To investigate the effects of bonito oligopeptide SEP-3 on the repair of liver damage and regulation of liver biorhythm in sleep-deprived mice (SDM), C57BL/6 male mice were subjected to sleep deprivation by modified multi-platform water environment method, and were given different doses of bonito oligopeptide SEP-3 in groups. To determine the liver organ index, liver tissue-related apoptotic protein levels, Wnt/β-Catenin pathway-related protein expression levels, serum alanine transaminase (ALT), glutamicum transaminase (AST), glucocorticoid (GC), and adrenocorticotropin (ACTH) content in each group of mice, four time points were selected to examine the mRNA expression levels of circadian clock-related genes in mouse liver tissue. The results showed that low, medium, and high doses of SEP-3 significantly increased SDM, ALT, and AST (p < 0.05), and medium and high doses of SEP-3 significantly reduced SDM liver index and GC and ACTH. As SEP-3 increased the apoptotic protein and Wnt/β-Catenin pathway, mRNA expression gradually tended to normal (p < 0.05). This suggests that sleep deprivation can cause excessive oxidative stress in mice, which can lead to liver damage. Additionally, oligopeptide SEP-3 achieves the repair of liver damage by inhibiting SDM hepatocyte apoptosis, activating liver Wnt/β-Catenin pathway, and promoting hepatocyte proliferation and migration, and suggests that oligopeptide SEP-3 is closely related to repair of liver damage by regulating the biological rhythm of SDM disorder.

Erythropoietin-producing human hepatocellular receptor (EPH) B6 (EPHB6) is a member of the receptor tyrosine kinase family. We previously demonstrated that EPHB6 knockout reduces catecholamine secretion in male but not female mice, and castration reverses this phenotype. We showed here that male EPHB6 knockout adrenal gland chromaffin cells presented reduced acetylcholine-triggered Ca2+ influx. Such reduction depended on the non-genomic effect of testosterone. Increased large conductance calcium-activated potassium channel current densities were recorded in adrenal gland chromaffin cells from male EPHB6 knockout mice but not from castrated knockout or female knockout mice. Blocking of the large conductance calcium-activated potassium channel in adrenal gland chromaffin cells from male knockout mice corrected their reduced Ca2+ influx. We conclude that the absence of EPHB6 and the presence of testosterone would lead to augmented large conductance calcium-activated potassium channel currents, which limit voltage-gated calcium channel opening in adrenal gland chromaffin cells. Consequently, acetylcholine-triggered Ca2+ influx is reduced, leading to lower catecholamine release in adrenal gland chromaffin cells from male knockout mice. This explains the reduced resting-state blood catecholamine levels, and hence the blood pressure, in male but not female EPHB6 knock mice. These findings have certain clinical implications.

Overview: The treatment of chronic renal failure (CRF) with traditional Chinese medicine has attracted much attention, but its mechanism is not clear. Network pharmacology is an effective strategy for exploring the interaction mechanisms between Chinese herbs and diseases, however, it still needs to be validated in cell and/or animal experiments due to its virtual screening characteristics. Herein, the anti-CRF mechanism of the Fushengong decoction (FSGD) was investigated using a dual-dimension network pharmacological strategy combined with in vivo experiment. Methods: The traditional Chinese medicine systems pharmacology (TCMSP) database (https://tcmspw.com) and UHPLC-MS/MS technology were used to identify the effective compounds of FSGD in theory and practice, such as quercetin, formononetin, and pachymic acid. The putative targets of FSGD and CRF were obtained from the Swisstarget prediction platform and the Genecards database, respectively. The common target pathways between FSGD and CRF were got from the dual-dimension network pharmacology analysis, which integrated the cross-common targets from the TCMSP components-Swisstarget-Genecards-Venn platform analysis in theory, and the UHPLC-MS/MS identified effective ingredients-Swisstarget screening, such as TNF and PI3K/AKT. Furthermore, system molecular determinations were used to prove the dual-dimension network pharmacology study through CRF rat models, which were constructed using adenine and treated with FSGD for 4 weeks. Results: A total of 121 and 9 effective compounds were obtained from the TCMSP database and UHPLC-MS/MS, respectively. After dual-dimension network pharmacology analysis, the possible mechanism of PTEN/PI3K/AKT/NF-κB pathway was found for FSGD in CRF. In vivo experiments indicated that FSGD can play a role in protecting renal function and reducing fibrosis by regulating the PTEN/PI3K/AKT/NF-κB pathway. These findings provide a reference for FSGD in CRF. Conclusion: Based on the theoretical and practical dual-dimension network pharmacology analysis for FSGD in CRF, the possible molecular mechanism of PTEN/PI3K/AKT/NF-κB was successfully predicted, and these results were verified by in vivo experiments. In this study, the dual-dimension network pharmacology was used to interpret the key signal pathway for FSGD in CRF, which also proved to be a smart strategy for the study of effective substances and pharmacology in FSGD.

Ephb6 gene knockout causes hypertension in castrated mice. EPHB6 controls catecholamine secretion by adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent way. Nicotinic acetylcholine receptor (nAChR) is a ligand-gated Ca2+/Na+ channel, and its opening is the first signaling event leading to catecholamine secretion by AGCCs. There is a possibility that nAChR might be involved in EPHB6 signaling, and thus sequence variants of its subunit genes are associated with hypertension risks. CHRNA3 is the major subunit of nAChR used in human and mouse AGCCs. We conducted a human genetic study to assess the association of CHRNA3 variants with hypertension risks in hypogonadic males. The study cohort included 1,500 hypogonadic Chinese males with (750 patients) or without (750 patients) hypertension. The result revealed that SNV rs3743076 in the fourth intron of CHRNA3 was significantly associated with hypertension risks in the hypogonadic males. We further showed that EPHB6 physically interacted with CHRNA3 in AGCCs, providing a molecular basis for nAChR being in the EPHB6 signaling pathway.

TGFβ-induced (TGFBI/βig-H3) is a protein inducible by TGFβ1 and secreted by many types of cells. It binds to collagen, forms part of the extracellular matrix (ECM), and interacts with integrins on cell surfaces. In this study, we investigated the role of TGFBI in tumorigenesis and the underlying mechanisms.

Next generation sequencing and bio-informatic analyses were conducted to investigate the mechanism of reactivation of p53 and induction of tumor cell apoptosis (RITA)-enhancing X-ray susceptibility in FaDu cells.

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP(+/-) mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP(+/-) mice were in the normal range. The functions of MLF1-IP(+/-) T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation.

ARMC5 is implicated in several pathological conditions, but its function remains unknown. We have previously identified CUL3 and RPB1 (the largest subunit of RNA polymerase II (Pol II) as potential ARMC5-interacting proteins. Here, we show that ARMC5, CUL3 and RBX1 form an active E3 ligase complex specific for RPB1. ARMC5, CUL3, and RBX1 formed an active E3 specific for RPB1. Armc5 deletion caused a significant reduction in RPB1 ubiquitination and an increase in an accumulation of RPB1, and hence an enlarged Pol II pool in normal tissues and organs. The compromised RPB1 degradation did not cause generalized Pol II stalling nor depressed transcription in the adrenal glands but did result in dysregulation of a subset of genes, with most upregulated. We found RPB1 to be highly expressed in the adrenal nodules from patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) harboring germline ARMC5 mutations. Mutant ARMC5 had altered binding with RPB1. In summary, we discovered that wildtype ARMC5 was part of a novel RPB1-specific E3. ARMC5 mutations resulted in an enlarged Pol II pool, which dysregulated a subset of effector genes. Such an enlarged Pol II pool and gene dysregulation was correlated to adrenal hyperplasia in humans and KO mice.

Several erythropoietin-producing hepatocellular receptor B family (EPHB) and their ligands, ephrinBs (EFNBs), are involved in blood pressure regulation in animal models. We selected 528 single nucleotide polymorphisms (SNPs) within the genes of EPHB6, EFNB2, EFNB3 and GRIP1 in the EPH/EFN signalling system to query the International Blood Pressure Consortium dataset. A SNP within the glutamate receptor interacting protein 1 (GRIP1) gene presented a p-value of 0.000389, approaching the critical p-value of 0.000302, for association with diastolic blood pressure of 60,396 individuals. According to echocardiography, we found that Efnb3 gene knockout mice showed enhanced constriction in the carotid arteries. In vitro studies revealed that in mouse vascular smooth muscle cells, siRNA knockdown of GRIP1, which is in the EFNB3 reverse signalling pathway, resulted in increased contractility of these cells. These data suggest that molecules in the EPHB/EFNB signalling pathways, specifically EFNB3 and GRIP1, are involved blood pressure regulation.

Several members of the EPH kinase family and their ligands are involved in blood pressure regulation, and such regulation is often sex- or sex hormone-dependent, based on animal and human genetic studies. EPHB6 gene knockout (KO) in mice leads to hypertension in castrated males but not in un-manipulated KO males or females. To assess whether this finding in mice is relevant to human hypertension, we conducted a human genetic study for the association of EPHB6 and its two ligands, EFNB1 and EFNB3, with hypertension in hypogonadic patients. Seven hundred and fifty hypertensive and 750 normotensive Han Chinese patients, all of whom were hypogonadic, were genotyped for single nucleotide polymorphisms (SNPs) within the regions of the genes, plus an additional 50 kb 5' of the genes for EPHB6, EFNB1 and EFNB3. An imputed insertion/deletion polymorphism, rs35530071, was found to be associated with hypertension at p-values below the Bonferroni-corrected significance level of 0.0024. This marker is located 5' upstream of the EFNB3 gene start site. Previous animal studies showed that while male EFNB3 gene knockout mice were normotensive, castration of these mice resulted in hypertension, corroborating the results of the human genetic study. Considering the significant associations of EFNB3 SNPs with hypertension in hypogonadic males and supporting evidence from castrated EFNB3 KO mice, we conclude that loss-of-function variants of molecules in the EPHB6 signaling pathway in the presence of testosterone are protective against hypertension in humans.

Pno1 is a protein that plays a role in proteasome and ribosome neogenesis in yeast. So far, its functions in mammalian cells have not been investigated. To understand its function in mammals, we performed in situ hybridization analysis of Pno1 expression in different development stages and generated Pno1 gene knockout (KO) and transgenic (Tg) mice lineages. The results showed early lethality of homozygous Pno1 KO lineage caused, as demonstrated in parallel by ex vivo experiments, by arrest of embryo development before compaction stage. Though, heterozygous (HET) mice with 50% of normal Pno1 mRNA concentration were fertile and showed no obvious anomalies. The lymphoid organs of HET mice were normal in size, weight and cellularity, with normal T and B cell subpopulations. TCR-triggered activation and proliferation of HET T cells were normal. Proteasome activities in HET organs were uncompromised. Tg mice with actin promoter-driven Pno1 expression were also fertile, with no apparent anomalies, although they expressed 2-5-fold higher Pno1 mRNA levels. The lymphoid organs of Tg mice were of normal size, weight and cellularity with normal T and B cell sub-populations. TCR-triggered activation and proliferation of Tg T cells were normal. Tg organs and tissues presented normal proteasome activity as did their wild type counterparts. Tagged Pno1 over-expression in L cells and density gradient fractionation established that Pno1 existed in large complexes with sedimentation rates between 20S and 26S, bigger than mature 26S proteasomes. Pno1 in fractions did not coincide with 40S or 60S ribosome subunits. Our study indicates that Pno1 is essential for cellular functions, but only a small percentage of its normal level is sufficient, and excessive amounts are neither harmful nor useful. The nature of the large complexes it associates with remains to be identified, but it is certain that they are not mature proteasomes or ribosomes.

Our earlier study revealed that STRA6 (stimulated by retinoic acid gene 6) was up-regulated within 3 h of TCR stimulation. STRA6 is the high-affinity receptor for plasma retinol-binding protein (RBP) and mediates cellular vitamin A uptake. We generated STRA6 knockout (KO) mice to assess whether such up-regulation was critical for T-cell activation, differentiation and function. STRA6 KO mice under vitamin A sufficient conditions were fertile without apparent anomalies upon visual inspection. The size, cellularity and lymphocyte subpopulations of STRA6 KO thymus and spleen were comparable to those of their wild type (WT) controls. KO and WT T cells were similar in terms of TCR-stimulated proliferation in vitro and homeostatic expansion in vivo. Naive KO CD4 cells differentiated in vitro into Th1, Th2, Th17 as well as regulatory T cells in an analogous manner as their WT counterparts. In vivo experiments revealed that anti-viral immune responses to lymphocytic choriomeningitis virus in KO mice were comparable to those of WT controls. We also demonstrated that STRA6 KO and WT mice had similar glucose tolerance. Total vitamin A levels are dramatically lower in the eyes of KO mice as compared to those of WT mice, but the levels in other organs were not significantly affected after STRA6 deletion under vitamin A sufficient conditions, indicating that the eye is the mouse organ most sensitive to the loss of STRA6. Our results demonstrate that 1) in vitamin A sufficiency, the deletion of STRA6 in T cells does no affect the T-cell immune responses so-far tested, including those depend on STAT5 signaling; 2) STRA6-independent vitamin A uptake compensated the lack of STRA6 in lymphoid organs under vitamin A sufficient conditions in mice; 3) STRA6 is critical for vitamin A uptake in the eyes even in vitamin A sufficiency.

We investigated whether renal hedgehog interacting protein (Hhip) expression contributes to the progression of diabetic nephropathy (DN) and studied its related mechanism(s) in vivo and in vitro. Here, we show that Hhip expression is highly elevated in glomerular endothelial cells of adult type 1 diabetic (T1D) Akita and T2D db/db mouse kidneys as compared to non-diabetic control littermates. Hyperglycemia enhances reactive oxygen species (ROS) generation via NADPH oxidase 4 (Nox4) activation and stimulates renal Hhip gene expression, and that elevated renal Hhip gene expression subsequently activates the TGFβ1- Smad2/3 cascade and promotes endothelial to mesenchymal transition associated with endothelial cell fibrosis/apoptosis in vivo and in vitro. Furthermore, kidneys of low-dose streptozotocin-induced diabetic heterozygous Hhip deficient (Hhip+/-) mice displayed a normal albumin/creatinine ratio with fewer features of DN (glomerulosclerosis/fibrosis and podocyte apoptosis/loss) and less evidence of renal compensation (glomerular hypertrophy and hyperfiltration) as compared to diabetic wild type controls (Hhip+/+). Thus, our studies demonstrated that renal Hhip expression is associated with nephropathy development in diabetes and that hyperglycemia-induced renal Hhip expression may mediate glomerular endothelial fibrosis and apoptosis in diabetes, a novel finding.

Eph kinases are the largest family of cell surface receptor tyrosine kinases. The ligands of Ephs, ephrins (EFNs), are also cell surface molecules. Ephs interact with EFNs transmitting signals in both directions, i.e., from Ephs to EFNs and from EFNs to Ephs. EFNB1 is known to be able to co-stimulate T cells in vitro and to modulate thymocyte development in a model of foetal thymus organ culture. To further understand the role of EFNB1 in T cell immunity, we generated T-cell-specific EFNB1 gene knockout mice to assess T cell development and function in these mice.

Hearing loss is one of the most common birth disorders in humans, with an estimated prevalence of 1-3 in every 1000 newborns. This study investigates the molecular etiology of a hearing loss cohort using a stepwise strategy to effectively diagnose patients and address the challenges posed by the genetic heterogeneity and variable mutation spectrum of hearing loss. In order to target known pathogenic variants, multiplex PCR plus next-generation sequencing was applied in the first step; patients which did not receive a diagnosis from this were further referred for exome sequencing. A total of 92 unrelated patients with nonsyndromic hearing loss were enrolled in the study. In total, 64% (59/92) of the patients were molecularly diagnosed, 44 of them in the first step by multiplex PCR plus sequencing. Exome sequencing resulted in eleven diagnoses (23%, 11/48) and four probable diagnoses (8%, 4/48) among the 48 patients who were not diagnosed in the first step. The rate of secondary findings from exome sequencing in our cohort was 3% (2/58). This research presents a molecular diagnosis spectrum of 92 non-syndromic hearing loss patients and demonstrates the benefits of using a stepwise diagnostic approach in the genetic testing of nonsyndromic hearing loss.

Congenital hearing loss is one of the most common birth defects. Early identification and management play a crucial role in improving patients' communication and language acquisition. Previous studies demonstrated that genetic screening complements newborn hearing screening in clinical settings.

Sublingual immunotherapy (SLIT) has been shown to be efficacious in patients with airway allergic diseases. However, less data have been demonstrated to show the efficacy of SLIT in patients with atopic dermatitis (AD).

Armadillo repeat containing 5 (ARMC5) is a cytosolic protein with no enzymatic activities. Little is known about its function and mechanisms of action, except that gene mutations are associated with risks of primary macronodular adrenal gland hyperplasia. Here we map Armc5 expression by in situ hybridization, and generate Armc5 knockout mice, which are small in body size. Armc5 knockout mice have compromised T-cell proliferation and differentiation into Th1 and Th17 cells, increased T-cell apoptosis, reduced severity of experimental autoimmune encephalitis, and defective immune responses to lymphocytic choriomeningitis virus infection. These mice also develop adrenal gland hyperplasia in old age. Yeast 2-hybrid assays identify 16 ARMC5-binding partners. Together these data indicate that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signalling pathways.