Cis-regulatory elements (CREs) drive tissue- and cell-specific gene expression and are essential for safe, sustainable genetic control strategies in pest and vector insects, including the engineering of gene drives in the primary human-malaria vector Anopheles gambiae. Yet CREs remain poorly defined in mosquitoes due to limited computational tools and practical methods for identification and validation. We present a systematic in silico approach for CRE discovery, correlating targeted DNA-motif searches with gene expression, followed by frequency and distribution analysis within putative promoter regions. Applied to the A. gambiae germline, this approach identified hundreds of putative CREs significantly correlated with germline expression in one or both sexes, often linked to distinct sperm developmental stages and chromosomal locations, suggesting roles in broader regulatory mechanisms such as dosage compensation and meiotic silencing. When mapped onto pre-characterised germline promoters, CRE distribution aligned with regions associated with experimental expression patterns. Finally, we validated a top-ranked testis-enriched CRE using an in vivo dual-reporter assay, showing that mutation of conserved nucleotides drastically altered male germline expression. To the best of our knowledge this work provides the first nucleotide-resolution regulatory genome annotation of the A. gambiae germline, offering a transferable framework to aid promoter design for genetic control strategies against malaria mosquitoes and other insect pests.
Visual impairment affects over 2.2 billion people worldwide and the major causes include age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy. For research in these areas, although animal models offer a more physiologically complex system than in vitro approaches, their use raises ethical considerations, and species-specific differences such as variations in protein sequences and signaling pathways. This can limit the direct translatability of the outcomes. Traditional 2-D cell cultures, in contrast, lack the multicellular organization and dynamic microenvironment necessary to replicate human retinal complexity. Retinal organoids (ROs), three-dimensional tissue constructs derived from pluripotent stem cells, have emerged as a promising model due to their human origin and complex cellular interactions that cannot be achieved in conventional 2-D/3-D co-culture models. In this review, we provide a brief overview of the evolution from 2-D to 3-D retinal models, highlight the structural and functional features of ROs including the presence of layered retinal architecture, photoreceptor outer segment formation, and light-responsive electrophysiological activity and summarize their applications in disease modeling, drug discovery, and gene and cell therapy. ROs represent a significant advancement over traditional models by enabling the recapitulation of human-specific retinal development, facilitating the study of patient-derived disease phenotypes, and providing a platform for personalized therapeutic screening. Their development has deepened understanding of pathological mechanisms in conditions such as retinitis pigmentosa and AMD, while enabling preclinical testing of targeted interventions like CRISPR-based gene editing and photoreceptor cell replacement. Nonetheless, challenges remain in fully replicating retinal vascularization, long-term functional maturation, and synaptic connectivity, underscoring the need for continued refinement and integration with complementary model systems.
Cytokine-mediated cross-talk between immune cells and fibroblasts is a driver of excessive ECM accumulation during fibrosis. In this study, we used a 3D in vitro model of a connective tissue to discern the roles of three pro-inflammatory cytokines; TNF-α, IL-18 and IL-1β, alone, and in combination with TGF-β1 to simulate the fibrotic environment. Ring-shaped tissues were formed by seeding human fibroblasts into circular molds of agarose, wherein the cells self-assembled, formed a 3D tissue and synthesized de novo a collagen-rich ECM. Cytokine treated tissues were analyzed at days 7 and 14 by histology and measured for thickness, collagen, DNA and strength and stiffness by tensile testing. Despite their pro-inflammatory nature, none of the cytokines increased collagen alone or in combination with TGF-β1. TNF-α and IL-1β reduced collagen, tissue strength and stiffness, and altered tissue morphology. When combined with TGF-β1, TNF-α and IL-1β counteracted TGF-β1-mediated increases in collagen, strength, and stiffness. In contrast, IL-18 had minimal effects alone or when combined with TGF-β1. These data suggest that IL-18 has no effect on fibroblast activation, whereas TNF-α and IL-1β may modulate TGF-β1's effects. This 3D model of a human collagen-rich tissue can help define cytokine-mediated cross-talk between immune cells and fibroblasts.
Human biomonitoring (HBM) is crucial for evaluating exposure to diet-related contaminants, whose effects may pose substantial health risks. Saliva is recognized as a promising non-invasive biological matrix due to its ease of collection and potential to reflect external and systemic exposure. However, suitability for monitoring dietary hazardous compounds remains uncertain. To assess the potential of saliva as a biomonitoring matrix for diet-related contaminants, identify compounds with robust diet-related associations, and highlight knowledge gaps. A systematic literature review was conducted to screen over 500 diet-related contaminants analyzed in saliva. Detailed information was extracted only for contaminants quantitatively measured in saliva, including concentration ranges, sample sizes, and analytical methods. Evidence of correlations with systemic concentrations, exposure pathways, and individual or lifestyle factors was compiled into a FAIR database to provide an integrated evaluation of saliva's biomonitoring potential. Only a limited subset of contaminant groups, including nitrite/nitrate, heavy metals, bisphenols, polycyclic aromatic hydrocarbons (PAHs), biogenic amines, pesticides, advanced glycation end products (AGEs), perchlorate, microplastics (MPs), parabens and phthalates, have been quantitatively measured in saliva. Compounds such as nitrate, arsenic, AGEs, pesticides and perchlorate demonstrate moderate to strong correlations between salivary and systemic levels, supporting saliva's potential to estimate exposure. Conversely, substances like PAHs, MPs, phthalates and parabens generally show weak or no correlation, reflecting recent or localised exposures rather than cumulative burden. Salivary composition is influenced by intrinsic and extrinsic factors, including diet, oral microbiota, physiology, and sampling conditions, resulting in high interindividual variability. Despite challenges, low salivary concentrations and lack of standardized collection protocols, saliva offers advantages for biomonitoring vulnerable populations, such as children and pregnant women. Harmonized collection procedures, validated sensitive methods, predictive models accounting for variability and exposure context, could establish saliva as a reliable complementary or alternative matrix for assessing human exposure to dietary and environmental contaminants. This systematic review synthesizes findings from 104 studies, covering over 500 diet-related contaminants measured in saliva, and compiles them into a FAIR database, providing the most comprehensive resource to date for saliva-based biomonitoring. Compounds such as nitrate, arsenic, advanced glycation end-products (AGEs), pesticides, and perchlorate show meaningful correlations with systemic levels, supporting saliva's potential as a non-invasive matrix for assessing human exposure. To fully realize saliva's potential, standardized collection protocols, validated analytical methods, and predictive models that account for interindividual variability and exposure context are urgently needed, enabling more accurate and ethical monitoring of vulnerable populations.
In this review we comprehensively discuss organic cation transporter novel 1 (OCTN1), encoded by the SLC22A4 gene as a member in the solute carrier 22 (SLC22) family, which facilitates the cellular transport of diverse cationic and zwitterionic substrates. OCTN1 is highly expressed in many vital organs in humans, where it facilitates absorption and distribution of both endogenous compounds and therapeutic drugs. Among its substrates, ergothioneine (EGT) serves as the primary antioxidant and anti-inflammatory molecule, underscoring the essential role of OCTN1 in cellular defense and inflammation control. Genetic polymorphisms in SLC22A4 significantly alter OCTN1 expression, substrate affinity, and drug pharmacokinetics, with strong associations to susceptibility and treatment outcomes in human diseases. Insights from knockout models revealed that OCTN1 deficiency leads to reduced EGT availability, heightened oxidative stress, and aggravated inflammation, particularly in the tissues such as intestine, liver and lung. Moreover, OCTN1 activity is dynamically regulated by epigenetic modifications, cytokines, and hormones, linking it to immune modulation and disease progression. Put together, OCTN1 plays a defined role via high-affinity EGT transport, while its broader transport capacity and pharmacological relevance remain under investigation, with possible - though not yet established - implications for inflammation-associated biomarker development.
Eukaryotic mRNAs typically encode a single functional polypeptide, a principle challenged by the discovery of widespread non-canonical peptide-coding ORFs within 5'UTRs. However, their functional significance at the protein level remains underexplored. Using a four-layered pipeline, we identify 14 human transcripts predominantly transcribed in polycistronic forms, each encoding two conserved proteins. Focusing on the SLC35A4 transcript, we show that its 5'UTR encodes a mitochondrial inner membrane-localized microprotein that we name STREMI (SLC35A4 stress response regulating MICOS interactor). Sharing topology and motifs with the MICOS core subunit MIC10, STREMI regulates mitochondrial cristae morphogenesis in mice and human cells. Additionally, the STREMI-encoding uORF mediates stress-responsive translation of SLC35A4-a Golgi nucleotide sugar transporter-upregulating its translation during the integrated stress response. Evolutionary analyses indicate that these bicistronic transcripts likely arose through transcriptional readthrough following retroposition. We propose a mechanism of "gene symbiosis" that enables functional partitioning and coordinated translation of protein pairs from bicistronic transcripts.
Cold preservation is a critical logistical step in liver transplantation but induces ischemia-reperfusion injury (IRI), a key driver of early graft dysfunction. While bulk tissue assays capture global damage, they obscure the cell-type-specific transcriptional programs engaged during hypothermic storage. We utilized a multicellular human liver-on-chip model comprising Patient-Derived Organoids (PDOs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), and macrophages. Chips were exposed to 24-h static cold storage using either the clinical standard University of Wisconsin (UW) solution or a hyperbranched polyglycerol (HPG)-based formulation, followed by normothermic reperfusion. Single-cell RNA sequencing (scRNA-seq) was performed to map transcriptional trajectories across the preservation-reperfusion axis. We identified candidate solution-dependent transcriptional differences across cell types. PDOs from UW-preserved chips showed comparatively higher mean expression of inflammatory and oxidative stress-associated transcripts (IFI27, SAA1, HMOX1) and mitochondrially-encoded genes (MT-ND5) relative to HPG-preserved samples, which retained comparatively higher expression of homeostatic epithelial markers (EPCAM, KRT18). HSCs and LSECs in the UW group showed comparatively elevated expression of fibrosis-associated (COL1A1, TAGLN) and endothelial adhesion (ICAM1) transcripts. Ligand-receptor interaction modelling identified candidate inflammatory communication axes, including chemokine signaling interactions (CXCL1, CCL20) between macrophages and epithelial compartments, with higher predicted activity under UW preservation. This study provides an exploratory, high-resolution map of cell-type-specific transcriptional patterns associated with hypothermic preservation in a liver-on-chip model. Our findings suggest that preservation solution chemistry is associated with distinct transcriptional signatures spanning stress response, mitochondrial, and intercellular signaling pathways. Transcriptional patterns in HPG-preserved cells were consistent with comparatively attenuated injury responses; however, these observations are hypothesis-generating and require independent biological replication and functional validation, including metabolic flux assays and ROS production measurements before conclusions regarding mitochondrial protection or clinical preservation efficacy can be drawn.
The gut microbiome supports digestion, immunity, and metabolism; its imbalance (dysbiosis) drives inflammation and metabolic dysfunction, contributing to chronic diseases such as diabetes, cardiovascular disease, inflammatory bowel disease, and autoimmune disorders. Medicinal plants provide a wide range of phytochemicals (such as polyphenols, flavonoids, alkaloids, saponins), which reach the colon and undergo two-sided interactions with microbes in the gut, acting as potential microbiome modulators and substrates of biotransformation into bioactive metabolites. This structured narrative review synthesises evidence from peer-reviewed studies indexed in PubMed, Scopus, and Web of Science over the last 10 years on the role of medicinal plants in microbiome-mediated chronic disease modulation. This literature is organised into three mechanistic axes: (i) perturbations, defined here as measurable shifts in microbial diversity or taxonomic composition relative to a baseline or healthy reference state, together with beneficial taxa enrichment; (ii) alterations in microbial metabolite output, especially short-chain fatty acids (SCFAs) and other immunometabolic mediators; and (iii) downstream host metabolic and immune signalling. Rather than broad descriptive summaries, the literature is organised using an axis-based mechanistic framework, highlighting key translational constraints such as botanical heterogeneity, dose/formulation variability, and inconsistent microbiome endpoint standardisation, that must be addressed to strengthen human evidence and clinical relevance. Illustrative microbiome-mediated processes involve botanicals such as turmeric (curcumin), ginseng (ginsenosides), and green tea (catechins), though evidence strength varies by study design. Future progress requires standardised phytochemical characterisation, microbiome-stratified trials, and integration of multi-omics with artificial intelligence analytics to enhance mechanistic insight, identify responders, and enable personalised plant-based microbiome therapies.
Cutaneous gene therapy has the potential to treat a wide range of skin disorders, but effective delivery remains limited by the skin's barrier properties and immune surveillance. Here, we identify AAVrh32.33 as a potent vector for targeting dermal stromal compartments. Following systemic administration in mice, AAVrh32.33 mediated robust and durable transgene expression, with preferential targeting of dermal fibroblasts and hair follicle bulge cells. Expression peaked at one month and persisted for up to two years, highlighting its suitability for chronic conditions. To reduce immunogenicity, a dominant CD8+ T cell epitope was disrupted, generating the IDPΔ variant. This modification attenuated peptide-specific T cell responses while preserving stromal transduction. In human skin explants, IDPΔ achieved high levels of gene expression, primarily in dermal fibroblasts and precursors, confirming translational relevance. Finally, vectors encoding CCL17, CCL20, and CCL22 demonstrated localized targeted therapeutic gene delivery in both healthy and inflamed skin, underscoring the feasibility of using this platform to reshape local immune responses. Together, these findings establish AAVrh32.33 and IDPΔ as promising platforms for durable cutaneous gene therapy, with direct applications in diseases such as vitiligo where long-term modulation of the dermal microenvironment is essential.
The progressive skeletal muscle degeneration observed in Duchenne Muscular Dystrophy (DMD) patients requires multiple cycles of satellite cells (SCs) activation to promote tissue regeneration. Dystrophic SCs present intrinsic defects, and the disrupting fibrotic niche hinders appropriate muscle recovery. Traditional 2D culture systems face challenges in modeling the DMD muscle niche and SCs behavior. Our aim was to validate a 3D culture of skeletal muscle spheroids (iSMS) for DMD modeling, as compared to the traditional 2D culture, while investigating the pathophysiological mechanisms of dystrophin deficiency in vitro. To compare iSMS with traditional 2D myogenic differentiation, we differentiated wild-type (WT), dystrophic (DMD) isogenic induced pluripotent stem cells (iPSCs), as well as iPSCs derived from DMD patients, characterized myogenic markers levels and assessed differences in proliferation and differentiation using RT-qPCR, immunofluorescence, and flow cytometry. Our data showed that iSMS improved PAX7 expression in vitro, while MYOD1, MYOG, MYF5, and MYH3 expression were significantly reduced. These findings suggest that, at three weeks of myogenic differentiation, iSMS cultures retained satellite-like cells in a less activated, progenitor-like state. Accordingly, we identified higher expression of canonical Notch signaling genes such as JAG1 and NOTCH1 in iSMS compared to 2D. We also characterized the response of 2D and iSMS to terminal differentiation medium, providing a valuable comparison with muscle fibers derived from human adult myoblasts. Additionally, we showed that DMD iSMS-derived progenitors proliferated at reduced levels compared with WT, a characteristic not observed in progenitors derived from 2D cultures. Finally, we performed iSMS and 2D myogenic differentiation of iPSC lines from three patients with DMD. Our results highlight important advantages of using the iSMS differentiation platform over 2D for DMD in vitro modeling. Exploring these 3D systems may help to gain a deeper understanding of SCs behavior to advance in novel treatments for DMD, which might be applicable to other forms of muscular disorders.
Zoonotic diseases are common threats to global health. A large number of infectious diseases are transmitted from animals to humans. The current study aimed to assess the community's knowledge, attitudes, and practices (KAP) regarding common zoonotic diseases in the Arbaminch district. A cross-sectional survey was carried out between November 2024 and June 2025. A total of 384 participants were interviewed in the study. Participants residing in these areas were randomly chosen. Data were collected using a structured questionnaire. The collected data were analyzed using Stata 17, and the results were reported using descriptive statistics and the chi-square test. The findings of this study revealed that a majority (55%) of participants had good knowledge about zoonotic diseases. Respondents know several modes of transmission for zoonotic diseases, with animal bites (32.5%) being the most recognized, followed by direct contact (15.5%), ingestion of raw products (10%), and inhalation (10%). Regarding attitudes, 63.2% of respondents exhibited a positive attitude towards the importance of zoonotic disease prevention and control, and 67.4% of respondents followed relatively good hygiene and preventive behaviors. However, risky practices were still common. Knowledge score showed a significant association with age. Attitudes of participants were significantly associated with education, age, occupation, and income. Similarly, practices were significantly associated with gender, education level, occupation, and income, with all associations being statistically significant (p < 0.05). The overall community knowledge, attitudes, and practices regarding zoonotic diseases were relatively good.
Chronobiology has advanced scientifically since 2000. Translating this knowledge and approach to medicine can alter diagnosis, treatment, and prevention, and improve health. Adding time-of-day (or time-of-year) information is both a concrete and conceptual change to clinical practice and public health relevant to humans and other animals, with low implementation costs. Successful translation of chronobiology to medicine requires new methods, training, and organizational and regulatory action.
To develop a semi-automated method to segment "black hole" lesions on post-gadolinium 2D T1-weighted images (GdT1) in multiple sclerosis (MS) that follows radiological intensity rules and perform multi-center validation. Multi-center spin-echo GdT1 images and accompanying proton-density (PD)/T2-weighted images and manual T2 lesion masks of the REFLEXION study (NCT00813709) of suspected/early MS were used. Briefly, the proposed method segments cortical gray matter (GM) to derive a T1-weighted intensity threshold, which is applied inside co-registered T2 lesion masks to segment black hole lesion voxels. It was optimized on a training set (N = 40, 57.5% female, mean age 31.4 ± 8.7 (standard deviation) years), and 274 patients formed the test set (61.3% female, age 31.8 ± 8.4 years). Performance was quantified by the Dice similarity coefficient (DSC) and the intraclass correlation coefficient (ICC) for absolute agreement with manual segmentations. Lesion-wise sensitivity and specificity were calculated. Optimization resulted in: (1) GM selection as minimally 0.8 total WM plus GM partial volume, masked by MNI cortex; (2) normalized mutual information-driven linear co-registration of T2 to GdT1 images, interpolating T2 lesion masks using trilinear interpolation and 0.6 threshold; (3) mean intensity inside GM mask used as upper intensity threshold. The optimized method had acceptable spatial accuracy (DSC: 0.39 ± 0.26) and good volumetric accuracy (ICC: 0.84, 95% CI [0.72, 0.90]. Lesion-wise sensitivity was 0.91 ± 0.19, and lesion-wise specificity was 0.62 ± 0.22. The proposed method to semi-automatically segment black holes from post-gadolinium T1-weighted images shows acceptable performance. As a potential aid to radiologists, the method is not recommended to be used entirely without human intervention. Question T1-hypointense "black hole" lesions reflect disease severity in multiple sclerosis but are not routinely quantified due to a lack of reliable analysis methods. Findings A rule-based semi-automated method for GdT1 "black hole" lesion segmentation was developed and optimized, and then validated in a large unseen multi-center test set. Clinical relevance This method adds quantitative information about GdT1 "black hole" lesions to the radiological assessment of multiple sclerosis disease severity, when false positives are manually removed. This can enhance the characterization of individual patients and advance the understanding of the disease.
The deltoid ligament (DL) is the primary stabilizer of the medial ankle, but its injury mechanisms remain poorly understood. This study aimed to investigate the injury risk and mechanisms of individual DL bundles under both acute and chronic conditions to inform prevention and treatment strategies. A validated finite element model of the human foot was used to examine peak stresses in DL bundles under four acute loading scenarios. Chronic loading was simulated by applying gait loads after transecting the lateral ligaments, and the resulting DL stresses were compared with those of the intact model. Additionally, thirty-nine rats were assigned to three groups: a lateral ligament rupture group (LR, n = 13), a tibialis posterior tendon rupture group (TPR, n = 13), and a sham group (n = 13). After 6 weeks of treadmill running, the mechanical properties and histological characteristics of the DL, along with ankle joint morphology and articular stresses, were evaluated to further verify the hypothesized mechanisms of chronic injury. Under acute loadings, the tibiocalcaneal ligament (TCL), anterior tibiotalar ligament (ATTL), and deep posterior tibiotalar ligament (dPTTL) showed the highest stress under pronation-external rotation loading. Lateral ligament rupture increased DL stress during gait. After 6 weeks of treadmill running, the LR and TPR groups showed roughened articular surfaces with osteophyte formation, increased articular stress, decreased talar bone volume fraction, lower failure load and stiffness ratios of the DL (p < 0.01), reduced fluorescence intensity of COL1, and elevated levels of COL3, MMP-2 and IL-1β compared with the sham group (p < 0.01). The TCL, ATTL, and dPTTL bundles are particularly susceptible to acute injury, with pronation-external rotation posing the greatest risk. Chronic degeneration of the DL occurs following rupture of the lateral ligament or tibialis posterior tendon, with a more pronounced effect after lateral ligament rupture.
Propofol is a widely employed intravenous general anesthetic that can induce neurotoxic effects on neurons. Previous research has indicated dysregulation of miR-140-3p in the hippocampal tissues of propofol-treated mice. This research was designed to investigate the function and underlying mechanism of miR-140-3p in propofol-induced neurotoxicity. To simulate propofol-induced neurotoxicity, human SH-SY5Y cells and mice were treated with propofol. Commercial kits were used to measure LDH, MDA, SOD, GSH-Px, and BDNF levels. Cells were transfected with miR-140-3p mimics, inhibitor, or BACE1 overexpression plasmids. Gene expression was assessed by RT-qPCR, cell viability by CCK-8, and apoptosis by flow cytometry. Dual-luciferase and RIP assays confirmed that miR-140-3p targets BACE1. The results confirmed that as the concentration of propofol increased, miR-140-3p levels were progressively downregulated, while BACE1 was correspondingly upregulated. Upregulation of miR-140-3p rescued propofol-treated SH-SY5Y cells from cytotoxicity, as evidenced by enhanced viability, suppressed apoptosis, and ameliorated oxidative stress. Consistently, miR-140-3p overexpression also attenuated propofol-induced neurotoxicity in vivo. Furthermore, BACE1 was confirmed to be a direct target of miR-140-3p through experimental validation, and this post-transcriptional repression was shown to mediate the observed neuroprotection. miR-140-3p attenuates propofol-induced neurotoxicity via BACE1 in vitro and in vivo, providing new insights and a potential biomarker for managing propofol-associated neurotoxicity.
Fast-track and outpatient surgery have significantly reduced postoperative hospital stays across many surgical specialties. As a result, patients are increasingly discharged with strong opioid prescriptions, contributing to the global opioid crisis. Careful follow-up and opioid tapering are essential. While multidisciplinary Transitional Pain Services (TPS), involving pain specialists, psychologists, and physiotherapists, have shown promise, their widespread implementation is limited by costs and complexity. To address these barriers, we implemented a nurse-led TPS, supervised by a pain specialist and embedded within a multidisciplinary pain clinic. The aim of this study was to evaluate its effectiveness in clinical practice, including a mechanism-based treatment approach to postsurgical pain aimed at opioid tapering and optimizing the use of adjuvant analgesics. This observational cohort study included postoperative patients discharged with >20 mg oral oxycodone equivalents and/or those experiencing or at risk for neuropathic pain. Referred patients received telephone consultations by a nurse practitioner (NP) one to two weeks post-discharge. Each consultation included assessment of pain severity, neuropathic characteristics (using the first two items of the DN4 questionnaire), current analgesic use, and willingness to taper opioids. Patient education and motivational interviewing techniques were employed to support opioid tapering. Descriptive statistics and paired t-tests were used to analyze the data. Between June 2019 and July 2025, 243 patients were enrolled in the TPS. Following nurse-led counseling, 73 % of patients discontinued opioid use entirely, 23 % significantly tapered their dosage (from mean 101-43 mg oral oxycodone equivalent), and 4 % continued at the same dose. Anti-neuropathic medications were initiated in 22 % of patients. A nurse-led Transitional Pain Service is a feasible and effective approach to support opioid tapering in postoperative patients. In addition, early screening for neuropathic pain allows for targeted treatment. This model offers a scalable alternative to traditional multidisciplinary TPS programs.
Functional validation of candidate genes in congenital anomalies of the kidneys and urinary tract (CAKUT) and other disorders is essential for translating genetic discoveries into clinical applications. Conditional knockout mouse models are indispensable for studying gene function in complex organ systems. The Short Conditional intrON (SCON) system accelerates the generation of such models by inserting the artificial SCON into a coding exon. SCON is designed to be spliced out after transcription, without affecting gene expression. Upon Cre activity, SCON is converted into the ΔSCON allele which cannot be spliced out, introducing premature termination codons (PTCs) to inactivate the gene. Previous validation of the SCON system in mice has focused primarily on phenotypic outcomes. Here, we provide a molecular characterization of the SCON system in Cdh12-a candidate gene implicated in kidney damage in CAKUT. We found that both Cdh12SCON and Cdh12ΔSCON alleles caused unintended skipping of the exon downstream of the insertion site, culminating in a frameshift and PTC. Consequently, the Cdh12SCON allele led to a ~ 25% reduction in mRNA expression, indicating that it was not transcriptionally inert as designed. Despite unintended exon skipping, the Cdh12ΔSCON allele still effectively suppressed mRNA expression. These findings highlight the importance of transcript-level characterization of engineered alleles prior to functional studies, as artefactual splicing events may occur across multiple gene-targeting strategies, including artificial intron-based conditional alleles as shown here.
Although roles of CD44 in the genetic predisposition for bronchopulmonary dysplasia (BPD) has been recognized, the immune characteristics of CD44+ monocytes in BPD are unclear. We aimed to (1) compare the expression and function of CD44 on monocytes in BPD and non-BPD infants; (2) explore the roles of CD44 on monocytes in hyperoxia-induced inflammation. Flow cytometry assessments of the expression pattern and cytokine-secreting response upon LPS stimulation of CD44+ monocytes were conducted using peripheral blood samples from BPD infants (n = 80) and non-BPD infants (n = 106). The role of CD44 on monocytes was validated in hyperoxia exposure. CD44 expression and inflammatory cytokine secretion ability increased with postmenstrual age. The ability of CD44+ monocytes to secrete IL-6 and TNF-α was significantly greater than that of CD44-monocytes. Compared with those of non-BPD infants, the response capacity of the monocytes in BPD infants to secret IL-6 and TNF-α, especially TNF-α sourced from CD44+ intermediate monocytes, was greater upon LPS stimulation. CD44 expression significantly increased in hyperoxia, and CD44 knockdown significantly ameliorated the inflammation induced by hyperoxia. CD44+ monocytes played important roles in mediating the hyper-inflammatory response in BPD. One specific subpopulation, TNF-α+/CD44+intermediate monocytes, might be a valuable marker for identifying BPD. 1. This study revealed the key role of an immune cell subtype-CD44+ monocytes-in hyperoxia-induced bronchopulmonary dysplasia (BPD) and characterized the expression and function of CD44+ monocytes between premature infants with BPD and those without BPD. 2. CD44 was closely associated with the proinflammatory cytokine secretion capacity of monocytes. A specific subpopulation-TNF-α+/CD44+ intermediate monocytes- might serve as a valuable marker for the identification of BPD in the future. 3. Our findings added new evidence of the association between CD44+ monocytes and BPD pathogenesis, which provided new insights into the immune mechanisms for BPD.
There have been discussions as to the time of elective induction of labour to curb the continuation of pregnancy that might endanger the lives of both the mother and child. This research was conducted to assess foetal and maternal consequences of planned delivery at 40 and 41weeks in women with low-risk singleton pregnancy. A randomised controlled trial with equal allocation of participants (96 pregnant women in each arm) into 40weeks and 41weeks. Participants were randomised at the antenatal clinic at 39 weeks for induction of labour. The main outcome was the caesarean section rate. Secondary outcomes were maternal (genital tract laceration rate) and foetal (rates of meconium staining of amniotic fluid, SCBU admission, perinatal mortality, birth trauma, birth weight, and neonatal APGAR score at 1 and 5 minutes). Student t-test and chi-square test were used for inter-group comparison. Incidence of caesarean delivery (26.6% vs. 21.3%; p=0.406), and genital laceration (2.1% vs. 5.6%; p=0.268) did not differ between groups. Significantly higher birth weight was noted among women induced at 41weeks (3.41 ± 0.37kg) than 40weeks (3.28 ± 0.46kg) (p=0.043). Also, there was significant variation in meconium staining of amniotic fluid between 40weeks (11.7%) and 41weeks (25.8%) (p=0.014). Other foetal outcomes showed no significant difference. Inducing labour at 40weeks is safe for low-risk women as it does not significantly increase the cesarean delivery rate and adverse perinatal outcomes. Therefore, elective induction of labour at 40weeks should be recommended and introduced into obstetric practice without the fear of adverse outcomes.
Central nervous system (CNS) metastases from Wilms tumor (WT) are exceedingly rare. Intracerebral hemorrhage secondary to metastatic WT is even less common, and the management of such cases is further complicated when patients are receiving a direct oral anticoagulant (DOAC) like Rivaroxaban, for which pediatric reversal guidelines are lacking. We report on the case of a 5-year-old boy with relapsed stage IV Wilms tumor who presented with rapidly progressive neurological deterioration caused by brain metastases with extensive intraparenchymal and intraventricular hemorrhage while receiving Rivaroxaban due to prior thrombosis. An emergent craniotomy and tumor resection was safely performed after emergent reversal of anticoagulation with Rivaroxaban using Andexanet alfa, administered in this pediatric patient with off-label consent in the setting of a life-threatening intracranial hemorrhage requiring emergent neurosurgical intervention. No excessive intraoperative bleeding was noted. Treatment for relapsed WT according to the SIOP-UMBRELLA-Protocol was initiated. Three weeks after Andexanet alfa treatment, a thrombotic event in the left iliac veins occurred, requiring anticoagulation with unfractionated heparin. This case highlights the therapeutic challenges of managing intracranial hemorrhage in a pediatric patient requiring emergent neurosurgical debulking in the setting of Rivaroxaban anticoagulation. To our knowledge, this is the second case reporting on Rivaroxaban reversal through Andexanet alfa in children. Early multidisciplinary intervention, meticulous neurosurgical management and continuation of oncologic therapy can lead to favorable outcomes even in such complex presentations.