Gene Knockout Mice Research Articles

ZG16 impacts gut microbiota-associated intestinal inflammation and pulmonary mucosal function through bacterial metabolites

Zymogen granule 16 (ZG16) is a secretory glycoprotein found in zymogen granules, which also plays an important role in colorectal inflammation and cancer. Herein, a ZG16 gene knock-out (ZG16−/−) mouse line was established and we found that ZG16 deletion damaged the intestinal mucosal barrier and gut microbiota, which resulted in low-level inflammation and further promoted the development of ulcerative colitis and inflammation-related colorectal cancer. Meanwhile, a metabolomics analysis on mouse feces showed that the metabolites significantly differed between ZG16−/− and WT mice, which were important mediators of host-microbiota communication and may impact the pulmonary inflammation of mice. Indeed, ZG16−/− mice showed more severe inflammation in a bronchial asthma model. Taken together, the results demonstrate that ZG16 plays a pivotal role in inhibiting inflammation and regulating immune responses in colorectum and lung of experimental animals, which may provide a better understanding of the underlying mechanism of human inflammatory diseases associated with ZG16.

ITGB6 modulates resistance to anti-CD276 therapy in head and neck cancer by promoting PF4+ macrophage infiltration

Enoblituzumab, an immunotherapeutic agent targeting CD276, shows both safety and efficacy in activating T cells and oligodendrocyte-like cells against various cancers. Preclinical studies and mouse models suggest that therapies targeting CD276 may outperform PD1/PD-L1 blockade. However, data from mouse models indicate a significant non-responsive population to anti-CD276 treatment, with the mechanisms of resistance still unclear. In this study, we evaluate the activity of anti-CD276 antibodies in a chemically-induced murine model of head and neck squamous cell carcinoma. Using models of induced and orthotopic carcinogenesis, we identify ITGB6 as a key gene mediating differential responses to anti-CD276 treatment. Through single-cell RNA sequencing and gene-knockout mouse models, we find that ITGB6 regulates the expression of the tumor-associated chemokine CX3CL1, which recruits and activates PF4+ macrophages that express high levels of CX3CR1. Inhibition of the CX3CL1-CX3CR1 axis suppresses the infiltration and secretion of CXCL16 by PF4+ macrophages, thereby reinvigorating cytotoxic CXCR6+ CD8+ T cells and enhancing sensitivity to anti-CD276 treatment. Further investigations demonstrate that inhibiting ITGB6 restores sensitivity to PD1 antibodies in mice resistant to anti-PD1 treatment. In summary, our research reveals a resistance mechanism associated with immune checkpoint inhibitor therapy and identifies potential targets to overcome resistance in cancer treatment.

Selenomethionine supplementation mitigates fluoride-induced liver apoptosis and inflammatory reactions by blocking Parkin-mediated mitophagy in mice

As an environmental pollutant, fluoride-induced liver damage is directly linked to mitochondrial alteration and oxidative stress. Selenium's antioxidant capacity has been shown to alleviate liver damage. Emerging research proves that E3 ubiquitin ligase Park2 (Parkin)-mediated mitophagy may be a therapeutic target for fluorosis. The current study explored the effect of diverse selenium sources on fluoride-caused liver injury and the role of Parkin-mediated mitophagy in this intervention process. Therefore, this study established a fluoride-different selenium sources co-intervention wild-type (WT) mouse model and a fluoride-optimum selenium sources co-intervention Parkin gene knockout (Parkin−/−) mouse model. Our results show that selenomethionine (SeMet) is the optimum selenium supplementation form for mice suffering from fluorosis when compared to sodium selenite and chitosan nano‑selenium because mice from the F-SeMet group showed more closely normal growth and development levels of liver function, antioxidant capacity, and anti-inflammatory ability. Explicitly, SeMet ameliorated liver inflammation and cell apoptosis in fluoride-toxic mice, accomplished through downregulating the mRNA and protein expression levels associated with mitochondrial fusion and fission, mitophagy, apoptosis, inflammatory signalling pathway of nuclear factor-kappa B (NF-κB), reducing the protein expression levels of PARKIN, PTEN-induced putative kinase1 (PINK1), SQSTM1/p62 (P62), microtubule-associated protein light chain 3 (LC3), cysteinyl aspartate specific proteinase 3 (CASPAS3), as well as restraining the content of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ). The Parkin−/− showed comparable positive effects to the SeMet in the liver of fluorosis mice. The structure of the mitochondria, mRNA, protein expression levels, and the content of proinflammatory factors in mice from the FParkin−/− and F + SeMetParkin−/− groups closely resembled those in the F + SeMetWT group. Overall, the above results indicated that SeMet could alleviate fluoride-triggered inflammation and apoptosis in mice liver via blocking Parkin-mediated mitophagy.

MAPK13 controls structural remodeling and disease after epithelial injury.

All living organisms are charged with repair after injury particularly at epithelial barrier sites, but in some cases this response leads instead to structural remodeling and long-term disease. Identifying the molecular and cellular control of this divergence is key to disease modification. In that regard, stress kinase control of epithelial stem cells is a rational entry point for study. Here we examine the potential for mitogen-activated protein kinase 13 (MAPK13) regulation of epithelial stem cells using models of respiratory viral injury and post-viral lung disease. We show that Mapk13 gene-knockout mice handle acute infectious illness as expected but are protected against structural remodeling manifest as basal-epithelial stem cell (basal-ESC) hyperplasia-metaplasia, immune activation, and mucinous differentiation. In corresponding cell models, Mapk13-deficiency directly attenuates basal-ESC growth and organoid formation. Extension to human studies shows marked induction/activation of basal-cell MAPK13 in clinical samples of comparable remodeling found in asthma and COPD. Here again, MAPK13 gene-knockdown inhibits human basal-ESC growth in culture. Together, the data identify MAPK13 as a control for structural remodeling and disease after epithelial injury and as a suitable target for down-regulation as a disease-modifying strategy.

Eighteen genes primarily expressed in the testis are not required for male fertility in mice.

There are approximately 20,000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

Podocyte cell-specific Npr1 is required for blood pressure and renal homeostasis in male and female mice: role of sex-specific differences.

Atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase A/natriuretic peptide receptor A (GC-A/NPRA), stimulating natriuresis and diuresis and reducing blood pressure (BP), but the role of ANP/NPRA signaling in podocytes (highly specialized epithelial cells covering the outer surfaces of renal glomerular capillaries) remains unclear. This study aimed to determine the effect of conditional deletion of podocyte-specific Npr1 (encoding NPRA) gene knockout (KO) in male and female mice. Tamoxifen-treated wild-type control (PD Npr1 f/f; WT), heterozygous (PD-Cre-Npr1 f/+; HT), and KO (PD-Cre-Npr1 f/-) mice were fed a normal-, low-, or high-salt diet for 4 wk. Podocytes isolated from HT and KO male and female mice showed complete absence of Npr1 mRNA and NPRA protein compared with WT mice. BP, plasma creatinine, plasma sodium, urinary protein, and albumin/creatinine ratio were significantly increased, whereas plasma total protein, albumin, creatinine clearance, and urinary sodium levels were significantly reduced in the HT and KO male and female mice compared with WT mice. These changes were significantly greater in males than in females. On a normal-salt diet, glomerular filtration rate was significantly decreased in PD Npr1 HT and KO male and female mice compared with WT mice. Immunofluorescence of podocin and synaptopodin was also significantly reduced in HT and KO mice compared with WT mice. These observations suggest that in podocytes, ANP/NPRA signaling may be crucial in the maintenance and regulation of glomerular filtration and BP and serve as a biomarker of renal function in a sex-dependent manner.NEW & NOTEWORTHY Our results demonstrate that the podocyte-specific deletion of Npr1 showed increased blood pressure (BP) and altered biomarkers of renal functions, with greater magnitudes in animals fed a high-salt diet in a sex-dependent manner. The results suggest a direct and sex-dependent effect of Npr1 ablation in podocytes on the regulation of BP and renal function and reveal that podocytes may be considered an important target for the ANP-BNP/NPRA/cGMP signaling cascade.

CD248-expressing cancer-associated fibroblasts induce non-small cell lung cancer metastasis via Hippo pathway-mediated extracellular matrix stiffness.

Metastasis is a crucial stage in tumour progression, and cancer-associated fibroblasts (CAFs) support metastasis through their participation in extracellular matrix (ECM) stiffness. CD248 is a possible biomarker for non-small cell lung cancer (NSCLC)-derived CAFs, but its role in mediating ECM stiffness to promote NSCLC metastasis is unknown. We investigated the significance of CD248+ CAFs in activating the Hippo axis and promoting connective tissue growth factor (CTGF) expression, which affects the stromal collagen I environment and improves ECM stiffness, thereby facilitating NSCLC metastasis. In this study, we found that higher levels of CD248 in CAFs induced the formation of collagen I, which in turn increased extracellular matrix stiffness, thereby enabling NSCLC cell infiltration and migration. Hippo axis activation by CD248+ CAFs induces CTGF expression, which facilitates the formation of the collagen I milieu in the stromal matrix. In a tumour lung metastasis model utilizing fibroblast-specific CD248 gene knockout mice, CD248 gene knockout mice showed a significantly reduced ability to develop tumour lung metastasis compared to that of WT mice. Our findings demonstrate that CD248+ CAFs activate the Hippo pathway, thereby inducing CTGF expression, which in turn facilitates the collagen I milieu of the stromal matrix, which promotes NSCLC metastasis.

Vaccine Therapy for Heart Failure Targeting the Inflammatory Cytokine Igfbp7.

The heart comprises many types of cells such as cardiomyocytes, endothelial cells (ECs), fibroblasts, smooth muscle cells, pericytes, and blood cells. Every cell type responds to various stressors (eg, hemodynamic overload and ischemia) and changes its properties and interrelationships among cells. To date, heart failure research has focused mainly on cardiomyocytes; however, other types of cells and their cell-to-cell interactions might also be important in the pathogenesis of heart failure. Pressure overload was imposed on mice by transverse aortic constriction and the vascular structure of the heart was examined using a tissue transparency technique. Functional and molecular analyses including single-cell RNA sequencing were performed on the hearts of wild-type mice and EC-specific gene knockout mice. Metabolites in heart tissue were measured by capillary electrophoresis-time of flight-mass spectrometry system. The vaccine was prepared by conjugating the synthesized epitope peptides with keyhole limpet hemocyanin and administered to mice with aluminum hydroxide as an adjuvant. Tissue samples from heart failure patients were used for single-nucleus RNA sequencing to examine gene expression in ECs and perform pathway analysis in cardiomyocytes. Pressure overload induced the development of intricately entwined blood vessels in murine hearts, leading to the accumulation of replication stress and DNA damage in cardiac ECs. Inhibition of cell proliferation by a cyclin-dependent kinase inhibitor reduced DNA damage in ECs and ameliorated transverse aortic constriction-induced cardiac dysfunction. Single-cell RNA sequencing analysis revealed upregulation of Igfbp7 (insulin-like growth factor-binding protein 7) expression in the senescent ECs and downregulation of insulin signaling and oxidative phosphorylation in cardiomyocytes of murine and human failing hearts. Overexpression of Igfbp7 in the murine heart using AAV9 (adeno-associated virus serotype 9) exacerbated cardiac dysfunction, while EC-specific deletion of Igfbp7 and the vaccine targeting Igfbp7 ameliorated cardiac dysfunction with increased oxidative phosphorylation in cardiomyocytes under pressure overload. Igfbp7 produced by senescent ECs causes cardiac dysfunction and vaccine therapy targeting Igfbp7 may be useful to prevent the development of heart failure.

OTUD1 Deficiency Alleviates LPS-Induced Acute Lung Injury in Mice by Reducing Inflammatory Response.

The ovarian tumor (OTU) family consists of deubiquitinating enzymes thought to play a crucial role in immunity. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) pose substantial clinical challenges due to severe respiratory complications and high mortality resulting from uncontrolled inflammation. Despite this, no study has explored the potential link between the OTU family and ALI/ARDS. Using publicly available high-throughput data, 14 OTUs were screened in a simulating bacteria- or LPS-induced ALI model. Subsequently, gene knockout mice and transcriptome sequencing were employed to explore the roles and mechanisms of the selected OTUs in ALI. Our screen identified OTUD1 in the OTU family as a deubiquitinase highly related to ALI. In the LPS-induced ALI model, deficiency of OTUD1 significantly ameliorated pulmonary edema, reduced permeability damage, and decreased lung immunocyte infiltration. Furthermore, RNA-seq analysis revealed that OTUD1 deficiency inhibited key pathways, including the IFN-γ/STAT1 and TNF-α/NF-κB axes, ultimately mitigating the severity of immune responses in ALI. In summary, our study highlights OTUD1 as a critical immunomodulatory factor in acute inflammation. These findings suggest that targeting OTUD1 could hold promise for the development of novel treatments against ALI/ARDS.

ASM is a therapeutic target in dermatomyositis by regulating the differentiation of naive CD4 + T cells into Th17 and Treg subsets

BackgroundThis study aims to investigate the involvement of acid sphingomyelinase (ASM) in the pathology of dermatomyositis (DM), making it a potential therapeutic target for DM.MethodsPatients with DM and healthy controls (HCs) were included to assess the serum level and activity of ASM, and to explore the associations between ASM and clinical indicators. Subsequently, a myositis mouse model was established using ASM gene knockout and wild-type mice to study the significant role of ASM in the pathology and to assess the treatment effect of amitriptyline, an ASM inhibitor. Additionally, we investigated the potential treatment mechanism by targeting ASM both in vivo and in vitro.ResultsA total of 58 DM patients along with 30 HCs were included. The ASM levels were found to be significantly higher in DM patients compared to HCs, with median (quartile) values of 2.63 (1.80–4.94) ng/mL and 1.64 (1.47–1.96) ng/mL respectively. The activity of ASM in the serum of DM patients was significantly higher than that in HCs. Furthermore, the serum levels of ASM showed correlations with disease activity and muscle enzyme levels. Knockout of ASM or treatment with amitriptyline improved the severity of the disease, rebalanced the CD4 T cell subsets Th17 and Treg, and reduced the production of their secreted cytokines. Subsequent investigations revealed that targeting ASM could regulate the expression of relevant transcription factors and key regulatory proteins.ConclusionASM is involved in the pathology of DM by regulating the differentiation of naive CD4 + T cells and can be a potential treatment target.

Examination of the Effect of Dimethyl Trisulfide in Acute Stress Mouse Model with the Potential Involvement of the TRPA1 Ion Channel.

Polysulfides are endogenously produced in mammals and generally associated with protective functions. Our aim was to investigate the effect of dimethyl trisulfide (DMTS) in a mouse model of acute stress. DMTS activates transient receptor potential ankyrin 1 (TRPA1) channels and leads to neuropeptide release, potentially that of substance P (SP). We hypothesize that DMTS might inhibit the degrading enzymes of endocannabinoids, so this system was also investigated as another possible pathway for mediating the effects of DMTS. Trpa1 gene wild-type (WT) and knockout (KO) mice were used to confirm the role of the TRPA1 ion channel in mediating the effects of DMTS. C57BL/6J, NK1 gene KO, and Tac1 gene KO mice were used to evaluate the effect of DMTS on the release and expression of SP. Some C57BL/6J animals were treated with AM251, an inhibitor of the cannabinoid CB1 receptor, to elucidate the role of the endocannabinoid system in these processes. Open field test (OFT) and forced swim test (FST) were performed in each mouse strain. A tail suspension test (TST) was performed in Trpa1 WT and KO animals. C-FOS immunohistochemistry was carried out on Trpa1 WT and KO animals. The DMTS treatment increased the number of highly active periods and decreased immobility time in the FST in WT animals, but had no effect on the Trpa1 KO mice. The DMTS administration induced neuronal activation in the Trpa1 WT mice in the stress-related brain areas, such as the locus coeruleus, dorsal raphe nucleus, lateral septum, paraventricular nucleus of the thalamus, and paraventricular nucleus of the hypothalamus. DMTS may have a potential role in the regulation of stress-related processes, and the TRPA1 ion channel may also be involved in mediating the effects of DMTS. DMTS can be an ideal candidate for further study as a potential remedy for stress-related disorders.

Hippocampal Crhr1 conditional gene knockout ameliorated the depression-like behavior and pathological damage in male offspring mice caused by chronic stress during pregnancy

Numerous studies have demonstrated that chronic stress during pregnancy (CSDP) can induce depression and hippocampal damage in offspring. It has also been observed that high levels of corticotropin-releasing hormone (CRH) can damage hippocampal neurons, and intraperitoneal injection of a corticotropin releasing hormone receptor 1 (CRHR1) antagonist decreases depression-like behavior and hippocampal neuronal damage in a mouse depression model. However, whether CSDP causes hippocampal damage and depression in offspring through the interaction of CRH and hippocampal CRHR1 remains unknown and warrants further investigation. Therefore, hippocampal Crhr1 conditional gene knockout mice and C57/BL6J mice were used to study these questions. Depression-related indexs in male offspring mice were examined using the forced swim test (FST), sucrose preference test (SPT), tail suspension test (TST) and open field test (OFT). Serum CRH levels were measured by enzyme-linked immunosorbent assay (ELISA). Golgi-Cox staining was used to examine the morphological changes of hippocampal neuronal dendrites. Neuronal apoptosis in the hippocampal CA3 regions was detected by terminal deoxynucleotidy transferase dUTP nick end labeling (TUNEL) staining. The levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR) and protein kinase B (AKT) proteins were measured by Western blot analysis. This study showed that CSDP induces depression-like behavior, hippocampal neuronal dendrite damage and apoptosis in male offspring mice. Conditional gene knockout of hippocampal Crhr1 in mice reduced CSDP-induced depression-like behavior, hippocampal neuronal dendrite damage and apoptosis in male offspring, and counteracted the CSDP-induced decreased expression of p-Akt and mTOR activity in male offspring hippocampus. These findings demonstrated that CSDP might inhibit the Akt/mTOR pathway by increasing the levels of CRH, leading to increased CRH-mediated activation of hippocampal CRHR1, thereby inducing synaptic impairment and apoptosis in hippocampal neurons, which in turn leads to depression-like behavior in offspring.

Intestinal microbiota imbalance resulted by anti-Toxoplasma gondii immune responses aggravate gut and brain injury

BackgroundToxoplasma gondii infection affects a significant portion of the global population, leading to severe toxoplasmosis and, in immunocompromised patients, even death. During T. gondii infection, disruption of gut microbiota further exacerbates the damage to intestinal and brain barriers. Therefore, identifying imbalanced probiotics during infection and restoring their equilibrium can regulate the balance of gut microbiota metabolites, thereby alleviating tissue damage.MethodsVimentin gene knockout (vim−/−) mice were employed as an immunocompromised model to evaluate the influence of host immune responses on gut microbiota balance during T. gondii infection. Behavioral experiments were performed to assess changes in cognitive levels and depressive tendencies between chronically infected vim−/− and wild-type (WT) mice. Fecal samples were subjected to 16S ribosomal RNA (rRNA) sequencing, and serum metabolites were analyzed to identify potential gut probiotics and their metabolites for the treatment of T. gondii infection.ResultsCompared to the immunocompetent WT sv129 mice, the immunocompromised mice exhibited lower levels of neuronal apoptosis and fewer neurobehavioral abnormalities during chronic infection. 16S rRNA sequencing revealed a significant decrease in the abundance of probiotics, including several species of Lactobacillus, in WT mice. Restoring this balance through the administration of Lactobacillus murinus and Lactobacillus gasseri significantly suppressed the T. gondii burden in the intestine, liver, and brain. Moreover, transplantation of these two Lactobacillus spp. significantly improved intestinal barrier damage and alleviated inflammation and neuronal apoptosis in the central nervous system. Metabolite detection studies revealed that the levels of various Lactobacillus-related metabolites, including indole-3-lactic acid (ILA) in serum, decreased significantly after T. gondii infection. We confirmed that L. gasseri secreted much more ILA than L. murinus. Notably, ILA can activate the aromatic hydrocarbon receptor signaling pathway in intestinal epithelial cells, promoting the activation of CD8+ T cells and the secretion of interferon-gamma.ConclusionOur study revealed that host immune responses against T. gondii infection severely disrupted the balance of gut microbiota, resulting in intestinal and brain damage. Lactobacillus spp. play a crucial role in immune regulation, and the metabolite ILA is a promising therapeutic compound for efficient and safe treatment of T. gondii infection.Graphical

Dectin-1 induces TRPV1 sensitization and contributes to visceral hypersensitivity of irritable bowel syndrome in male mice.

Visceral hypersensitivity is considered the core pathophysiological mechanism that causes abdominal pain in patients with irritable bowel syndrome (IBS). Fungal dysbiosis has been proved to contribute to visceral hypersensitivity in IBS patients. However, the underlying mechanisms for Dectin-1, a major fungal recognition receptor, in visceral hypersensitivity are poorly understood. This study aimed to explore the role of Dectin-1 in visceral hypersensitivity and elucidate the impact of Dectin-1 activity on the function of transient receptor potential vanilloid type 1 (TRPV1). Visceral hypersensitivity model was established by the intracolonic administration of 0.1 mL TNBS (130 μg/mL in 30% ethanol) in the male mice. Fluconazole and nystatin were used as fungicides. Laminarin, a Dectin-1 antagonist and gene knockout (Clec7a-/-) mice were used to interrupt the function of Dectin-1. Colorectal distension-electromyogram recording was performed to assess visceral sensitivity. Immunostaining experiment was performed to determine the localization of Dectin-1 in dorsal root ganglion (DRG) neurons. Calcium imaging study was performed to assay TRPV1-mediated calcium influx in acutely dissociated DRG neurons. Pretreatment with fungicides, administration of laminarin or genetic deletion of Clec7a alleviated TNBS-induced visceral hypersensitivity in male mice. The expression of Dectin-1 was upregulated in the DRG and colon of TNBS-treated mice. Colocalization of Dectin-1 and TRPV1 was observed in DRG neurons. Importantly, pretreatment with curdlan, a Dectin-1 agonist, increased TRPV1-mediated calcium influx. Dectin-1 contributes to visceral hypersensitivity in IBS or in inflammatory bowel disease in remission and activation of Dectin-1 induces TRPV1 sensitization. This work provides direct evidence for the functional regulation of TRPV1 channel by Dectin-1 activity, proposing a new mechanism underlying TRPV1 sensitization. Control of intestinal fungi might be beneficial for the treatment of refractory abdominal pain in patients with IBS or IBD in remission.

Modeling Fragile X Syndrome: Characterizing Fmr1 Gene Knockout Mice across Genotype, Behavior, and Morphology

ABSTRACT Objective: Fragile X syndrome (FXS) is a hereditary condition resulting from dynamic mutations in the Fmr1 gene, leading to reduced or absent fragile X mental retardation protein (FMRP). Although molecular genetic diagnostics for FXS have advanced, there is currently a lack of effective clinical treatment. Hence, identifying a suitable FXS animal model is crucial for FXS therapeutic studies. This study primarily aims to evaluate whether Fmr1 mice display aberrant behaviors similar to those observed in human FXS patients, aiming to validate their feasibility as a model for FXS. Research Design and Methods: Polymerase chain reaction and immunoblotting were employed to genotype Fmr1 knockout (KO) mice and wild-type (WT) mice. Using hematoxylin and eosin staining and Golgi staining, testicular and dendritic spine morphology in both animal groups was observed. The animals were subjected to audiogenic seizure (AGS) observation, evaluating their learning and memory levels using the water maze and light avoidance methods, while their exploratory behavior capacity was assessed using the open-field and autonomous activity experiments. Anxiety behavior was evaluated using the elevated plus maze. Results: WT mice produced a 500 bp DNA fragment, while KO mice yielded an 800 bp fragment. KO mice exhibited a lack of FMRP expression, notably increased dendritic spine length and density, and presented with macroorchidism. KO mice showed increased susceptibility to AGS. In the Morris water maze experiment, KO mice displayed significantly increased escape latency and platform crossings. The spatial search experiment revealed that KO mice spent significantly less time in the target quadrant compared to WT mice. The light-avoidance and platform experiments indicated a significant reduction in latency for KO mice but an increase in errors. In the open-field experiment, KO mice spent significantly more time and made more entries into the central zone compared to WT mice. KO mice exhibited increased total travel distance and speed. The elevated plus maze experiment showed that KO mice spent significantly more time, made more entries, and traveled further in the open area compared to WT mice. The autonomous activity experiment revealed that KO mice had significantly more activity counts than WT mice but fewer standing counts. Conclusions: Findings from genotype, protein expression spectrum, morphology, and behavioral results suggest that Fmr1 gene KO mice demonstrate phenotypes similar to those observed in human FXS, making them a potential model for future FXS research. These phenotypes are associated with the absence of FMRP expression.

Solute Transporter OCTN1/Slc22a4 Affects Disease Severity and Response to Infliximab in Experimental Colitis: Role of Gut Microbiota and Immune Modulation

Abstract Background Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial etiology, still incompletely understood. OCTN1, an organic cation transporter, could have a role in modulating the inflammatory response, and some genetic polymorphisms of this molecule have been associated with increased risk of inflammatory bowel diseases. Until now, limited information exists on its potential in predicting/modulating patient’s response to therapies. The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and mucosal immunity in response to infliximab therapy in murine colitis. Methods A dextran sodium sulphate model of colitis was used to assess the clinical efficacy of infliximab administered intravenously in ocnt1 gene knockout mice and their C57BL/6 controls. Stool, colon, and mesenteric lymph node samples were collected to evaluate differences in gut microbiota composition, histology, and T cell populations, respectively. Results Octn1 -/- influences the microbiota profile and is associated with a worse dysbiosis in mice with colitis. Infliximab treatment attenuates colitis-associated dysbiosis, with an increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1-/- mice have milder disease and a higher baseline percentage of Treg, Tmemory, Th2 and Th17 cells. Conclusions Our data support the murine model to study OCTN1 genetic contribution to inflammatory bowel diseases. This could be the first step towards the recognition of this membrane transporter as a biomarker in inflammatory conditions and a predictor of response to therapies.

Hypersensitivity to type I interferon as a cause of hydrocephalus development

Ubiquitin specific protease 18 (USP18) serves as a potent inhibitor of Type I interferon (IFN) signaling. Previous studies have shown that Usp18 deficient (homozygous Usp18 gene knockout) mice exhibit hydrocephalus; however, the precise molecular mechanism underlying hydrocephalus development remains elusive. In this study, we demonstrate that mice lacking both type I IFN receptor subunit 1 (Ifnar1) and Usp18 (Ifnar1/Usp18 double knockout mice) are viable and do not display a hydrocephalus phenotype. Moreover, we observed that suppression of USP18 in ependymal cells treated with IFN significantly increased cell death, including pyroptosis, and decreased proliferation. These findings suggest that heightened sensitivity to type I IFN during brain development contributes to the onset of hydrocephalus. Furthermore, they imply that inhibition of IFN signaling may hold promise as a therapeutic strategy for hydrocephalus.

Anti-Inflammatory Responses Produced with Nippostrongylus brasiliensis-Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model.

Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that Nippostrongylus brasiliensis (Nb) infection or its derived products could inhibit AS development by inducing an anti-inflammatory response. We performed a metabolic analysis to screen Nb-derived metabolites with anti-inflammation activity and evaluated the AS-prevention effect. We observed that the metabolite uridine had higher expression levels in mice infected with the Nb and ES (excretory-secretory) products and could be selected as a key metabolite. ES and uridine interventions could reduce the pro-inflammatory responses and increase the anti-inflammatory responses in vitro and in vivo. The apolipoprotein E gene knockout (ApoE-/-) mice were fed with a high-fat diet for the AS modeling. Following the in vivo intervention, ES products or uridine significantly reduced serum and liver lipid levels, alleviated the formation of atherosclerosis, and reduced the pro-inflammatory responses in serum or plaques, while the anti-inflammatory responses showed opposite trends. After blocking with 5-HD (5-hydroxydecanoate sodium) in vitro, the mRNA levels of M2 markers were significantly reduced. When blocked with 5-HD in vivo, the degree of atherosclerosis was worsened, the pro-inflammatory responses were increased compared to the uridine group, while the anti-inflammatory responses decreased accordingly. Uridine, a key metabolite from Nippostrongylus brasiliensis, showed anti-inflammatory and anti-atherosclerotic effects in vitro and in vivo, which depend on the activation of the mitochondrial ATP-sensitive potassium channel.

Deciphering potential causative factors for undiagnosed Waardenburg syndrome through multi-data integration

BackgroundWaardenburg syndrome (WS) is a rare genetic disorder mainly characterized by hearing loss and pigmentary abnormalities. Currently, seven causative genes have been identified for WS, but clinical genetic testing results show that 38.9% of WS patients remain molecularly unexplained. In this study, we performed multi-data integration analysis through protein-protein interaction and phenotype-similarity to comprehensively decipher the potential causative factors of undiagnosed WS. In addition, we explored the association between genotypes and phenotypes in WS with the manually collected 443 cases from published literature.ResultsWe predicted two possible WS pathogenic genes (KIT, CHD7) through multi-data integration analysis, which were further supported by gene expression profiles in single cells and phenotypes in gene knockout mouse. We also predicted twenty, seven, and five potential WS pathogenic variations in gene PAX3, MITF, and SOX10, respectively. Genotype-phenotype association analysis showed that white forelock and telecanthus were dominantly present in patients with PAX3 variants; skin freckles and premature graying of hair were more frequently observed in cases with MITF variants; while aganglionic megacolon and constipation occurred more often in those with SOX10 variants. Patients with variations of PAX3 and MITF were more likely to have synophrys and broad nasal root. Iris pigmentary abnormality was more common in patients with variations of PAX3 and SOX10. Moreover, we found that patients with variants of SOX10 had a higher risk of suffering from auditory system diseases and nervous system diseases, which were closely associated with the high expression abundance of SOX10 in ear tissues and brain tissues.ConclusionsOur study provides new insights into the potential causative factors of WS and an alternative way to explore clinically undiagnosed cases, which will promote clinical diagnosis and genetic counseling. However, the two potential disease-causing genes (KIT, CHD7) and 32 potential pathogenic variants (PAX3: 20, MITF: 7, SOX10: 5) predicted by multi-data integration in this study are all computational predictions and need to be further verified through experiments in follow-up research.

FKBP10-inhibited β-catenin ubiquitination in the malignant transformation of breast epithelial cells.

e12580 Background: Current model of breast cancer (BCa) progression suggests a linear and three-stage process from normal epithelial tissue to ductal carcinoma in situ (DCIS), and then invasive ductal carcinoma (IDC). Since the detailed mechanisms for BCa oncogenesis remain largely unclear and there is still a lack of root-cause level interventions, exploring the underlying molecular alterations during three-stage process of BCa carcinogenesis is urgently needed. Methods: Three-stage specimens from the very same patients were collected. High-throughput sequencing was applied to obtain the differentially expressed genes among normal, DCIS, and IDC samples. Expression level and prognostic value of the selected gene were evaluated by PCR, western blot, tissue chip, and bioinformatics analysis. MCF10DCIS and MCF10Ca cell lines were respectively used to establish DCIS and IDC tumor models in breast epithelial cell-specific gene knockout mice. Patient-matched DCIS and IDC organoids were also constructed. Signaling pathways associated with malignant transformation of breast epithelial cells were detected by using high-throughput sequencing in mouse models and organoids. Protein binding was evaluated by protein mass spectrometry and co-immunoprecipitation. Results: By using high-throughput sequencing, we found that FKBP10 levels were gradually increased in three-stage specimens from the very same patients and were associated with poor prognosis. By establishing DCIS and IDC models in FKBP10flox/floxCre+/- and FKBP10flox/floxCre-/- control mice, we confirmed that FKBP10 deficiency could suppress BCa formation. Besides, FKBP10 knockdown could reduce oncogenic potentials in patient-matched DCIS and IDC organoids. Wnt/β-catenin signaling pathway was related to malignant transformation of breast epithelial cells, when MCF10DCIS+FKBP10flox/floxCre+/- model, MCF10Ca+FKBP10flox/floxCre+/- model, FKBP10-knockdown DCIS and IDC organoids, and their corresponding controls were comprehensively compared. Moreover, FKBP10 was found to inhibit β-catenin ubiquitination with the help of deubiquitinase Trim13, thus activating Wnt/β-catenin signaling pathway and accelerating malignant transformation of breast epithelial cells. Increased level of β-catenin was able to promote FKBP10 expression in a feedback loop. We also confirmed that exosome-loaded FKBP10-targeting peptide could suppress tumor growth both in vitro and in vivo. Conclusions: FKBP10-inhibited β-catenin ubiquitination was responsible in the malignant transformation from normal tissue to DCIS, and then IDC. Our study would uncover novel regulatory mechanisms of FKBP10 in three-stage process of BCa carcinogenesis and provide a new therapeutic approach for clinical treatment.