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PubMed2026

A doubly robust framework for addressing outcome-dependent selection bias in multi-cohort EHR studies.

Selection bias can hinder accurate estimation of association parameters in binary disease risk models using non-probability samples like electronic health records (EHRs). The issue is compounded when participants are recruited from multiple clinics/centers with varying selection mechanisms that may depend on the disease/outcome of interest. Traditional inverse-probability-weighted (IPW) methods, based on constructed parametric selection models, often struggle with misspecifications when selection mechanisms vary across cohorts. This paper introduces a new Joint Augmented Inverse Probability Weighted (JAIPW) method, which integrates individual-level data from multiple cohorts collected under potentially outcome-dependent selection mechanisms, with data from an external probability sample. JAIPW offers double robustness by incorporating a flexible auxiliary score model to address potential misspecifications in the selection models. We outline the asymptotic properties of the JAIPW estimator, and our simulations reveal that JAIPW achieves up to 6 times lower relative bias and 5 times lower root mean square error (RMSE) compared to the best performing joint IPW methods under scenarios with misspecified selection models. Applying JAIPW to the Michigan Genomics Initiative (MGI), a multi-clinic EHR-linked biobank, combined with external national probability samples, resulted in cancer-sex association estimates closely aligned with national benchmark estimates. We also analyzed the association between cancer and polygenic risk scores (PRS) in MGI to illustrate a situation where the exposure variable is not measured in the external probability sample.

Selection Bias↗

PubMed2026

Robust prioritization of genomic features with stability selection.

MOTIVATION: The heterogeneity of complex diseases including cancer leads to heavy-tailed distributions in the disease traits. In such settings, non-robust variable selection methods are inherently susceptible to data contamination and can yield unstable or misleading results. This vulnerability becomes more severe for recently proposed approaches that introduce pseudo-features as negative controls, as these methods further amplify the curse of dimensionality by expanding the genotype matrix in the presence of outliers and high-dimensional genomic features. RESULTS: We develop a robust variable selection framework with stability selection to prioritize genomic features in the presence of contamination. In contrast to existing approaches that rely on pseudo-features for error control, the proposed method achieves double robustness. First, it adopts least absolute deviation (LAD) LASSO to ensure robustness against outliers and heavy-tailed errors in disease traits. Second, it avoids augmenting the genotype matrix with pseudo-features, thereby mitigating the curse of dimensionality that is particularly problematic in high-dimensional genomic data. The proposed method has been extensively evaluated in simulation studies to demonstrate its effectiveness over multiple competing methods for variable selection. In addition, we have applied the proposed method and competing approaches to two real-data case studies: the The Cancer Genome Atlas (TCGA) Skin Cutaneous Melanoma (SKCM) dataset and an eQTL dataset. The results demonstrate that the proposed method achieves superior performance by identifying genomic features with higher reproducibility. AVAILABILITY AND IMPLEMENTATION: The source code for implementing the proposed methods is publicly available at https://github.com/cenwu/RSS with an archival DOI https://doi.org/10.6084/m9.figshare.32306883.

Genomics↗

PubMed2025

Semiparametric efficient estimation of small genetic effects in large-scale population cohorts.

Population genetics seeks to quantify DNA variant associations with traits or diseases, as well as interactions among variants and with environmental factors. Computing millions of estimates in large cohorts in which small effect sizes and tight confidence intervals are expected, necessitates minimizing model-misspecification bias to increase power and control false discoveries. We present TarGene, a unified statistical workflow for the semi-parametric efficient and double robust estimation of genetic effects including $ k $-point interactions among categorical variables in the presence of confounding and weak population dependence. $ k $-point interactions, or Average Interaction Effects (AIEs), are a direct generalization of the usual average treatment effect (ATE). We estimate genetic effects with cross-validated and/or weighted versions of Targeted Minimum Loss-based Estimators (TMLE) and One-Step Estimators (OSE). The effect of dependence among data units on variance estimates is corrected by using sieve plateau variance estimators based on genetic relatedness across the units. We present extensive realistic simulations to demonstrate power, coverage, and control of type I error. Our motivating application is the targeted estimation of genetic effects on trait, including two-point and higher-order gene-gene and gene-environment interactions, in large-scale genomic databases such as UK Biobank and All of Us. All cross-validated and/or weighted TMLE and OSE for the AIE $ k $-point interaction, as well as ATEs, conditional ATEs and functions thereof, are implemented in the general purpose Julia package TMLE.jl. For high-throughput applications in population genomics, we provide the open-source Nextflow pipeline and software TarGene which integrates seamlessly with modern high-performance and cloud computing platforms.

Humans↗

PubMed2026

Tetraploid Caenorhabditis elegans embryos exhibit enhanced tolerance to osmotic stress.

Polyploidy is a widespread phenomenon in development and evolution and is frequently associated with altered cellular physiology and developmental robustness. However, how genome doubling influences an embryonic robustness to environmental stress remains poorly understood. Here, we investigated developmental traits and osmotic responses in tetraploid Caenorhabditis elegans embryos. Tetraploid animals exhibited increased body and tissue sizes and produced larger embryos than diploids, accompanied by moderately delayed development and partial embryonic arrest. When exposed to a range of osmotic environments, both diploid and tetraploid embryos swelled or shrank in response to external osmolarity. Strikingly, tetraploid embryos at the early stage maintained normal cell division across a broader range of osmotic conditions than diploids. Quantitative analyses further revealed that tetraploid embryos exhibited reduced cytoplasmic mass density, primarily reflecting lower protein concentration, while lipid and RNA levels remain unaffected. These compositional differences likely buffer cellular size fluctuations and underlie the enhanced osmotic tolerance of tetraploid embryos. Together, our findings demonstrate that genome doubling reshapes embryonic cellular physiology in a non-proportional manner to ploidy, thereby enhancing robustness during early development.

Animals↗

PubMed1987

Behavioral determination of refractory periods of the brainstem substrates of self-stimulation.

The objective of this study was to estimate the refractory periods of the brainstem neurons responsible for self-stimulation behavior in the rat. In a first experiment, we tested the robustness of the double pulse technique used to estimate the refractory periods of reward-relevant neurons. We obtained estimates of the relative T-pulse effectiveness at a wide range of stimulation frequencies. The results of this experiment suggest that the refractory period estimates obtained with the behavioral version of the double pulse technique are not dependent on the arbitrary choice of the stimulation frequency. However, the use of stimulation frequencies higher than 100 Hz should preferably be avoided. In a second experiment, we applied the double pulse technique using C-pulse intensity higher than T-pulse intensity to estimate the refractory periods of the brainstem reward-relevant neurons. Using moveable electrodes, we tested 9 metencephalic and 7 mesencephalic sites in 4 animals. In the metencephalon, the most excitable reward-relevant neurons have absolute refractory periods of less than 0.6 and 0.8 ms and have a supernormal period that occurs at least between 5 and 10 ms after the initial excitation. The mesencephalic reward-relevant neurons were found to have more heterogeneous physiological characteristics. The most excitable cells in the mesencephalon have absolute refractory periods of less than 0.4 ms and have a supernormal period occurring as soon as 2.4 ms after the initial excitation. At some mesencephalic sites, we observed first an abrupt initial recovery followed by a plateau, followed by a renewed and continuous recovery, a pattern that was never observed in the metencephalon. The hypothesis of the contribution of two distinct sub-populations of reward-relevant neurons is proposed and the implication of monoaminergic pathways in reward is discussed.

Animals↗

PubMed2026

De novo transcriptome meta-analysis reveals candidate genes involved in life-stage transitions for RNAi-mediated management of the citrus root weevil (Diaprepes abbreviatus).

BACKGROUND: The citrus root weevil, Diaprepes abbreviatus, is a destructive agricultural pest for which molecular control options remain limited due to historically sparse genomic resources. Leveraging a comprehensive de novo transcriptome, we investigated developmental gene regulation across larval, pupal, and adult stages and identified essential targets for RNA interference (RNAi)-based intervention. RESULTS: Stage-resolved transcriptomic analyses revealed extensive transcriptional reprogramming associated with metabolism, detoxification, cuticle biosynthesis, endocrine signaling, and sensory perception. Among these, chitin synthase (DaCHS) emerged as a critical developmental gene, exhibiting pronounced up-regulation during late larval and pupal stages corresponding to intensive cuticle synthesis. Phylogenetic and structural analyses demonstrated that DaCHS is highly conserved among insects and retains canonical catalytic domains and transmembrane topology. Alpha Fold-based structural modeling and molecular docking confirmed stable interaction of DaCHS with its substrate, N-acetylglucosamine, supporting functional conservation of enzymatic activity. Oral delivery of DaCHS double-stranded RNA induced robust transcript suppression, leading to significant mortality and severe developmental defects, including larval and pupal abnormalities, and adults with disrupted wing and abdominal morphogenesis. CONCLUSION: These findings establish DaCHS as an indispensable gene for D. abbreviates development and validate transcriptome-guided RNAi as a powerful framework for target discovery. This work provides a strong molecular foundation for developing RNAi-based strategies that can be integrated into sustainable management programs for citrus root weevil control. © 2026 Society of Chemical Industry.

Animals↗

PubMedDate not supplied

An in vitro skin corrosivity test--modifications and validation.

An in vitro rat epidermal slice technique has been developed for identifying chemicals with potential to cause a corrosive lesion in animal skin in vivo. This potential has been correlated with the ability to lyse stratum corneum and has been measured as a lowering of the electrical resistance of the skin slice. Initial validation of the technique with 63 chemicals resulted in a high sensitivity for corrosive chemicals but a lower specificity for irritant chemicals. Subsequent modification relating to chemical contact resulted in an improved specificity (i.e. fewer false positives) at the expense of a small loss in sensitivity (i.e. an increase in the number of false negatives). An intralaboratory double blind trial with 34 corrosive chemicals and 36 irritants showed the technique to have total sensitivity (i.e. no false negatives) and a specificity of 88%. The results of the initial validation and the double blind trial illustrate the robust nature and high reproducibility of this in vitro technique for identifying skin-corrosive chemicals. Overall the model has considerable potential as a pre-screen for conventional animal tests with the additional advantage of providing objective and quantifiable information.

Animals↗

PubMed2025

PLNMFG: Pseudo-label guided non-negative matrix factorization model with graph constraint for single-cell multi-omics data clustering.

The development of single-cell multi-omics sequencing technologies has enabled the simultaneous analysis of multi-omics data within the same cell. Accurate clustering of these cells is crucial for downstream analyses of complex biological functions. Despite significant advances in multi-omics integration approaches, current methodologies exhibit two major limitations. First, they inadequately incorporate prior biological knowledge from various omic layers. Second, these methods often conduct independent dimensionality reduction on individual omic datasets, thereby failing to capture the intrinsic complementary information and potentially overlooking crucial cross-platform interactions. Motivated by these, this study investigates a non-negative matrix factorization model called PLNMFG, which integrates the unified latent representation learning that retains the features between and within omics and the cluster structure learning that retains the intrinsic structure of the data into one joint framework. Specially, PLNMFG performs adaptive imputation to handle dropout events and uses prior pseudo-labels as constraints during the process of collective non-negative matrix factorization, as a result, a more robust latent representation that preserves the double similarity information is obtained. Graph Laplacian constraint is applied during clustering which further preserves structure characteristic of multi-omics data. In addition, the weight of each omic is adaptively learned based on the omic contribution. A series of experiments on 8 benchmark datasets show that our model performs well in terms of clustering accuracy and computational efficiency.

Single-Cell Analysis↗

PubMed2025

Efficiency of DNA repair mechanisms of domestic dog primary fibroblasts isolated from small and large breeds of different ages in response to double stranded breaks (DSB).

Aging is associated with increased genomic instability, a phenomenon largely driven by the accumulation of DNA damage over time, and large species of mammals seem to have more robust DNA repair systems associated with longer lives. Among DNA lesions, double-strand breaks (DSBs) are particularly deleterious and have been implicated in age-related functional decline and disease. In this study, we investigated how age and body mass affect the efficiency of DSB repair (DSBr) in primary fibroblast cells isolated from domestic dogs, a species that exhibits significant intraspecies variation in lifespan and body mass. Primary fibroblast cells were isolated from puppies and senior dogs of both large and small breeds. Cells were treated with 100 µM etoposide to induce DSBs and subsequently analyzed at two post-treatment recovery intervals (2 and 24 h) to correlated with the two pathways associated with DSBr, the fast, non-homologous end joining (NHEJ) and the much slower, homologous recombination (HR). Cells were stained for γ-H2AX foci and images were collected using confocal microscopy. We found that mean fluorescence per cell was higher in older dogs of both size classes in the 2 h recovery, indicating higher amounts of DNA damage but suggesting similar efficiencies through the NHEJ repair mechanism in older dogs despite size class. We also show that mean fluorescence per cell was higher in the older large breed dogs in the 24 h recovery, suggesting that the slower phase associated with HR seems to be deficient in cells from older, larger breeds of dogs. These findings support the broader theory that aging is associated with impaired genomic maintenance and establish domestic dogs as a valuable model for studying the cellular mechanisms of age-related genomic instability.

Animals↗

PubMed1992

Effects of alprazolam on pituitary-adrenal and catecholaminergic responses to metabolic stress in humans.

Concurrent effects of benzodiazepines on stress-induced activation of the three classical "stress" systems: pituitary-adrenal, adrenomedullary, and sympathoneural systems have not been extensively investigated in humans. In the present study, the effects of alprazolam (1.5 mg) on plasma levels of adrenocorticotropin hormone (ACTH), epinephrine, norepinephrine, dihydroxyphenylglycol (DHPG, the intraneuronal metabolite of norepinephrine), and mood states were examined in 10 healthy volunteers undergoing glucoprivic stress. Glucoprivic stress was induced by intravenous administration of the glucose analog, 2-deoxyglucose (2DG), at a dose (50 mg/kg) that impairs cellular glucose metabolism and produces a state comparable to hypoglycemia. Alprazolam and 2DG were administered in a double-blind, placebo-controlled manner. 2DG produced robust elevations in plasma ACTH and epinephrine levels, modest elevations in plasma norepinephrine levels, and decreases in plasma DHPG levels. Alprazolam significantly attenuated the 2DG-induced increases in plasma ACTH and epinephrine, but did not significantly effect plasma norepinephrine and DHPG. These data suggest that benzodiazepines attenuate metabolic stress-induced activation of the pituitary-adrenal and adrenomedullary systems but do not effect 2DG-related effects on peripheral sympathoneural function. The possible mechanisms involved are discussed.

Adrenocorticotropic Hormone↗

PubMed2026

Venetoclax added to dose-adjusted EPOCH-R for newly diagnosed double-hit lymphomas: phase 2 results from ALLIANCE A051701, an open-label, randomised, controlled, phase 2-3 trial.

BACKGROUND: High-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6, known as double-hit lymphoma, is a highly aggressive malignancy with poor outcomes after standard chemoimmunotherapy. We aimed to study whether the addition of the BCL2-inhibitor venetoclax to chemoimmunotherapy in patients with double-hit lymphoma resulted in superior efficacy compared with chemotherapy alone. METHODS: ALLIANCE A051701 is an open-label, randomised, controlled, phase 2-3 trial in separate cohorts of patients with double-hit lymphoma and patients with double-expressor lymphoma. In this analysis, we report phase 2 results from the double-hit lymphoma cohort. Patients aged 18-80 years with newly diagnosed double-hit lymphoma and Eastern Cooperative Oncology Group (ECOG) performance status 0-2 were recruited from 41 hospitals and outpatient clinics in the USA. Patients were randomly assigned (1:1) to receive DA-EPOCH-R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) either alone (DA-EPOCH-R group) or with venetoclax (DA-EPOCH-R plus venetoclax group) using permuted block randomisation schedule. All patients and investigators were aware of group assignment. DA-EPOCH-R was administered on a 21-day schedule for up to six total cycles. Venetoclax was given as 600 mg by mouth daily on days 4-8 of cycle 1 and on days 1-5 of cycles 2-6. The primary endpoint was progression-free survival in the modified intent-to-treat population inclusive of all eligible patients with centrally confirmed double-hit lymphoma. The safety analysis population consisted of all evaluable patients who received at least one dose of protocol treatment. This trial is registered with ClinicalTrials.gov (NCT03984448) and is closed to enrolment. FINDINGS: 36 patients were randomly assigned to the DA-EPOCH-R group and 37 to the DA-EPOCH-R plus venetoclax group between Oct 22, 2019, and Sept 18, 2020. Median age was 65 years (IQR 56-73) and baseline demographic factors were well balanced between groups, with 30 (45%) female and 36 (55%) male patients. Most patients (59 [89%]) were white, two (3%) were Asian, one (2%) was Black or African American, and four (6%) had unknown or unreported ethnicity. The majority of patients had MYC-BCL2 double-hit lymphoma (59 [89%] patients), advanced stage disease (57 [86%] patients), and high-intermediate/high-risk IPI score (42 [64%] patients). Median follow-up was 34·7 months (IQR 30·1-36·8). Median progression-free survival was 28·4 months (95% CI 5·2-not estimable) in the DA-EPOCH-R group (n=30) and 7·7 months (95% CI 4·7-NE) in the DA-EPOCH-R plus venetoclax group (n=36; hazard ratio [HR] 1·13, 95% CI 0·53-2·37; p=0·75). Deaths on treatment occurred in one (3%) patient in the DA-EPOCH-R group (due to dyspnoea; possibly related to treatment) and six (17%) patients in the DA-EPOCH-R plus venetoclax group (four due to sepsis [three at least possible related and one unrelated], two due to cardiac arrest [at least possibly related]), prompting early closure of the double-hit lymphoma cohort. The most common grade 3-4 non-haematological adverse event was febrile neutropenia, occurring in 15 (43%) of 35 patients in the DA-EPOCH-R plus venetoclax group and 11 (37%) of 30 patients in the DA-EPOCH-R group. The median overall survival has not been reached in either group. The 24-month overall survival estimates were 72% (95% CI 52-85) in the DA-EPOCH-R group compared with 52% (95% CI 33-68) in the DA-EPOCH-R plus venetoclax group (HR 2·49, 95% CI 1·03-6·04; p=0·038). INTERPRETATION: The addition of venetoclax to DA-EPOCH-R resulted in excess mortality, prompting early study closure. Robust accrual shows that prospective multicentre trials are feasible in double-hit lymphoma, and the outcomes in the DA-EPOCH-R group serve as a benchmark for future studies. FUNDING: National Cancer Institute of the National Institutes of Health.

Humans↗

PubMed2026

Causal associations between hormone replacement therapy and brain structure: Evidence from large-scale Mendelian randomization and double machine learning.

BACKGROUND: Hormone replacement therapy (HRT) is widely prescribed for the management of hormone deficiency, particularly during menopause, yet its causal effects on human brain structure remain incompletely understood. Observational studies have reported heterogeneous associations, underscoring the need for robust causal inference. METHODS: We applied an integrated causal framework combining two-sample Mendelian Randomization (MR) and Double Machine Learning (DML) to evaluate the effects of four HRT-related exposures-age at initiation, age at cessation, ever-use of HRT, and a composite medication-based phenotype-on 1366 brain imaging-derived phenotypes from the UK Biobank. Genetic instruments were derived from large-scale GWAS summary statistics, and causal estimates were validated using non-parametric DML models with cross-fitting and performance evaluation. RESULTS: Genetic instruments for age at HRT initiation, age at cessation, and ever-use of HRT were strong (median F-statistics 16.29-36.66). MR analyses identified a causal association between later initiation of HRT and lower orientation dispersion in the right inferior cerebellar peduncle (ubm-a-542; primary finding, no pleiotropy detected). An additional association with the left tapetum FA (ubm-a-243) was identified but exhibited significant directional horizontal pleiotropy (MR-Egger intercept P = 0.001) and is excluded from primary conclusions (Supplementary Note S2). Later cessation of HRT was associated with increased cortical thickness in the left middle occipital gyrus, reduced surface area in the left frontopolar cortex, and increased orientation dispersion in the splenium of the corpus callosum. Ever-use of HRT was causally linked to larger volumes of the right inferior frontal gyrus and right nucleus accumbens. These associations were corroborated by independent DML validation, which provided causally debiased estimates robust to high-dimensional confounding. Results for ukb-b-8080 (median F = 1.45) are provided in Supplementary Note S1 only; weak-instrument bias precludes causal inference. CONCLUSIONS: This study provides genetic-instrument-based and machine-learning-validated evidence for causal associations between HRT exposure-particularly its timing and lifetime use-and specific features of human brain structure, including white-matter microarchitecture, cortical thickness, and regional brain volume. These findings are FDR-controlled within exposures and independently replicated by DML, but require replication in external neuroimaging GWAS cohorts to establish definitive causal conclusions. They highlight the neurobiological relevance of sex steroid exposure and inform future research on brain aging and personalized hormone-based interventions.

Humans↗

PubMed2026

Efficacy of a high-frequency repetitive transcranial magnetic stimulation for craving reduction in adolescents with gaming disorder: a 4-week randomized control trial with 24-week follow-up.

BACKGROUND: With the widespread popularity of online gaming, gaming addiction has come under scrutiny. While there is ongoing research on the diagnosis and treatment of gaming disorder among adolescents, the clinical robustness and reliability of intervention strategies remain uncertain. METHODS: A 4-week, double-blind, randomized, sham-controlled clinical trial was conducted to evaluate the efficacy of noninvasive, high-frequency repetitive transcranial magnetic stimulation (rTMS) in alleviating psychological craving in adolescents diagnosed with gaming disorder. Sham rTMS was administered to the control group using the tilted-coil method. Both groups of participants were treated with SSRI medications. A total of 80 adolescents with gaming disorder participated in this study, and 73 ultimately completed the 24-week follow-up. The primary outcome was the change in craving levels before and after the rTMS intervention, as assessed by the Visual Analogue Scale (VAS). Secondary outcomes included changes in anxiety and depression levels before and after the intervention, as assessed by the HAMA and HAMD. RESULTS: We assessed levels of psychological craving, anxiety, and depression among adolescents with gaming disorder at baseline, after completing a 4-week intervention, and at a 24-week follow-up post-intervention. Our repeated-measures MANOVA results, adjusted for course variables, revealed a significant main effect of rTMS intervention on psychological craving levels in adolescents addicted to online games (F(11, 781) = 11.238, P < 0.001, partial η² = 0.142), as well as significant main effects on time (F(11, 781) = 6.809; P < 0.001, partial η² = 0.091) and group effects (F(1, 71) = 26.707, P < 0.001, partial η² = 0.282). In addition, repeated-measures ANOVA results showed significant time effects for anxiety (F(2, 142) = 20.747, P < 0.001, partial η² = 0.234) and depression levels (F(2, 142) = 22.277, P < 0.001, partial η² = 0.285) among adolescents with gaming disorder, with nonsignificant between-group effects and no intergroup interaction. In the active stimulation group, changes in psychological craving levels after 4 weeks of treatment were significantly and positively correlated with changes in anxiety levels after 4 weeks of treatment in adolescents addicted to online games (r = 0.335, P < 0.05). CONCLUSION: Our findings indicate that high-frequency rTMS targeting the left dorsolateral prefrontal cortex may be a promising approach for reducing psychological craving in adolescents with gaming disorder. TRIAL REGISTRATION: ChiCTR2500102979 in chictr.org.cn, registered on May 22, 2025.

Humans↗

PubMed1992

Paracrine communication regulates adrenocorticotropin secretion.

Local communication among cells of the anterior pituitary appears to play an important role in the regulation of ACTH secretion. Dissociated pituitary cells were plated as a monolayer at decreasing concentrations of cells (increasing the distance between cells and, thus, decreasing their potential interactions), and ACTH secretion was measured from individual corticotropes using a specific reverse hemolytic plaque assay. There was a critical intercell distance above which significant changes in the number of CRF-responsive corticotropes were observed. Provided that this critical distance was not exceeded the number of secretory corticotropes in response to CRF (10 nM) was relatively constant, thereby defining a fraction of corticotropes that was robustly CRF responsive. In contrast, when this critical distance between cells was exceeded, the number of CRF-responsive corticotropes progressively increased to almost double their original number, thereby defining a second fraction of CRF-responsive corticotropes that was previously repressed. These observations suggest the presence of a paracrine factor that profoundly inhibits CRF-stimulated ACTH secretion from a repressed fraction of corticotropes. Further independent studies confirmed and extended these observations. We identified the cellular source of the inhibitory factor as the robustly CRF-responsive fraction of corticotropes. Pituitary cells were identified by reverse hemolytic plaque assay and then destroyed using a laser photoablation procedure that did not compromise the remaining cells. The pituitary cells were separated by a distance at which the inhibitory factor was fully effective. Destruction of the cellular source of the paracrine inhibition would, therefore, allow secretion from the previously repressed fraction of corticotropes. Accordingly, when robustly CRF-responsive corticotropes were destroyed, a significant number of previously repressed corticotropes appeared in a second assay. Destruction of somatotropes or a cell adjacent to a robustly CRF-responsive corticotrope did not alter the number of CRF-stimulated corticotropes among the remaining cells. We conclude that a paracrine factor liberated by the robustly CRF-responsive corticotropes inhibits ACTH secretion from the repressed fraction of corticotropes. The robustly CRF-responsive corticotropes appear unresponsive to the effects of the factor, and the repressed corticotropes are unlikely to secrete it. A role of this paracrine communication is to hold corticotropes in reserve and, therefore, prevent the severe depletion of hormone. This form of paracrine communication may be a specialized adaption among cells where the physiological setting demands robust secretory responses to multiple stimuli. The experimental paradigms developed here may be extremely useful for 1) screening potential paracrine factors and 2) determining whether the secretion of the paracrine factor is regulated by adrenal or hypothalamic hormones.

Adrenocorticotropic Hormone↗

PubMed2025

Well-differentiated systemic mastocytosis: Genetics, mast cell immunophenotypes, and KIT autophosphorylation.

BACKGROUND: Well-differentiated systemic mastocytosis (WDSM) is a rare myeloid neoplasm where the genetic etiology is often unknown. OBJECTIVE: We aimed to assess WDSM patients for novel KIT variants, mast cell (MC) aberrant immunophenotypes, and KIT autophosphorylation patterns. METHODS: Next-generation sequencing, MC immunophenotyping, and KIT autophosphorylation studies were performed. RESULTS: Among 454 SM patients, there were 432 with KIT p.D816V+ SM and 4 with KIT p.D816Y+ SM-notably, none of these patients had WDSM. Of the remaining patients, we identified 7 with WDSM (1.5%) and 2 relatives with mastocytosis in skin. Next-generation sequencing revealed that 6 of 9 subjects carried known or novel germline KIT variants corresponding to regions outside of codon 816. Three patients had germline KIT p.K509I; 2 had germline KIT p.A533D; 1 had two germline KIT variants p.F681L and p.M541L; and 3 had no KIT mutation. Intracellular expression of CD2 and CD25 and less robust expression of CD30 was observed in MCs from WDSM patients. By developing a novel transient transfection assay in 293T cells, we found that unlike KIT p.D816F/V/Y variants that exhibit nearly exclusive intracellular localization and strong ligand-independent autophosphorylation (class II), WDSM-associated KIT variants showed enhanced ligand-dependent autophosphorylation relative to wild type (class I). CONCLUSIONS: Our study doubles the number of KIT variants identified in WDSM patients. No KIT p.D816V+ SM patient had WDSM. Intracellular CD2 and CD25 expression was more robustly detected in MCs from WDSM patients compared to CD30.

Humans↗

PubMed2026

Origin flexibility governs robust ssDNA engagement by the DnaA initiator.

In model bacteria, initiation of chromosome replication requires engagement of single-stranded DNA by oligomers of the DnaA-family initiator assembled within the origin DNA. Although arrays of double-strand motifs recognized by DnaA are a general feature of the origins, the DnaA-binding single-strand elements are elucidated in only a limited number of species, and the mechanical principles governing their recognition remain elusive. Using the Alphaproteobacterium Caulobacter crescentus, we identify a previously uncharacterized GA-rich single-stranded element in the origin that directly engages DnaA oligomers and is essential for robust initiation. This element is positioned at a subkilobase distance from the DnaA oligomerization region and is brought into proximity through dynamic structural rearrangements. Moreover, DnaA oligomers exhibit an unexpectedly broad yet constrained capacity to accommodate single-stranded sequence variation. These findings provide the molecular basis for origin plasticity, highlighting how origins can diverge while preserving initiation logic.

DNA, Single-Stranded↗

PubMed2025

Impact of RNA extraction on respiratory microbiome analysis using third-generation sequencing.

BACKGROUND: The respiratory microbiome, which comprises bacteria, fungi, and viruses, plays a crucial role in respiratory health and disease. However, its study is limited by the low microbial biomass in respiratory samples and the dominance of host RNA. Metatranscriptomics offers comprehensive insights into active microbial communities and their interactions with the host but requires optimized RNA extraction protocols for robust and unbiased analysis. This study evaluated two RNA extraction kits—one employing chemical lysis (CL) and another combining chemical and mechanical lysis (CML)—to determine their effectiveness for metatranscriptomic analysis of respiratory samples. RESULTS: The CML protocol significantly increased double-stranded DNA (dsDNA) library yields, leading to higher sequencing read counts for both sample types (p < 0.0001). The read length was unaffected by the lysis protocol for the BAL and NPS samples. Taxonomic profiling revealed that CML enhanced the detection of robust microorganisms, such as gram-positive bacteria and fungi, without compromising viral detection. CONCLUSIONS: The CML protocol demonstrated superior recovery of genetic material, particularly for fungi and gram-positive bacteria, making it better suited for comprehensive metatranscriptomic analyses. These findings underscore the need for tailored RNA extraction strategies on the basis of sample type and research objectives. Optimized metatranscriptomic protocols are pivotal for advancing our understanding of the respiratory microbiome and its role in health and disease.

Microbiota↗

PubMed2026

Rapid derivation of cloning-competent cells from peripheral blood advances conservation biobanking.

Establishing viable cell lines from endangered species is essential for conservation, yet traditional fibroblast derivation from skin biopsies faces challenges including contamination risk and extended culture timelines. Here, we demonstrate that endothelial progenitor cells (EPCs) and pericytes isolated from peripheral blood represent robust alternatives to fibroblasts for biobanking. Compared to canid fibroblasts, canid blood-derived cells exhibit 2- to 3-fold faster doubling rates (15 to 20 h vs. ~35 h for fibroblasts) and reduced time to banked cell lines (1.5 to 2 wks vs. 3 to 4 wks for fibroblasts). Proteomic profiling of 32 canonical markers confirmed EPCs and pericytes represent distinct populations with lineage-specific molecular signatures. Optical genome mapping demonstrated equivalent genomic stability across cell types with no detectable structural variants or aneuploidies. Finally, interspecific somatic cell nuclear transfer (iSCNT) experiments confirmed both EPCs and pericytes generate viable canid embryos with efficiency meeting or exceeding fibroblasts. As a proof of concept for conservation cloning, iSCNT embryos made with gray wolf blood-derived cells had a 15% implantation rate following embryo transfer and resulted in six viable fetuses. These findings support integrating blood-derived cell banking into conservation programs, which enables opportunistic genetic preservation during standard management activities and expands options for genetic rescue through assisted reproductive technologies.

Animals↗