Yiqi Kaimi prescription regulates protein phosphorylation to promote intestinal motility in slow transit constipation.

ETHNOPHARMACOLOGICAL RELEVANCE: The clinical efficacy of the Yiqi Kaimi prescription has been confirmed in slow transit constipation. However, the effects and biological mechanism of Yiqi Kaimi prescription are still unclear. AIMS OF THE STUDY: To identify the effects of Yiqi Kaimi prescription on intestinal motility; To reveal the potential key targets and pathways of Yiqi Kaimi prescription for the treatment of slow transit constipation. MATERIALS AND METHODS: The effects of Yiqi Kaimi prescription on slow transit constipation were investigated in a mouse model. The terminal ink propulsion experiment and fecal indocyanine green imaging was used to measure the intestinal transit time. Protein phosphorylation changes in colon tissues treated with Yiqi Kaimi prescription were detected using a Phospho Explorer antibody microarray. Bioinformatic analyses were performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) and the Search Tool for the Retrieval of Interacting Genes (STRING). Western blot analysis and immunohistochemistry confirmed the observed changes in phosphorylation. RESULT: s: Yiqi Kaimi prescription significantly increased the intestinal transit rate (P < 0.05 vs. model) and reduced the time to first discharge of feces containing fecal indocyanine green imaging in mice (P < 0.05 vs. model). The administration of Yiqi Kaimi prescription induced phosphorylation changes in 41 proteins, with 9 upregulated proteins and 32 downregulated proteins. Functional classification of the phosphorylated proteins with DAVID revealed that the critical biological processes included tyrosine protein kinases, positive regulation of calcium-mediated signaling and response to muscle stretch. The phosphorylation of the spleen tyrosine kinase (SYK) at Tyr348 increased 2.19-fold, which was the most significant change. The phosphorylation level of the transcription factor p65 (RELA) at Thr505 was decreased 0.57-fold. SYK was a hub protein in the protein-protein interaction network and SYK and RELA formed the core of the secondary subnetwork. The key protein phosphorylation after treatment with Yiqi Kaimi prescription were verified by Western blot analysis and immunohistochemistry. CONCLUSION: Yiqi Kaimi prescription significantly enhanced intestinal motility. This effect was attributed to alterations in the phosphorylation levels of various target proteins. The observed changes in protein phosphorylation, including SYK and RELA, may serve as crucial factors in the treatment of slow transit constipation.

Gasdermin-E-Dependent Non-Canonical Pyroptosis Promotes Drug-Induced Liver Failure by Promoting CPS1 deISGylation and Degradation.

Drug-induced liver injury (DILI) is a significant global health issue that poses high mortality and morbidity risks. One commonly observed cause of DILI is acetaminophen (APAP) overdose. GSDME is an effector protein that induces non-canonical pyroptosis. In this study, the activation of GSDME, but not GSDMD, in the liver tissue of mice and patients with APAP-DILI is reported. Knockout of GSDME, rather than GSDMD, in mice protected them from APAP-DILI. Mice with hepatocyte-specific rescue of GSDME reproduced APAP-induced liver injury. Furthermore, alterations in the immune cell pools observed in APAP-induced DILI, such as the replacement of TIM4+ resident Kupffer cells (KCs) by monocyte-derived KCs, Ly6C+ monocyte infiltration, MerTk+ macrophages depletion, and neutrophil increase, reappeared in mice with hepatocyte-specific rescue of GSDME. Mechanistically, APAP exposure led to a substantial loss of interferon-stimulated gene 15 (ISG15), resulting in deISGylation of carbamoyl phosphate synthetase-1 (CPS1), promoted its degradation via K48-linked ubiquitination, causing ammonia clearance dysfunction. GSDME deletion prevented these effects. Delayed administration of dimethyl-fumarate inhibited GSDME cleavage and alleviated ammonia accumulation, mitigating liver injury. This findings demonstrated a previously uncharacterized role of GSDME in APAP-DILI by promoting pyroptosis and CPS1 deISGylation, suggesting that inhibiting GSDME can be a promising therapeutic option for APAP-DILI.

Spatially modulated light harvesting with plasmonic crescent metasurface.

Harvesting light by metallic structures with sharp corners, or the so-called photonic singularities, has exhibit their potential in nanophotonics, sensing, and bio-medical applications. The high-quality light confinement of the light energy mainly relies on the precise preparation of nanoscale photonic singularities. However, the realization of massive photonic singularities still meets the challenges on integration and low-cost mask multiplexing. Here, we show an angle-dependent elevated nanosphere lithography to achieve massive photonic singularities for spatially modulated light harvesting at the near-infrared regime. The photonic geometrical singularity is constructed by the gold crescent array of plasmonic materials. The numerical simulation shows that the light can be localized at the spatially distributed singularities. This phenomenon is verified experimentally through the infrared spectral measurement. Our work provides the possibility to produce integrated light-harvesting devices for numerous optical applications in illumination, display, and enhanced nonlinear excitation.

Germline FOXJ2 overexpression causes male infertility via aberrant autophagy activation by LAMP2A upregulation.

Spermatogenesis is a complex biological process that produces haploid spermatozoa and requires precise regulation by many tissue-specific factors. In this study, we explored the role and mechanism of Fork head box J2 (FOXJ2, which is highly expressed in spermatocytes) in the regulation of spermatogenesis using a germline-specific conditional Foxj2 knock-in mouse model (Stra8-Cre; Foxj2 tg/tg mouse). Foxj2 overexpression in mouse testes led to spermatogenesis failure, which started at the initiation of meiosis, and resulted in male infertility. Lysosomes and autophagy-related genes were upregulated in Stra8-cre; Foxj2 tg/tg mouse testes and the number of autolysosomes in the spermatocytes in Stra8-cre; Foxj2 tg/tg mice was increased. Chromatin immunoprecipitation-PCR and Dual-luciferase reporter assays showed that Lamp2 (encoding lysosome-associated membrane protein-2) was a target of FOXJ2. Foxj2 overexpression increased the expression levels of Lamp2a and Hsc70 (70-kDa cytoplasmic heat shock protein) in the Stra8-cre; Foxj2 tg/tg mouse testes. Our results suggested that Foxj2 overexpression in the germ cells of mouse testes affects chaperone-mediated autophagy by upregulating LAMP2A, leading to spermatogenesis failure at the initiation of meiosis, thus resulting in male infertility. Our findings provide a new insight into the function of FOXJ2 in spermatogenesis and the significance of autophagy regulation in spermatogenesis.

[Impact of overexpressed FOXJ2 on mouse spermatogenesis and its action mechanism].

OBJECTIVE: To analyze the phenotype of the male reproductive system in the germline-specific conditional Foxj2 knock-in mouse model (Stra8-cre; Foxj2tg/+), identify a target gene of the transcription factor FOXJ2, and investigate the effect of the overexpression of Foxj2 on mouse spermatogenesis and its action mechanism. METHODS: Based on the Cre-loxP recombination system, we generated a germline-specific conditional Foxj2 knock-in mouse model (Stra8-cre; Foxj2tg/+). We determined male fertility by counting the number of pups per litter and the fertilization rate after intracytoplasmic sperm injection (ICSI), observed the morphology of the testes and epididymides by HE staining, examined the sperm quality by computer assisted sperm analysis (CASA), detected the expression and localization of Cx43 in the testis by RT-qPCR, Western blot and immunohistochemistry, and verified the binding site of FOXJ2 to the Cx43 promoter using ChIP-PCR and dual luciferase reporter assay. RESULTS: The number of pups per litter and fertilization rate after ICSI were lower in the Stra8-cre; Foxj2tg/+ male mice than in the controls, and so were the size and weight of the testis. HE staining exhibited obvious exfoliation of germ cells and dramatically decreased spermatocytes and spermatids in the seminiferous tubules of the Stra8-cre; Foxj2tg/+ mice. Moreover, sperm concentration in the cauda epididymides was reduced, and the transcription and expression levels of Cx43 in the testis were increased. ChIP-PCR and dual luciferase reporter assay showed direct binding of FOXJ2 to the Cx43 promoter in the testis. CONCLUSIONS: Overexpressed FOXJ2 may lead to spermatogenic failure and subfertility in Stra8-cre; Foxj2tg/+ male mice by upregulating the expression of Cx43.

Control of multiciliogenesis by miR-34/449 in the male reproductive tract through enforcing cell cycle exit.

Multiciliated cells (MCCs) are terminally differentiated postmitotic cells that possess hundreds of motile cilia on their apical surface. Defects in cilia formation are associated with ciliopathies that affect many organs. In this study, we tested the role and mechanism of the miR-34/449 family in the regulation of multiciliogenesis in EDs using an miR-34b/c-/-; miR-449-/- double knockout (dKO) mouse model. MiR-34b/c and miR-449 depletion led to a reduced number of MCCs and abnormal cilia structure in the EDs starting from postnatal day (P)14. However, abnormal MCC differentiation in the dKO EDs could be observed as early as P7. RNA-seq analyses revealed that the aberrant development of MCCs in the EDs of dKO mice was associated with the upregulation of genes involved in cell cycle control. Using a cyclin-dependent kinase inhibitor to force cell cycle exit promoted MCC differentiation, and partially rescued the defective multiciliogenesis in the EDs of dKO mice. Taken together, our results suggest that miR-34b/c and miR-449 play an essential role in multiciliogenesis in EDs by regulating cell cycle exit.

Intranasal Transplantation of Human Neural Stem Cells Ameliorates Alzheimer's Disease-Like Pathology in a Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory impairments, which has no effective therapy. Stem cell transplantation shows great potential in the therapy of various disease. However, the application of stem cell therapy in neurological disorders, especially the ones with a long-term disease course such as AD, is limited by the delivery approach due to the presence of the brain blood barrier. So far, the most commonly used delivery approach in the therapy of neurological disorders with stem cells in preclinical and clinical studies are intracranial injection and intrathecal injection, both of which are invasive. In the present study, we use repetitive intranasal delivery of human neural stem cells (hNSCs) to the brains of APP/PS1 transgenic mice to investigate the effect of hNSCs on the pathology of AD. The results indicate that the intranasally transplanted hNSCs survive and exhibit extensive migration and higher neuronal differentiation, with a relatively limited glial differentiation. A proportion of intranasally transplanted hNSCs differentiate to cholinergic neurons, which rescue cholinergic dysfunction in APP/PS1 mice. In addition, intranasal transplantation of hNSCs attenuates ß-amyloid accumulation by upregulating the expression of ß-amyloid degrading enzymes, insulin-degrading enzymes, and neprilysin. Moreover, intranasal transplantation of hNSCs ameliorates other AD-like pathology including neuroinflammation, cholinergic dysfunction, and pericytic and synaptic loss, while enhancing adult hippocampal neurogenesis, eventually rescuing the cognitive deficits of APP/PS1 transgenic mice. Thus, our findings highlight that intranasal transplantation of hNSCs benefits cognition through multiple mechanisms, and exhibit the great potential of intranasal administration of stem cells as a non-invasive therapeutic strategy for AD.

[Effects of miR-34b/c and miR-449 on the morphology and function of efferent ductules in mice: A transcriptomics-based analysis].

OBJECTIVE: To investigate the role of miR-34b/c and miR-449 in maintaining the normal structure and function of efferent ductules and explore the molecular mechanism of infertility in miR-34b/c－/－ and miR-449－/－ dKO mice. METHODS: We observed the morphology of mouse efferent ductules by HE staining and analyzed the gene expressions in the efferent ductules of the wild-type and miR-34b/c－/－ and miR-449－/－ dKO mice by RNA sequencing. Then we screened the possible target genes of these two miRNA clusters and analyzed them along with the differentially expressed genes, followed by verification of the sequencing results by qRT-PCR. RESULTS: Compared with the wild-type, the dKO mice showed morphologically abnormal efferent ductules and significantly decreased expressions of the genes involved in the formation of cilia and related to the transportation of water, ion and protein in the efferent ductules. CONCLUSIONS: The deletion of miR-34b/c and miR-449 led to morphological abnormality of efferent ductules and dysfunction of aberrant cilia motility and reabsorption in the efferent ductules of dKO mice, resulting in infertility.

Mutations in the PKM2 exon-10 region are associated with reduced allostery and increased nuclear translocation.

PKM2 is a key metabolic enzyme central to glucose metabolism and energy expenditure. Multiple stimuli regulate PKM2's activity through allosteric modulation and post-translational modifications. Furthermore, PKM2 can partner with KDM8, an oncogenic demethylase and enter the nucleus to serve as a HIF1α co-activator. Yet, the mechanistic basis of the exon-10 region in allosteric regulation and nuclear translocation remains unclear. Here, we determined the crystal structures and kinetic coupling constants of exon-10 tumor-related mutants (H391Y and R399E), showing altered structural plasticity and reduced allostery. Immunoprecipitation analysis revealed increased interaction with KDM8 for H391Y, R399E, and G415R. We also found a higher degree of HIF1α-mediated transactivation activity, particularly in the presence of KDM8. Furthermore, overexpression of PKM2 mutants significantly elevated cell growth and migration. Together, PKM2 exon-10 mutations lead to structure-allostery alterations and increased nuclear functions mediated by KDM8 in breast cancer cells. Targeting the PKM2-KDM8 complex may provide a potential therapeutic intervention.

A promising non-invasive index for predicting liver inflammation in chronic hepatitis B patients with alanine aminotransferase ≤2 upper limit of normal.

Inexpensive and simple non-invasive indexes for predicting liver inflammation are urgently required, but have been poorly studied in chronic hepatitis B (CHB) patients with alanine transaminase (ALT) ≤2 times the upper limit of normal (ULN). A total of 356 CHB patients with ALT ≤2 ULN who presented at Huashan Hospital (n=181) and the First Hospital of Quanzhou (n=175) were enrolled and randomly divided into an experimental assessment cohort (n=238) and validation cohort (n=118) at a ratio of 2:1. Histological analysis of liver tissue was performed to determine the pathological stage according to the Scheuer scoring system. For the experimental assessment cohort, univariate and multivariate analysis identified aspartate aminotransferase (AST) and albumin (ALB) as independent predictors of liver necroinflammation [liver necroinflammation grade (G)≥2] in patients with ALT ≤2 ULN. Therefore, a novel index, the AST-to-ALB ratio (ATAR), was proposed, which had a better diagnostic performance [area under receiver operating characteristic curve (AUC)=0.721] than that of ALB (AUC=0.632; P=0.039 vs. ATAR) and AST (AUC=0.682; P=0.082 vs. ATAR). In the validation cohort, the AUC of ATAR (0.728) to identify patients with a G≥2 was slightly greater than that of AST (0.660; P=0.149 vs. ATAR) and ALB (0.672; P=0.282 vs. ATAR). Furthermore, a similar diagnostic superiority was also demonstrated in patients with ALT ≤1 ULN. Thus, ATAR may be a promising non-invasive surrogate marker for liver necroinflammation CHB patients with ALT ≤2 ULN and thereby determine whether anti-viral treatment should be initiated.

[Metabolic syndrome and chronic kidney disease in a rural adult population of Hunan province, China].

OBJECTIVE: To explore the relationship between metabolic syndrome and chronic kidney disease (CKD) in a rural adult population of Hunan province. METHODS: 1953 residents (older than 18 years) from the same village were randomly selected, using a stratified, multistage sampling method. All residents were interviewed and tested for albuminuria with morning spot urine albumin to creatinine ratio (abnormal: >/= 30 mg/g), reduced renal function with estimated glomerular filtration rate by modified MDRD equation [abnormal: < 60 ml/min (1.73 m(2))]. The associations of kidney damage indicators with demographic characteristics (age, gender, smoking status), indicators on health (diabetes, hypertension) and metabolic syndrome traits were examined. RESULTS: Eligible data of 1709 subjects were enrolled in the study. After the adjustment of age, gender and other metabolic syndrome traits, participants with metabolic syndrome had a higher prevalence of CKD (19.3% vs. 13.2%, P < 0.001) than those without the syndrome. As the number of metabolic syndrome traits increased, so did the prevalence of CKD. There seemed to be a strong and independent association between metabolic syndrome and chronic kidney disease. For participants without hypertension and diabetes, metabolic syndrome was also associated with CKD (OR value 1.733, 95%CI: 1.20 - 2.41, P = 0.004). CONCLUSION: In these 1709 adults under this study from a village of southern China, metabolic syndrome seemed to be associated with CKD.

Expression of the retinoic acid-metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development.

AIM: To study the expression pattern of the retinoic acid metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development at both mRNA and protein levels. METHODS: Real-time polymerase chain reaction and Western blot analysis were performed to determine the relative quantity of RALDH2 and CYP26b1 at both mRNA and protein levels at postnatal day 1, 5, 10, 20, and in adult mice (70 days testes). Testicular localization of RALDH2 and CYP26b1 during mouse postnatal development was examined using immunohistochemistry assay. RESULTS: Aldh1a2 transcripts and its protein RALDH2 began to increase at postnatal day 10, and remained at a high level through postnatal day 20 to adulthood. Cyp26b1 transcripts and CYP26b1 protein did not change significantly during mouse postnatal testis development. RALDH2 was undetectable in the postnatal 1, 5 and 10 day testes using immunohistochemistry assay. At postnatal day 20 it was detected in pachytene spermatocytes. Robust expression of RALDH2 was restricted in round spermatids in the adult mouse testis. In the developing and adult testis, CYP26b1 protein was confined to the peritubular myoepithelial cells. CONCLUSION: Our results indicate that following birth, the level of retinoic acid in the seminiferous tubules might begin to increase at postnatal day 10, and maintain a high level through postnatal day 20 to adulthood.

[Exogenous nitric oxide inhibits the proliferation of mouse neural progenitor cells].

OBJECTIVE: To explore the inhibitive effect of exogenous nitric oxide (NO) on the proliferation of mouse neural progenitor cells (NPCs). METHODS: NPCs were cultured in medium containing EGF, and SNP was added as a donor of NO in vitro. The concentration of NO was measured by the Griess assay; The amount of living cells was measured by MTT assay. The NPCs were induced to differentiate in medium supplemented with serum. RESULT: The NO(-)2 concentrations in the supernatants of the experimental groups 1, 2, and 3 were 64.701 +/- 0.606, 82.659 +/- 0.394 and 93.648 +/- 0.394 micro mol/L respectively, 3.099 +/- 0.420 micro mol/L in th control group. After culture with SNP for 24 hours, MTT assay showed the A values of 0.546 +/- 0.016, 0.484 +/- 0.007, and 0.357 +/- 0.007 in the experimental groups 1, 2, and 3 respectively, 0.642 +/- 0.021 in th control group (all P < 0.01). After further culture in the medium without SNP for 24 hours, MTT assay showed the A values of 1.243 +/- 0.036, 1.064 +/- 0.097, and 0.834 +/- 0.031 in the experimental groups 1, 2, and 3 respectively, 1.581 +/- 0.072 in the control group (all P < 0.01). The differences between the A values of the supernatants in the same wells filled with the medium with and without SNP were all significant (P < 0.01). In the medium with serum the NPCs still proliferated actively and differentiated into neuron and glial cell after SNP was removed. CONCLUSION: Exogenous nitric oxide inhibits the proliferation of mice NPC in vitro.

[Endovascular treatment of intracranial aneurysms with mechanical detachable spiral-neurological].

OBJECTIVE: To evaluate the efficacy of endovascular treatment of intracranial aneurysms with mechanical detachable spiral-neurological (MDS-N). METHODS: Thirty-six patients with intracranial aneurysms were treated with MDS-N by Seldinger's approaches through femoral artery puncture. A catheter was sent into the aneurysm with proper-size MDS-N system and a spiral was pushed into the aneurysmal lumen. RESULTS: The aneurysms were completely occluded in 32 cases while partial occlusion was obtained in 4 patients. The parent arteries remained unobstructed. Follow-up of 3 - 6 months showed that all patients recovered well. No death or permanent serious complication occurred. CONCLUSION: Simple, safe, and low-cost, treatment of intracranial aneurysm with MDS-N is effective and suitable for use in larger hospital.

Experiment and observation on invasion of brain glioma in vivo.

Research on invasion and metastasis of glioma in vivo was performed by implanting C6 glioma cells transfected with enhanced green fluorescent protein (EGFP) gene into the brain of SD rats. Firstly, C6 glioma cells were transfected with a plasmid vector (pEGFP-N3) containing the EGFP gene. Stable EGFP-expressing clones were isolated and examination for these cells by flow cytometry and electron microscope was done. Secondly, EGFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections were examined using fluorescence microscopy after adjacent sections were examined by immunohistochemistry or routine hematoxylin and eosin staining for the visualization and detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of EGFP gene in vivo. The results showed that EGFP-transfected C6 glioma cells maintained stable high-level EGFP expression in the central nervous system during their growth in vivo. EGFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the visualization of distant micrometastasis and invasion on the single-cell level. Small locally invasive foci, including those immediately adjacent to the leading invasive edge of the tumor, were virtually undetectable by routine hematoxylin and eosin staining and immunohistochemistry. These results suggested that EGFP-transfected C6 cells can be visualized by fluorescence microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion and metastasis of brain glioma in vivo.