Developmental & Comparative Immunology

Amoebic gill disease (AGD), caused by the parasite Neoparamoeba perurans, is a major welfare and economic burden for the Atlantic salmon aquaculture industry. However, the spatial organisation of immune functions within the gill, the site of infection, remains poorly resolved. Here, we present the first spatial transcriptomic atlas of the Atlantic salmon gill affected by AGD, generated using 10x Genomics Visium on four AGD-positive fish, confirmed by gill scoring and quantitative PCR for parasite load. As no uninfected gills were included, this study maps the anatomical and immunological organisation of AGD-affected gills rather than defining AGD-specific pathology. Unsupervised clustering of spatial gene expression resolved distinct transcriptional domains that grouped into four principal anatomical compartments of the gill, including (i) epithelial and lamellar regions defined by keratin and osmoregulatory gene expression, (ii) gill arch regions enriched for connective tissue and smooth muscle markers, (iii) an erythroid-associated vascular cluster, and (iv) a skeletal muscle cluster with signatures of glycolysis and oxidative phosphorylation. Across these domains, we identified a spatially organised and diffused immune landscape, with pattern recognition receptors, antimicrobial effectors and chemokines concentrated at the filament surface, and immunoglobulin light chain transcripts consistent with B lymphocyte activity enriched toward the gill arch. Integration of the spatial data with a published single-nucleus reference atlas using reference-based deconvolution resolved the cellular identity underlying these domains and revealed frequent co-localisation of immune and structural cell types, suggesting regulated cell to cell communication within the gill-associated lymphoid tissue. Overall, our results show that the Atlantic salmon gill is not a transcriptionally uniform surface but a regionally specialised organ, with immune functions distributed across, and specialised within, distinct anatomical compartments

Interferon gamma (IFN-{gamma}) is a potent cytokine secreted following primary varicella-zoster virus (VZV) infection. Our previous studies demonstrated that pretreatment with IFN-{gamma} completely inhibited VZV replication in lung fibroblast MRC-5 and retinal epithelial ARPE-19 but not in melanoma MeWo cells, suggesting that IFN-{gamma}-stimulated protein(s) inhibit viral replication. Our microarray analysis revealed that 34 interferon-stimulated genes (ISGs) were upregulated by greater than 2.5-fold at 8 h post-treatment in both ARPE-19 and MRC-5 cells compared to those of MeWo cells. The depletion of CTSS, GBP4, MX1, IFIT1, IFITM1 and IRF1 by siRNA in IFN-{gamma}-treated cells significantly increased VZV yields. Ectopic overexpression of interferon-induced myxovirus resistance 1 (MX1) or transmembrane protein 1 (IFITM1) significantly reduced VZV yield both in ARPE-19 and MeWo cells. IFITM1 abrogated viral major immediate-early IE62 protein-mediated trans-activation. IFITM1 reduced the level of VZV immediate-early IE62 protein, but not the IE62 mRNA, suggesting that IFITM1 expression reduces the expression level of IE62 by post-transcriptional regulation. Cell-free VZV failed to infect human primary epidermal keratinocytes, in which the basal level of endogenous MX1 gene expression is very high. Pharmacologic inhibition of JAK signaling reduced MX1 expression and enhanced VZV replication in calcium chloride-induced differentiated keratinocytes. Collectively, these findings identify MX1 and IFITM1 as key IFN-{gamma}-induced restriction factors that inhibit VZV replication and suggest that suppression of JAK-STAT signaling can promote VZV replication in differentiated keratinocytes.

Chronic hepatitis B virus (HBV) infection persists under current antiviral therapy because covalently closed circular DNA (cccDNA) remains in the infected hepatocytes as a durable transcriptional template. Although type I interferon (IFN-I) can reduce HBV cccDNA gene expression, the host effectors and mechanisms that restrict cccDNA remain incompletely defined. Using a minicircle-based HBV cccDNA expressing a Gaussia luciferase reporter gene (mcHBV-GLuc), we investigated how promyelocytic leukemia protein (PML) suppresses HBV cccDNA activity. Multiple PML isoforms reduced cccDNA-driven reporter activity and HBsAg production, and HBx-deficient ({Delta}X) mcHBV cccDNA was more susceptible than wild-type cccDNA to PML-mediated inhibition. Interestingly, efficient antiviral activity required the major PML SUMOylation sites. Disruption of SMC5/6 function attenuated PML-mediated repression of HBx-deficient cccDNA, associated with reduced PML SUMOylation and PML nuclear-body organization. These findings identify PML as a SUMO-dependent inhibitor of HBV cccDNA activity and reveal a context-dependent functional relationship between PML and SMC5/6 that is modulated by HBx.

Introduction: Thyroid diseases are major health problems and among the most common endocrine disorders worldwide, including in Ethiopia. They are characterized by alterations in hormone secretion, enlargement of the thyroid gland, or both. Fine needle aspiration cytology is an important initial screening tool, and histopathologic examination is essential for the definitive diagnosis of thyroid disease. This study aimed to compare the diagnostic accuracy of fine needle aspiration cytology with histopathology in identifying thyroid diseases. Methods: A cross-sectional study was conducted at three health facilities in Hawassa City, Ethiopia, involving 304 patients who underwent both thyroid fine needle aspiration cytology and histopathologic examination of the same site between 30th October 2024 and 29th October 2025. FNAC results were classified using the Bethesda system, with histopathology as the gold standard. Data were entered into Kobo Toolbox, exported to SPSS version 27, and analyzed using cross-tabulation and chi-square ({chi}{superscript 2}) tests to determine diagnostic accuracy. Result: 304 patient records were included in the study. The fine needle aspiration cytology was determinate/diagnostic in 81.6% of cases. The study revealed the sensitivity and specificity of fine needle aspiration cytology to be 88.2% (CI 77.6-95.1%) and 94.4% (CI 90.6-97.1%), respectively. The false negative and false positive rates in this study were 2.4% and 4.4%, respectively. The positive predictive value was 80.3% (CI 68.7-89.3%), and the negative predictive value was 96.8% (CI 93.8-98.7%). The accuracy of fine needle aspiration cytology in differentiating benign from malignant thyroid lesions was 93.1%. Conclusion: FNAC demonstrated strong agreement with histopathology and remains a reliable, cost-effective first-line diagnostic tool for thyroid disease, particularly in resource-limited settings. Strengthening sample handling, ultrasound guidance, and adherence to Bethesda criteria may further improve diagnostic precision

Fibroblasts are readily obtained and expanded and may provide an alternative cell source for cultivated seafood if their latent myogenic potential can be accessed. We examined whether transient exposure to 5-azacytidine (AZA), a cytidine analogue known to convert mouse embryonic fibroblasts into myoblasts, elicits a persistent muscle gene program in embryo-derived zebrafish (Danio rerio) Z3 fibroblasts. Cells were exposed to 0, 2.5, 5 or 10 M AZA for 48 h and maintained in drug-free medium for 3 weeks. RNA-seq identified 744 genes that increased and 670 that decreased consistently across the concentration series. Increased genes encompassed myogenic regulators, fusion factors, sarcomeric components and excitation-contraction machinery, whereas decreased genes were enriched for extracellular-matrix and adhesion functions. In a separate six-dose RT-qPCR experiment (0-20 M AZA) with three biological replicates per concentration, all 19 selected muscle genes were significantly increased at 1.25 M, the lowest concentration tested, whereas 9 of 12 extracellular-matrix/fibroblast genes were significantly decreased. Several muscle genes plateaued or declined at higher concentrations, and multinucleated myotubes were not observed. A separate 5-aza-2'-deoxycytidine (decitabine) series showed a comparable muscle-associated response at 0.25 M. These findings indicate that zebrafish fibroblasts retain access to a myogenic-like transcriptional program, and they provide a model for identifying food-compatible approaches to control fish-cell lineage plasticity for cultivated seafood.

Epithelial cells of the female reproductive tract (FRT) form a protective barrier and serve as the first line of defense against sexually transmitted infections (STIs). They recognize pathogens, release cytokines and initiate innate immune responses. Sex hormones 17{beta}-estradiol (E2) and progesterone (P4) fluctuate throughout the menstrual cycle and influence epithelial cell functions. Generally, E2 confers protection against viral STIs like HIV-1 and HSV-2, whereas P4 increases susceptibility. However, the underlying mechanisms remain incompletely understood. MicroRNAs, small non-coding RNAs that regulate gene expression by inhibiting translation of target mRNAs, significantly influence viral infections, by promoting or restricting viral pathogenesis. Here, we examined the effect of sex hormones on microRNA expression in vaginal epithelial cells (VECs), the first responders to pathogens in the FRT. Using next generation sequencing analysis, the effect of physiological concentrations of E2 or P4 on microRNA transcription profile of VECs grown in air-liquid interface cultures was characterized. Thirteen miRNAs were uniquely regulated by E2 and 102 by P4, with 29 miRNAs that overlapped between both. The top four differentially expressed miRNAs from both treatments were validated by RT-qPCR. The mRNA targets of unique E2-miRNAs were involved in adherens junction, tight junction and chemokine signaling pathways; whereas the mRNA targets of unique P4-miRNAs were involved in MAPK signaling pathway. Interestingly, several miRNAs regulated by E2- (eg. hsa-miR-9-3p, hsa-miR-200c- 5p, hsa-miR-548c-5p) and by P4 (eg. hsa-let-7f-5p, hsa-miR-16-5p, hsa-miR-27b-3p) targeted genes related to cell cycle pathway. Together, these findings indicate that microRNAs regulated by E2 and P4 influence mRNAs in the pathways associated with cell adhesion, cell growth and development and innate immune responses, critical for maintenance of epithelial barrier integrity and regulating pathogen susceptibility in the vaginal tract. These hormone-regulated miRNAs may serve as potential targets for therapeutic interventions.

Background: Neutrophils migrate to the respiratory epithelium during rhinovirus (RV) infection, but whether RV directly activates neutrophils or whether activation arises from epithelial responses remains unclear. Objective: To determine whether RV directly activates human neutrophils or indirectly activates them through RV infected airway epithelial cells. Methods: Human neutrophils were exposed to purified RV, and activation was assessed by myeloperoxidase activity, CD11b/CD66b expression, and reactive oxygen species (ROS) production. Neutrophils were co-cultured with RV-infected air liquid interface (ALI) bronchial epithelium evaluated for effects on epithelial integrity. At 48 to 72 hours post-infection, we used live-cell imaging to measure ROS responses and neutrophil-tracking assays to measure migration velocity and ROS responses to basolateral supernatants, clarified apical washes, and apical debris from RV-infected epithelial cells. Plaque assays were used to assess for RV infectivity after incubation with apical neutrophils. Results: RV did not directly activate neutrophils. Adding neutrophils to the apical surface of airway epithelial cells increased neutrophil ROS production at 48 to 72 hrs post-infection but did not alter epithelial cytotoxicity. Conversely, basolateral supernatants from infected cells enhanced neutrophil migration but not ROS, consistent with cytokine-mediated priming. Clarified apical washes had no effect on migration or ROS. By contrast, apical debris from infected cultures triggered a robust ROS response. Basolateral fractions from medium control or RV infected cultures did not induce ROS. Infectivity is not impacted by apical neutrophil presence. Conclusion: RV does not directly activate neutrophils. Instead, RV infected epithelial cells release basolateral factors that enhance neutrophil migration, while apical debris from infected cells drives ROS activation. These findings indicate that epithelial injury, not RV itself, is the dominant trigger of neutrophil activation during infection.

Traditional veterinary pathology investigations on wild animals generally conclude a primary cause of death based on a combination of observed clinical signs, gross and microscopic lesions, microbial culture and PCR. However, these methods may fail to capture the broader microbial community of the host. We employed metatranscriptomic sequencing to characterise the potential pathobiome of necropsied marine turtles, comparing these findings with corresponding veterinary pathology reports. Tissue samples were collected from 76 individuals spanning three species of marine turtles -- the green turtle (Chelonia mydas), the hawksbill turtle (Eretmochelys imbricata), and the loggerhead turtle (Caretta caretta) -- across the coastline of New South Wales, Australia. Reported causes of mortality ranged from infectious disease to trauma. We identified a diverse range of parasites within the genera Neospirochis, Hapalotrema, Paraorgymatobothrium, Eimeria and Callitetrarhynchus, and bacteria including the genera Aeromonas, Clostridium, and Vibrio. Novel viruses of the families Astroviridae, Caliciviridae, Hepaciviridae, Hepeviridae, Nyamivirdae, Orthomyxoviridae, Picobirnaviridae and Rhabdoviridae were also identified. In cases where infectious disease was noted in the pathology report, we frequently observed high pathogen read counts consistent with the listed primary diagnoses. However, we also detected a range of incidental infections involving eukaryotic parasites, bacteria, and viruses. The diversity of infectious agents detected in marine turtles suggests that the observed disease outcomes are unlikely to be driven by a single causative agent, but rather by complex interactions between host health, the pathobiome, and environmental stressors. We discuss the potential implications for turtle health and conservation.

Marmosets (MAR, Callithrix jacchus) are nonhuman primates extensively studied in a broad array of preclinical biomedical research areas. However, the limited number of available immunological reagents remains a critical shortcoming of these models. We successfully developed monoclonal antibodies (mAbs) pairs that can be used in immunoassays for the identification and quantification of the biomarkers of inflammation C-reactive protein (CRP), CXCL-10 (IP-10), interleukin (IL)-4, IL-6, and IL-10. Recombinant MAR protein variants were produced in mammalian cells, purified and used for mice immunization. Hybridoma clones were selected first for their capacity to bind the recombinant proteins. All possible pair combinations of these mAbs were then tested for their capacity to identify their targets in MAR-derived samples. These MAR samples consisted of supernatant of PBMCs exposed ex vivo to different stimulants, and plasma samples from animals stimulated in vivo with a low-dose intravenous LPS inoculation. The majority of the mAbs that recognized the recombinant proteins failed to bind to the MAR-derived homologs. However, we found MAR-derived reactive mAbs and selected pairs with optimal sensitivity and signal-to-noise ratio for immunoassays that included Luminex multiplexing assays, ELISA, and ELISpot assays. These immunoreagents and assays will improve the translational value of different MAR biomedical models.

Objective: Hyperthyroidism and hypothyroidism related to iodine deficiency are major public health concerns in Afghanistan. This study aimed to assess the frequency of thyroid dysfunction among male patients referred for thyroid testing and its association with age, and to examine monthly trends in thyroid dysfunction at Asia Med Laboratory in Herat, Afghanistan, from July 2021 to January 2022. Methods: A retrospective analysis was conducted on 250 male patients aged 0-69 years. We measured Serum TSH, total T4, and total T3 levels, and thyroid status was classified using age specific reference ranges. In addition, the frequency of thyroid dysfunction was analyzed across age groups with monthly trends of thyroid state. Results: Overall, the euthyroid state consisted of 69.2% of participants, 24.8% with overt hypothyroidism, 3.2% with overt hyperthyroidism, and 2.8% with subclinical hyperthyroidism. Thyroid status differed significantly by age (p = 0.0135), with hypothyroidism increasing in older age groups and reaching its highest proportion among men aged 60-69 years (55.6%). Euthyroidism predominated in patients aged 10-39 years, while hyperthyroidism across age groups remained relatively infrequent. After September 2021, a threefold increase was observed in the total number of male patients referred for thyroid testing. During this period, the proportion of hyperthyroidism increased slightly, whereas hypothyroidism cases declined. Conclusion: In conclusion, hypothyroidism was more frequent with older age. The rise in absolute case numbers after September 2021 likely reflects increased patient referrals, underscoring the need for ongoing monitoring of thyroid function. The study may assist in the early management of thyroid disorders and in reducing their complications.

Core fucosylation (alpha1,6-fucosylation) of N-glycans, catalyzed solely by fucosyltransferase 8 (FUT8), plays important roles in development, immunity, and antibody effector functions. Here we report the generation of Fut8 floxed mice using a CRISPR/Cas9-based Extreme Genome Editing (EGE) system, in which loxP sites flank exon 8 of the Fut8 gene and a LoxP-STOP-LoxP-RFP cassette was knocked in downstream of the promoter, rendering the targeted allele functionally null before Cre-mediated recombination. Crossing floxed mice with CMV-Cre transgenic mice yielded complete knockout mice carrying the Cre-recombined allele. A PCR-based genotyping method was established that unambiguously distinguishes the wild-type, heterozygous, and homozygous genotypes (+/+, delta/+, and delta/delta). Intercrossing of heterozygous mice revealed that homozygous knockout mice die during the early postnatal period, consistent with previous reports, whereas heterozygous mice are viable. Flow cytometric analysis of AAL binding to splenic B cells and AAL lectin blotting both demonstrated markedly reduced core fucosylation in heterozygous mice (60.0 +/- 6.8% and approximately 48% of wild-type levels, respectively). These results show that disruption of a single Fut8 allele substantially reduces core fucosylation, and the mouse model described here provides a practical tool for functional studies of core fucosylation.

Objective: Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) frequently present as overlap syndromes, characterized by shared sexual dimorphism and elevated intestinal Veillonella dispar (V. dispar) abundance, yet exhibiting distinct hallmark pathological manifestations. This study aimed to elucidate the shared pathogenesis, phenotypic differentiation mechanisms and sex-specific predisposition of AIH and PBC induced by V. dispar. Methods: Clean-grade and germ-free (GF) mice were employed to establish AIH and PBC models. Model induction involved oral gavage of V. dispar and intraperitoneal injection of either liver-specific antigen S100 (for AIH) or a pyruvate dehydrogenase complex E2 subunit (PDC-E2) mimetic peptide (for PBC). Single-cell RNA sequencing, metabolomics, transmission electron microscopy, western blotting, and flow cytometry were utilized to elucidate the underlying mechanisms. Results: V. dispar successfully induced autoimmune liver disease-like features in mice, characterized by significant intrahepatic B cell infiltration. Mechanistically, V. dispar caused systemic arginine reduction, leading to intrinsic apoptosis of gonadal stromal cells and a subsequent reduction in testosterone. This decreased testosterone weakened its inhibitory effect on B cell activating factor (BAFF) secretion, thereby amplifying B cell-mediated immune responses. Furthermore, in the presence of S100 or PDC-E2 mimetic peptide, V. dispar also facilitated Th17 cell activation, which guided the phenotypic differentiation into AIH or PBC, respectively. Consistent with these findings, our clinical observations revealed that patients with AIH and PBC tended to harbor enriched V. dispar, along with lower serum arginine and testosterone. In addition, elevated serum levels of BAFF and IL-17 were also observed in these patients. Conclusion: V. dispar induces reduced testosterone levels and upregulation of BAFF, leading to intrahepatic B cell expansion, which serves as the common pathogenic basis for AIH and PBC. The presence of S100 and PDC-E2 subsequently drives further maturation of these expanded B cells and promotes Th17 cell activation, thereby determining the phenotypic divergence between AIH and PBC.

Hematopoiesis sustains lifelong blood production and immunity and is strongly influenced by biological sex. Males and females differ in their hematopoietic composition and susceptibility to infection and hematologic disease. Because current models cannot readily disentangle the independent contributions of sex chromosomes versus hormones, the mechanisms underlying sexual dimorphism remain poorly understood. Zebrafish have proven to be a powerful, translatable system for studying hematopoiesis, and offer a unique opportunity to isolate hormonal contributions due to their absence of sex chromosomes. However, whether sex is regulated in the zebrafish hematopoietic system remained unknown. Here, we discovered pronounced sexual dimorphism in the adult zebrafish marrow, with compositional differences peaking at reproductive age. Transcriptome analysis of marrows revealed conservation of sex-specific programs between zebrafish and human datasets. Upon immune challenge with lipopolysaccharide, all major blood lineages exhibited sex-biased changes in their composition and transcriptomes with the largest responses in erythroid and myeloid cell types. Among these sex differences was an unexpected immunoregulation of the erythroid lineage across maturation states. These findings establish biological sex as a major regulator of zebrafish hematopoiesis and immunity, paralleling humans, and lay the groundwork necessary for future mechanistic studies on sexual dimorphism in hematologic health and disease.

The longitudinal effects of initial influenza exposure on the magnitude, persistence, and antigen specificity of antibody responses during infancy remain incompletely understood. We investigated humoral antibody responses following primary influenza infection or seasonal vaccination in a prospective US birth cohort with weekly respiratory surveillance for influenza infection. Influenza-specific serum IgG binding to H1 and H3 haemagglutinin (HA) antigens, HA inhibition (HAI), and live-virus neutralizing antibody responses were assessed longitudinally. To minimize influence of maternally derived antibodies, analyses of primary exposure responses were restricted to infants for whom influenza-specific maternal antibodies had declined below the assay detection limit before exposure. Antibody responses were compared following primary infection and vaccination and examined after subsequent annual vaccination. We found that maternal influenza-specific IgG declined rapidly during early infancy. Among infants without detectable pre-exposure influenza-specific antibodies, primary influenza infection was associated with greater post-exposure antibody responses and less measurable decline over follow-up than primary vaccination. These groups differed in age, calendar season, and exposure characteristics, limiting direct attribution of these differences to route of exposure. In infants with previous influenza infection followed by vaccination, responses after subsequent vaccination initially showed greater binding to the previously infecting subtype among the tested HA antigens. With repeated vaccination, responses became more distributed across the tested antigens. Our findings reveal differences in the magnitude, persistence, and antigen specificity of antibody responses according to the nature and sequence of influenza exposure. By prospectively capturing early-life exposures, this study provides longitudinal evidence linking exposure history to subsequent humoral antibody responses.

Phenuiviruses which include Rift Valley Fever Virus (RVFV), classified by WHO as one of the top priority pathogens with outbreak potential, are emerging arthropod-borne viruses that pose an increasing threat to public health. In line with the prototype pathogen framework advanced by the NIAID, the sandfly-transmitted Toscana virus (TOSV) has been adopted as a prototype to advance understanding of relevant phenuiviruses and inform the development of broadly applicable medical countermeasures. In this study, we sought to isolate and characterize monoclonal antibodies (mAbs) elicited by natural TOSV infection to identify shared antigenic determinants. By employing the conserved envelope Gc glycoprotein as bait for antigen-specific B cell sorting, we recovered more than 1,450 memory B cells and identified 112 Gc-specific neutralizing mAbs. Over one-third of these mAbs exhibited cross-reactivity with RVFV, a genetically distinct phenuivirus. Selected mAbs provided prophylactic protection in a murine model of TOSV infection. Together, our findings establish Gc as an important target of protective neutralizing antibodies and provide insights that may guide the rational design of widely effective interventions, strengthening readiness against future phenuivirus outbreaks.

Chagas disease, caused by the parasite Trypanosoma cruzi, remains a major health concern, with effective immune control relying heavily on CD8+ T cells. However, the metabolic factors that regulate CD8+ T cell function during infection are not well understood. Acute T. cruzi infection induced metabolic remodeling in parasite-specific and effector CD8+ T cells, characterized by increased reliance on glycolysis, altered mitochondrial homeostasis, oxidative stress, and increased CD38 expression. Nicotinamide riboside (NR), an NAD+ precursor, improved mitochondrial function, reduced mitochondrial reactive oxygen species (mROS) and CD38 expression, decreased apoptosis, and enhanced CD8+ T cell effector responses in vitro and in infected mice. In vivo, NR treatment increased intracellular NAD+ levels in CD8+ T cells and improved parasite control during acute infection, reducing parasitemia and skeletal muscle parasite burden. Furthermore, transient NR supplementation was associated with persistent metabolic and functional changes in memory CD8+ T cells during chronic infection. These findings identify NAD+ metabolism as a modulator of CD8+ T cell metabolic fitness during T. cruzi infection and support its further investigation as a host-directed strategy to enhance parasite control.

Primordial germ cells (PGCs) are the stem cell lineage responsible for transmitting genetic information across generations and are therefore central to studies of germline development, reproduction, and evolution. However, a lack of molecular tools in jawless vertebrates such as lampreys and hagfish has limited comparative insights into the evolutionary origins of vertebrate germline cells. Here, we report the development of the first germline-specific reporter (GSR) in the sea lamprey (Petromyzon marinus), Tg(piwil1: egfp-UTRnanos1), a piwil1-based germline reporter that is driven by the sea lamprey piwil1 regulatory region that directs Green Fluorescent Protein (GFP) expression in early PGCs. Promoter activity was validated by robust GFP expression in PGCs of zebrafish embryos co-injected with Tol2 transposase mRNA. Injection of the construct into sea lamprey embryos resulted in early, persistent, and gonad-localized GFP-positive cells. These results demonstrate stable germline-specific transgene expression in a jawless vertebrate and suggest deep evolutionary conservation of germline regulatory mechanisms between jawless and jawed vertebrates. Furthermore, the piwil1-based germline reporter line enabled lifetime visualization of zebrafish germline cells, revealing that increased PGC abundance during embryogenesis biases toward female sexual differentiation. Together, this study establishes the first GSR system in a jawless vertebrate and provides a useful platform for investigating germline biology and vertebrate germline evolution.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Genetics shape cocaine behavior and abuse liability, but the underlying neurophysiological effects remain understudied. In male C57BL/6J, B6129SF2/J, and DO/J mice, we report conserved and divergent cocaine induced neuroplasticity in the accumbens shell following 5 days of cocaine and 10 to 14 days of abstinence.

Primary sulcal patterns, the spatial arrangement of the earliest cortical folds, emerge prenatally and remain essentially stable after birth. Alterations are associated with cognitive outcomes and hold promise as clinical biomarkers. However, detecting abnormalities is challenging due to the high topological variability and complexity of normal individual sulcal patterns. Here, we introduce an unsupervised generative model that quantifies individual sulcal pattern deviations against a normative baseline learned from 10,349 typically developing subjects spanning early childhood through adulthood. We combine a generative reconstruction error with a discriminator trained on diffusion-generated pseudo-atypical graphs to leverage their complementary strengths in sensitivity and robustness. The discriminator captures subtle anomalies and is robust to demographic and data quality variations, whereas the generative model enables severity stratification and demonstrates cross-site generalizability. Across two clinical cohorts spanning subtle to overt sulcal abnormalities (congenital heart disease and polymicrogyria), our model captured global and localized condition-related deviations and linked them to neurodevelopmental outcomes. By enabling population-scale, individualized quantification of sulcal deviations, this framework provides a foundation for future clinical risk stratification.