of traits, and investigated gene-environment interactions. Methods: This study included 12,460 adults aged 18-90 with a history of depression from the Australian Genetics of Depression Study. Regression models included predictors for latitude (distance from equator) and polygenic scores for eight traits; major depressive disorder, bipolar disorder, anxiety disorders, chronotype, sleep duration, body mass index, vitamin D levels, and educational attainment. Outcomes were SAD status and general seasonality score. Results: SAD was positively associated with latitude (OR[95%CI] = 1.05[1.03-1.06], padjusted<0.001), and there was nominal evidence of additive and multiplicative interactions between chronotype genetic risk and latitude (OR = 0.99[0.99-0.99], padjusted=0.381; OR=0.98[0.97-0.99], padjusted=0.489). General seasonality score was associated with latitude (IRR=1.01[1.01-1.01], padjusted 0.001) and genetic risk for major depressive disorder (IRR =1.02[1.01-1.03], padjusted<0.001), bipolar disorder (IRR=1.02[1.01-1.03], padjusted=0.001), anxiety disorders (IRR=1.03[1.01-1.04], padjusted<0.001), vitamin D levels (OR=0.89[0.80-0.95], padjusted=0.048), and educational attainment (IRR=0.97[0.96-0.99], padjusted<0.001). Conclusions: These findings enhance understanding of SAD etiology, highlighting contributions of psychiatric genetic risk and geographic measures on seasonal behavior, and support examining seasonality as a continuous dimension.

We completed a video-based, four-night, in-home, level 3 sleep apnea study of healthy, low-risk pregnant participants and their bed partners in order to characterize sleep physiology in the third trimester of pregnancy. Demographic, anthropometric, and baseline sleep health characteristics were recorded, and the NightOwl home sleep apnea test device was used to measure sleep breathing, posture, and architecture parameters. Symptoms of restless legs syndrome were elicited in the exit interview. Forty-one pregnant participants and 36 bed partners completed the study. Bed partners had a significantly higher prevalence of sleep apnea than their pregnant co-sleepers (31% vs. 5.9%). Bed partners also had more severe sleep apnea than their pregnant co-sleepers, and this persisted on an adjusted analysis for baseline differences in factors known to increase risk of sleep apnea. In pregnant participants, increasing gestational age was found to be protective against mild respiratory events but not more severe events. While the correlation between STOP-Bang score and measures of sleep apnea severity was weak, an affirmative response to the witnessed apneas item on the STOP-Bang questionnaire was a strong predictor of more severe sleep apnea for all participants. Smoking history also increased sleep apnea risk. Pregnant participants had lower sleep efficiency and longer self-reported sleep onset latency. Restless legs syndrome was experienced by 39.5% of the pregnant participants but no bed partners. From a sleep breathing perspective, people with healthy, low-risk pregnancies have better sleep than their bed partners despite lower sleep efficiency and higher rates of restless legs syndrome.

Alternative-splicing events (ASE) increase transcriptomic variability and play key roles in biological functions. The contribution of ASE to bipolar disorder (BD) remains largely unexplored. We performed a Transcriptome-Wide Alternative-Splicing Analysis (TWASA) to identify ASEs and genes potentially involved in BD. The study comprised 635 individuals: a discovery sample (DS) of 31 individuals from eight multiplex BD families (16 BD cases; 15 unaffected relatives), and a replication sample (RS) of 604 subjects (372 BD cases; 232 controls). Sequencing was conducted on RNA from lymphoblastoid cell lines (DS) and whole blood (RS). TWASA was performed using VAST-TOOLS (VT), rMATS (RM), and MAJIQ/MOCCASIN (MCC). Gene-set association analyses of genes containing ASEs were performed across six psychiatric disorders. Novel ASE (nASE) were investigated in the DS using FRASER. Limited gene overlap was observed across TWASA tools. MCC identified 2,031 complex ASEs involving 1,508 genes, showing the strongest genetic association with BD across psychiatric phenotypes. Prioritization of MCC-identified ASE genes yielded 441 candidates, including DOCK2 as top candidate from the DS. Replication was obtained for 98 genes, five with an identical ASE, and four (RBM26, QKI, ANKRD36, and TATDN2) showing a concordant percentage-spliced-in direction with the DS. Finally, 578 nASE were identified in the DS, with no evidence of familial segregation or differences in ASE types. This first TWASA in BD reveals tool-specific variability, complex ASE for genes specifically associated with BD, and novel candidate genes for BD. Alternative transcript isoform abundance may represent a mechanism contributing to BD pathophysiology.

Markhor (Capra falconeri) is a charismatic, threatened, large, high-altitude bovid found in parts of central and south Asia. The species faces threats such as habitat loss, hunting, poaching, livestock competition, hybridisation, and disease, yet research on wild populations is challenging. Various biological aspects, including surveys, diet, population dynamics, interactions with livestock, hybridisation, and disease, have been studied locally, along with behavior and reproductive biology, but details such as pregnancy, oestrus, and parturition timing remain unestablished. We conducted the first systematic, detailed, and fine-scale characterization of the reproductive steroid profiles of two males and five female markhors (Capra falconeri heptneri) in a captive population at the Padmaja Naidu Himalayan Zoological Park (PNHZP), West Bengal, India. We collected weekly fecal samples, standardized and validated measurements of progesterone (fP4M) and testosterone (fTM) metabolites, and conducted reproductive profiling to assess reproductive stages in both sexes. Analyses of annual fP4M and fTM data from male and female markhor individuals showed similar profiles and synchronicity, with individual variation, and peaks and baselines were evident for both hormones. In both sexes, significantly higher hormone titres were observed during the sexually active and inactive phases. Non-invasive measurement of reproductive hormones accurately reflected ovarian function in females, helping establish mating, gestation, and parturition timelines in female markhors and determine the breeding season in males. These approaches support husbandry and breeding management by identifying optimal pairing, diagnosing pregnancy, and predicting parturition in both captive and wild populations. When applied correctly, these tools could greatly aid population monitoring of other endangered species across high-altitude regions worldwide.

Objectives: Non-suicidal self-injury (NSSI) and self-harming sexual behaviours share functional and behavioural overlaps. However, the relationship between NSSI and problematic sexual behaviour (PSB) remains underexplored. This study aimed to investigate the association between NSSI and PSB in two cohorts - a non-clinical university cohort and a clinical PSB patient cohort. Methods: Data were collected from 2,189 university participants and 477 clinical PSB patients. NSSI was assessed via self-report, and PSB was measured with the Sexual Addiction Screening Test-Revised (SAST-R) Core. The four core addictive dimensions of PSB: relationship disturbance, loss of control, preoccupation, and affect disturbance, were also evaluated. Logistic regression analyses were conducted to examine the association between PSB (presence/absence and severity) and NSSI, looking at effects of gender and contributions of addictive dimensions of PSB. Results: Rates of NSSI were similar in the university (7.1%) and patient (5.7%) cohorts; stratified by gender, a higher proportion of women PSB patients had NSSI compared to in the university cohort (29.3% vs 9.3%). In the university group, who had milder PSB than patients, PSB was associated with NSSI (OR=2.11, p<0.001); a significant gender by PSB interaction was found showing that women with PSB were over four times more likely to have NSSI than men without PSB (OR=4.44, p=0.037). In contrast, PSB severity was not associated with NSSI in PSB patients (OR=1.10, p=0.25). Associations of the addictive dimensions of PSB with NSSI were observed only in the subgroup of university women, in the 'preoccupation' dimension (p<0.001). Conclusions: Our findings highlight gender-specific patterns in the association between PSB and NSSI, suggesting the need for further research and possibly targeted prevention and intervention strategies in women.

Regional volumetric assessment of perinatal brain development is currently limited by the lack of consistent high quality multi-regional segmentation methods applicable to both fetal and neonatal MRI. We present Multi-BOUNTI, a deep learning pipeline for automated multi-lobe segmentation of fetal and neonatal T2w brain MRI. The method is based on a dedicated 43-label parcellation protocol and a 3D Attention U-Net trained on brain MRI datasets of subjects spanning 21-44 weeks gestational/postmenstrual age. The pipeline integrates preprocessing, segmentation and volumetric analysis, and was evaluated on independent datasets, demonstrating fast (< 10 min/case) and accurate performance with high agreement to manually refined labels. We demonstrate the application of the framework with 267 fetal and 593 neonatal MRI datasets from the developing Human Connectome Project without reported clinically significant brain anomalies to derive normative volumetric growth models across 21-44 weeks GA/PMA. These models were used to characterise developmental trajectories, assess differences between fetal and preterm neonatal cohorts, and analyse longitudinal changes. The resulting normative models were integrated into an automated reporting framework enabling subject-specific volumetric assessment via centiles and z-scores. Multi-BOUNTI provides a unified and scalable approach for perinatal brain segmentation and volumetry, supporting large-scale studies and facilitating future clinical translation. The full pipeline is publicly available at https://github.com/SVRTK/perinatal-brain-mri-analysis.

The ovary contains two major somatic lineages, granulosa cells and interstitial cells, that arise from progenitors within the coelomic epithelium. However, how these two lineages diverge during ovarian development remains unclear. By analyzing joint single-nucleus transcriptomic and chromatin accessibility profiles of murine ovarian cells at the onset of ovary formation, we identified two somatic progenitor populations from the coelomic epithelium distinguished by expression of the nuclear receptors Nr5a1 and Nr2f2. Based on their transcriptomic trajectories, the Nr5a1+ epithelial cells preferentially transitioned toward the granulosa lineage whereas the Nr2f2+ epithelial cells differentiated into mesenchymal populations. This lineage relationship was supported by Nr2f2 lineage tracing experiments that fetal Nr2f2+ progenitors contribute to ovarian interstitial cells postnatally. To define the molecular features underlying this divergence, we performed differential gene expression and chromatin accessibility analyses and found that Nr2f2+ epithelial cells, but not Nr5a1+ cells, were enriched for Notch pathway components and Notch effector motifs. Consistently, lineage tracing of Notch-responsive cells marked Nr2f2+ interstitial cells in postnatal ovaries, whereas ectopic Notch activation in Nr5a1+ cells promoted expansion of the interstitial population accompanied by reduced granulosa cells. By integrating motif analysis with accessible chromatin-gene linkage, we also identified downstream targets regulated by Notch effectors in Nr2f2+ cells, which showed concordant changes upon ectopic Notch activation. These findings demonstrate that somatic cell fate is established early during ovarian development, with active Notch signaling specifying the interstitial lineage and a balanced Notch activity required for proper somatic lineage establishment. Significance StatementProper differentiation of somatic cell types in the fetal ovary lays the foundation for future ovarian function in adulthood. Understanding how each cell type is formed is essential for developing methods to intervene in ovarian diseases caused by cellular dysfunction. Given that common somatic progenitors give rise to both supporting and interstitial lineages, a main unanswered question is how these two lineages diverge apart from each other during ovarian development. By integrating joint single-nucleus transcriptomic and chromatin accessibility assays with lineage tracing, single cell RNA-sequencing, and mouse genetic models, we demonstrate the role of Notch signaling in specifying the interstitial lineage and separating it from the supporting cell fate.

This report identifies a bidirectional signaling axis connecting lipid metabolism to nuclear mechanotransduction, with the potential to control fatty acid/triglyceride metabolism. The sterol regulatory element-binding (SREBP) family of transcription factors control fatty acid, triglyceride and cholesterol synthesis and metabolism. The family consists of three members: SREBP1a, SREBP1c, and SREBP2, that are regulated by intracellular cholesterol levels and insulin signaling. The SREBP2-dependent control of the LDL receptor gene is a well-established target for cholesterol-lowering therapeutics and the activity of SREBP1c is an attractive target in metabolic disease. In the current report, we identify SYNE4 (nesprin-4), a component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, as a direct target of the SREBP family of transcription factors, and show that nesprin-4 in turn supports SREBP1c function. We identify functional SREBP binding sites in the human SYNE4 promoter and demonstrate that these are required for the sterol- and SREBP-dependent regulation of the promoter. Furthermore, we show that the endogenous SYNE4 gene is also regulated by SREBP1/2 and intracellular sterol levels. Interestingly, SREBP2 is responsible for the sterol regulation of the SYNE4 gene in HepG2 cells, while SREBP1 is the major regulator in MCF7 cells, demonstrating that diberent cell types use diberent SREBP paralogs to regulate the same promoter/gene. Importantly, we find that nesprin-4 is a positive regulator of SREBP1c expression and function in HepG2 cells and during the diberentiation of human adipose-derived stem cells. In summary, the current report identifies a novel regulatory interaction between lipid metabolism and the LINC complex. Importantly, we demonstrate that this signaling axis is bidirectional, forming a closed loop that has the potential to control SREBP1c activity and thereby fatty acid and triglyceride synthesis/metabolism. Based on our data, we propose that the nesprin-4-dependent regulation of SREBP1c could represent a novel therapeutic target in metabolic disease.

AO_SCPLOWBSTRACTC_SCPLOWSpatially resolved characterization of nanomaterial (NM) distribution within cellular ultrastructure is essential for understanding NM fate and activity in biological systems. Volume electron microscopy (vEM) is uniquely positioned to address this challenge, yet fully documented quantitative pipelines that simultaneously segment NMs and cellular structures remain scarce. Here, an end-to-end analytical pipeline is presented based on the example of serial block-face scanning electron microscopy (SBF-SEM) data of tumor spheroids containing nanoparticles (NPs). A hybrid segmentation strategy is adopted: a fine-tuned Cellpose-SAM model for cells and nuclei, and an empirical Bayes approach for AuNPs. The fine-tuned model outperforms both the pre-trained baseline and benchmark experiments in Amira, and shows good generalization to 2D EM datasets of varying sample types, suggesting potential as a general-purpose segmentation model for electron microscopy. Full 3D reconstruction of NP distributions reveals preferential clustering in the perinuclear region, with a median nucleus-to-NP distance of 2.57 {micro}m and NM uptake spanning several orders of magnitude across cells. Furthermore, morphological analysis of segmented cells and nuclei using 3D shape descriptors and local curvature metrics provides quantitative access to features inaccessible from single sections. Together, these results establish a reproducible, open framework for the joint quantitative analysis of NM distribution and cellular morphology in vEM data.

Toxoplasma gondii is a widespread human pathogen that has multiple, clinically relevant stages in its complex life cycle, including fast-replicating tachyzoites and latent bradyzoites. Bradyzoite differentiation is triggered by stress responses that lead to changes in transcription, translation, and metabolism. Two aspects of this process are addressed in this report: first, whether proteins that play roles in bradyzoite differentiation are specific to T. gondii and other bradyzoite-forming parasites of the Sarcocystidae family, and second, whether new bradyzoite differentiation proteins can be identified in T. gondii. To answer these questions, a phylogenetic approach was used, comparing proteomes of select members of the Sarcocystidae family that form morphologically different bradyzoite cysts and members of the Eimeriidae family that do not form cysts. This approach resulted in 8 distinct clusters of T. gondii proteins that reflected different conservation patterns; for example, one cluster showed conservation among all organisms, while another showed conservation in bradyzoite cyst-forming organisms. Known T. gondii proteins involved in bradyzoite differentiation were found in all clusters, indicating that this process uses both highly conserved pathways as well as bradyzoite-specific pathways. Importantly, the cluster containing proteins that are conserved in bradyzoite-forming organisms contained several known regulators of bradyzoites, and will be a source for identifying novel T. gondii proteins that are involved in bradyzoite differentiation.

The theory of island biogeography and its trophic extensions predict that both species richness and food-web complexity should increase with increasing ecosystem surface area. Accordingly, Species-Area Relationships (SARs) and Network-Area Relationships (NARs) are often observed to be positively-sloped, an observation that came to be considered as a law, and on which rest many area-based conservation plans for biodiversity. However, our mechanistic understanding of the driving mechanisms of SARs and NARs slopes remains limited, undermining our ability to predict how biodiversity will respond to habitat gain or loss. We show in 180 rural ponds sampled across five years that invasive alien predators reversed the SAR and NARs from positive in invader-free ponds, to negative in invaded ponds. Relationship reversal resulted from a higher prevalence of invasive alien predators driving magnified prey extinctions and simplified food webs in larger ponds. The ability of invasive alien predators to reverse SAR and NARs presumably reflected disproportionately high predation rates combined with a low sensitivity to prey extinction conferred by a wide trophic generalism. In a world where virtually all ecosystems face biological invasions, omnipresent invasive alien predators stress the pivotal role played by predation in shaping biocomplexity-area relationships, and highlight a growing need to preserve small ecosystems where invasive alien predators are less prevalent.

Cellular organelle content is fairly constant within a given cell type. This is accomplished in part by ensuring equitable organelle partitioning during division. Much of our understanding of organelle inheritance has come from investigating cells that divide in half producing two daughter cells. However, more elaborate division strategies that give rise to multiple daughters are not uncommon in nature. Here, we present the first characterization of organelle inheritance in a fungus that grows by multi-budding, producing several (2-20) daughter cells in a single cell cycle. We find that some organelles (mitochondria and ER) are evenly delivered to all growing buds, while others (vacuole and peroxisomes) are more variably inherited. We discuss the implications of even and uneven inheritance for this polyextremotolerant fungus capable of growing in dynamic, and diverse, environments.

Diabetes mellitus is characterized by chronic hyperglycemia and loss of pancreatic {beta}-cell function and mass. Current therapies focus on {beta}-cell protection and regeneration, led by GLP-1 receptor agonists. The G protein -subunit (Gs) acts as a key signaling node downstream of numerous GPCRs, integrating diverse signals that impact {beta}-cell mass and function. Elucidating the integrative role of pancreatic Gs signaling is thus crucial for understanding {beta}-cell biology. Our map of the pancreatic Gs-coupled GPCR landscape reveals sophisticated, cell-type-specific networks, positioning Gs as a central hub for intra-pancreatic communication. Previous studies in mice with {beta}-cell-specific or whole-pancreatic Gs deletion demonstrated reduced {beta}-cell mass, impaired insulin secretion, and glucose intolerance. The stronger phenotype in the whole-pancreas model--marked by -cell expansion and abnormal distribution--points to a crucial role for Gs in differential control of postnatal - and {beta}-cell proliferation. Here, we analyze the organ-wide consequences of Gs deletion using pancreas-specific Gs knockout mice (PGsKO). Consistent with prior findings, PGsKO mice exhibit reduced weight gain from four weeks and severe diabetes due to decreased {beta}-cell mass and concomitant -cell expansion. Furthermore, Gs loss induces profound architectural and functional defects in the exocrine pancreas, linked to YAP reactivation in acinar cells. Importantly, we observed attempted {beta}-cell regeneration in PGsKO mice. Although insufficient to reverse diabetes, our results delineate the full pancreatic phenotype that may facilitate these regenerative efforts and suggest that strategically biasing GPCR signaling network away from Gs could be a viable strategy to promote {beta}-cell regeneration from other pancreatic cell types. ARTICLE HIGHLIGHTSO_LIGs is a central signaling hub that integrates diverse GPCR inputs across pancreatic cell types, yet its organ-wide role remained poorly defined. C_LIO_LIWe addressed how pancreas-wide Gs deletion disrupts both endocrine and exocrine compartments, and whether regenerative programs are engaged. C_LIO_LIGs loss caused severe diabetes through {beta}-cell loss and -cell expansion, induced profound exocrine defects with YAP reactivation, and triggered attempted {beta}-cell regeneration from ducts and potentially other cell types. C_LIO_LIOur findings suggest that strategically biasing GPCR signaling away from Gs could promote regeneration from non-{beta}-cell sources, offering new therapeutic avenues for diabetes. C_LI