Two C-terminal isoforms of Aplysia tachykinin-related peptide receptors exhibit phosphorylation-dependent and phosphorylation-independent desensitization mechanisms.

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.

Increased expression of Myosin X contributes to the metastasis in patients with laryngeal squamous cell carcinoma.

Laryngeal Squamous Cell Carcinoma (LSCC) is one of the most common malignancy in Head and neck cancer for which the mechanism underlying its metastasis is poorly understood. Myosin X, a molecular motor in cells has been demonstrated to play an important role in cell migration. However, whether Myosin X is involved in the metastasis of LSCC remains unclear. To investigate the expression of Myosin X and its implication in the metastasis of LSCC, we recruited 30 patients with LSCC and 6 patients with vocal cord polyp range from October 2016 to October 2018. Tissue samples were obtained during surgery and the expression of Myosin X, Cortactin, MMP2, MMP9, E-cadherin, and ß-catenin in tissue samples were evaluated by RT-PCR, Western blot, immunohistochemistry or ELISA. Patients with LSCC were further followed-up 2 year after surgery for metastasis analysis. We found that the level of Myosin X, Cortactin, MMP2, and MMP9 was much higher in poorly differentiated LSCC compared to that in moderately and highly LSCC, as well as the control tissues. In contrast, the expression of epithelial-mesenchymal transition related marker, E-cadherin, and ß-catenin, were much lower in poorly differentiated LSCC tissues compared to that in moderately and highly differentiated LSCC tissues, as well as the control tissues. Moreover, the expression of Myosin X was positively correlated with Cortactin, MMP2, and MMP9 levels. Increased expression of Myosin X in LSCC tissues was related to higher risk of metastasis. In conclusion, our findings showed that. Myosin X augments the expression of Cortactin, MMP2 and MMP9, which could upregulate the cell migration and the matrix degradation, and consequently reduce the expression of E-cadherin and ß-catenin, thereby activating epithelial-mesenchymal transformation and promoting the metastasis of LSCC. Targeting Myosin X may have potential therapeutic effect in the metastasis of LSCC.

Dysoxylactam A: A Macrocyclolipopeptide Reverses P-Glycoprotein-Mediated Multidrug Resistance in Cancer Cells.

A 17-membered macrocyclolipopeptide, named dysoxylactam A (1) comprising an unprecedented branched C19 fatty acid and an l-valine, was isolated from the plants of Dysoxylum hongkongense. The challenging relative configuration of 1 was established by means of residual dipolar coupling-based NMR analysis. The absolute configuration of 1 was determined by single-crystal X-ray diffraction on its p-bromobenzoate derivative (2). Compound 1 dramatically reversed multidrug resistance in cancer cells with the fold-reversals ranging from 28.4 to 1039.7 at the noncytotoxic concentration of 10 µM. The mode-of-action study of 1 revealed that it inhibited the function of P-glycoprotein (P-gp), a key mediator in multidrug resistance.

The truncated mutant HBsAg expression increases the tumorigenesis of hepatitis B virus by regulating TGF-ß/Smad signaling pathway.

BACKGROUND: It has been reported that the emergence of HBV rtA181T/sW172* mutant could result in a dominant secretion defect of HBsAg and increase the risk of HCC development. This study was designed to reveal the role and possible pathogenic mechanism of truncated mutant HBsAg in tumorigenesis of HBV rtA181T/sW172* mutant. RESULTS: As compared to wide type or substituted mutant HBsAg, the ratio of cell clones was significant higher in L02 cells stable expressing truncated mutant HBsAg. Injection of L02 cells stable expressing truncated mutant HBsAg into the dorsal skin fold of nude mice resulted in increased primary tumor growth compared to L02 cells stable expressing wide-type and substituted mutant HBsAg. In HBV replication L02 cell lines, the key molecular involved in TGF-ß/Smad pathway was also investigated. We found that the mRNA and protein levels of Smad3/2, CREB and CyclinD1 were significantly higher and TGFBI level was significantly lower in cells stably expressing truncated mutant HBsAg as compared to cells stably expressing wide-type and substituted mutant HBsAg. Additionally, after administration of TGF-ß1 (increasing TGFBI level), the volume of tumor is obviously reduced in nude mice with injection of L02 cells stable expressing truncated HBsAg. CONCLUSIONS: The emergence of sW172* mutant may increase the tumorigenesis of HBV, and its mechanism may be associated with down-regulated expression of TGFBI in TGF-ß/Smad signaling pathway.

Diterpenoids from Croton laui and their cytotoxic and antimicrobial activities.

Fourteen new diterpenoids including clerodane (1-12), labdane (13), and norlabdane (14) types, as well as nine known analogues were isolated from the aerial parts of Croton laui. Their structures were established on the basis of spectroscopic analysis, and that of crotonolide H (11) was confirmed by single-crystal X-ray crystallography. Crotonolide A (1) exhibited moderate cytotoxicity against two tumor cell lines, HL-60 (human premyelocytic leukemia, IC50 9.42 µM) and P-388 (murine leukemia, IC50 7.45 µM), and crotonolide G (10) displayed significant antibacterial activity against a panel of Gram-positive bacteria.

Eurifoloids A-R, structurally diverse diterpenoids from Euphorbia neriifolia.

Eighteen new diterpenoids, named eurifoloids A-R (1-18), including ingenane (1 and 2), abietane (3-7), isopimarane (8-12), and ent-atisane (13-18) types, along with four known analogues were isolated from Euphorbia neriifolia. Eurifoloid M (13) represents a rare class of ent-atisane-type norditerpenoid. Eurifoloids E (5) and F (6) exhibited significant anti-HIV activities, with EC50 values of 3.58 ± 0.31 (SI = 8.6) and 7.40 ± 0.94 µM (SI = 10.3), respectively.

Effect of social support on illness perception in patients with atrial fibrillation during "Blanking Period": Mediating role of sense of mastery.

AIM: To explore whether sense of mastery can mediate the relationship between social support and illness perception in patients with atrial fibrillation (AF) who were at the "Blanking Period." DESIGN: A cross-sectional design. METHODS: 405 patients with AF who were at the "Blanking Period" in the Affiliated Hospital of Qingdao University were recruited; they completed a set of questionnaires, including the Perceived Social Support Scale, the Personal Mastery Scale and the Brief Illness Perception Questionnaire. RESULTS: Social support and sense of mastery were both adversely connected to illness perception. The indirect effect of social support on illness perception through sense of mastery was negative, accounting for 86.04% of the total effect. CONCLUSION: During the "Blanking Period," better social support and sense of mastery contribute to a positive illness perception of AF patients. Social support also can influence patients' illness perception indirectly via the mediator of sense of mastery.

Identification of three elevenin receptors and roles of elevenin disulfide bond and residues in receptor activation in Aplysia californica.

Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.

IP6 reduces colorectal cancer metastasis by mediating the interaction of gut microbiota with host genes.

Inositol hexaphosphate (IP6) is a phytochemical widely found in grains and legumes that plays an anti-cancer role. However, the mechanism underlying the inhibition of colorectal cancer metastasis by IP6 through host genes, gut microbiota, and their interactions remain elusive. In this study, 16S rRNA sequencing was used to study the effect of IP6 on gut microbiota in an orthotopic transplantation model of colorectal cancer mice. The transcriptome was used to study the changes of host genes in metastasis and the relationship with gut microbiota. The results showed that the gut microbiota composition of model mice was significantly different from that of normal mice. The beta diversity partly tended to return to the normal level after IP6 intervention. Especially, Lactobacillus helveticus and Lactococcus lactis were recovered after IP6-treated. Enrichment analysis showed that the enrichment score of the Cytokine-Cytokine receptor interaction signal pathway decreased after IP6 treatment compared to the model group. Further analysis of differentially expressed genes (DEGs) in this pathway showed that IP6 reduced the expression of the Tnfrsf1b gene related to the area of liver metastasis, and the Tnfrsf1b gene was negatively correlated with the relative abundance of Lactobacillus helveticus. Our results presented that host gene, microbiome and their interaction may serve as promising targets for the mechanism of IP6 intervention in colorectal cancer metastasis.

Butyrate ameliorates alcoholic fatty liver disease via reducing endotoxemia and inhibiting liver gasdermin D-mediated pyroptosis.

BACKGROUND: Alcoholic fatty liver disease (AFLD) is characterized by hepatic steatosis and carries an elevated risk of cirrhosis and hepatocellular carcinoma. However, the mechanism of AFLD has not been elucidated thoroughly, and there are still no efficient therapies in clinic. Notably, butyrate, one kind of short-chain fatty acids produced by gut microbiota, has been shown to improve methionine-choline-deficient diet-induced non-alcoholic steatohepatitis. And our previous study found that butyrate ameliorated endotoxemia in db/db mice. In this study, we aimed to explore the role of butyrate in the development of AFLD. METHODS: C57BL/6 mice were treated with saline (normal control), alcohol with or without butyrate by gavage for 6 months. AFLD was evaluated by the levels of serum alcohol, aspartate aminotransferase (AST), alanine transaminase (ALT), triglyceride (TG) and intrahepatic TG. And the histology and inflammation in liver and colon were analyzed using hematoxylin-eosin (H&E) staining, immunohistochemistry and western blot. In addition, gut microbiota composition was analyzed using the V3-V4 regions of the bacterial 16S ribosomal RNA gene by sequence. Furthermore, we performed in vitro experiment to verify the role of butyrate in hepatocyte by western blot and transmission electron microscopy. RESULTS: We found that butyrate ameliorated alcohol-induced hepatic steatosis and inflammation. Furthermore, chronic alcohol feeding induced dysbiosis and dysfunction of the gut microbiota, disrupted the intestinal barrier, and increased serum endotoxin levels. Meanwhile, butyrate improved the intestinal barrier disruption and endotoxemia induced by alcohol, but did not significantly alleviate the microbiome dysfunction. Mechanistically, butyrate ameliorated AFLD by inhibiting gasdermin D (GSDMD)-mediated pyroptosis. CONCLUSIONS: In summary, we found butyrate ameliorated alcoholic fatty liver by down-regulating GSDMD-mediated pyroptosis. We speculate that butyrate improves AFLD mainly by maintaining intestinal barrier function and alleviating gut leakage. These findings suggest that butyrate may have the potential to serve as a novel treatment for AFLD.

Association analysis of the RET proto-oncogene with Hirschsprung disease in the Han Chinese population of southeastern China.

Hirschsprung disease (HSCR) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. This study investigated a possible role of the RET proto-oncogene in sporadic HSCR patients in the Han Chinese population. Our results indicated that rs1800858, rs1800860, rs1800863, and rs2075912, located in exons 2, 7, 15, and intron 19 of RET, are strongly associated with the disease (P < 0.01), with rs1800860 and rs1800863 playing a protective role in the pathogenesis of HSCR in the Chinese population. We also showed that the haplotype consisting of four SNPs is significantly associated with HSCR. We did not find a significant difference in the CA-repeat in intron 5 of RET between cases and controls. Our study provided further evidence that the RET gene is involved in the susceptibility to HSCR in the Han Chinese population.

Staphylococcal enterotoxin C injection in combination with ascorbic acid promotes the differentiation of bone marrow-derived mesenchymal stem cells into osteoblasts in vitro.

Staphylococcal enterotoxin C injection is established as a clinical therapy for delayed healing or disunion of bone fractures. In the present study, the effects of staphylococcal enterotoxin C injection in combination with ascorbic acid (SEC-AA) on the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) and their influences on the mineralization of osteoblasts were investigated. SEC-AA treatment induced increased levels of alkaline phosphatase activity in MSCs and increased numbers of alizarin red-stained calcified nodules, indicating enhanced differentiation of MSCs into osteoblasts. The findings demonstrated that SEC-AA promoted the differentiation of MSCs into osteoblasts and accelerated the cytopoiesis of osteoblasts. Our data provide a cytological model for bone fracture therapy aimed at shortening the time required for healing and improving the clinical outcome, and also provide a theoretical basis for inducible differentiation of MSCs, mineralization of osteoblasts and reconstruction of bone tissues.

Effect of taurine-conjugated ursodeoxycholic acid on endoplasmic reticulum stress and apoptosis induced by advanced glycation end products in cultured mouse podocytes.

BACKGROUND: Activations of death receptors and mitochondrial damage are well-described common apoptotic pathways. Recently, a novel pathway via endoplasmic reticulum (ER) stress has been reported. METHODS: We assessed the role of tauroursodeoxycholic acid (TUDCA) in inhibition of ER stress and its protective effect on advanced glycation end products (AGEs)-induced apoptosis in murine podocytes. Podocytes were incubated with increasing doses of AGEs for variable time periods. Apoptosis was quantitatively determined by flow cytometry detecting propidium iodide expression and annexin V binding simultaneously. Level of glucose-regulated protein 78 (ER stress marker) expression was determined by Western blot. Intracellular calcium concentration ([Ca(2+)](i)) was recorded by a laser confocal microscope and the Ca(2+) indicator Fluo-3 labeling. RESULTS: AGEs induced podocyte apoptosis and increased the expression of glucose-regulated protein 78 in a dose- and time-dependent manner as compared with bovine serum albumin. These changes were accompanied by a rapid rise in [Ca(2+)](i) of podocytes. TUDCA was capable of abolishing AGEs-induced expression of glucose-regulated protein 78 and subsequently inhibited apoptosis in a dose-dependent manner. CONCLUSION: We propose that ER stress plays an important role in AGEs-induced apoptosis and that TUDCA prevents apoptosis by blocking an ER stress-mediated apoptotic pathway. This novel mechanism of TUDCA action suggests new intervention methods for AGEs-induced apoptosis of mouse podocytes in diabetic nephropathy.

Chronic activation of neutral ceramidase protects beta-cells against cytokine-induced apoptosis.

AIM: To investigate the activity and expression of neutral ceramidase (N-CDase) in the insulin-secreting cell line INS-1 and its role in the cellular response to cytokines. METHODS: HPLC, Western blotting, and quantitative real-time PCR were performed to detect the activity and expression of N-CDase in INS-1 cells treated with a cytokine mixture (5 ng/mL interleukin-1beta, 10 ng/mL TNF-alpha, and 50 ng/mL interferon-gamma). The expression and activity of N-CDase in the INS-1 cells were specifically inhibited using N-CDase-siRNA transfection. Annexin V-fluorescein- isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in the INS-1 cells. RESULTS: The INS-1 cells exhibited some basal N-CDase activity, and cytokines induced a time-dependent delay in the activation of NCDase. As a result, the activation of N-CDase was first detectable at 8 h after stimulation. It peaked at 16 h and remained elevated at 24 h. Cytokines also upregulated the mRNA and protein expression of N-CDase in the INS-1 cells. Furthermore, when N-CDase activity was inhibited by RNA interference, cytokine-induced apoptosis in the INS-1 cells was markedly increased. CONCLUSION: The N-CDase pathway is active in INS-1 cells, and the chronic activation of N-CDase is involved in the pathological response of beta-cells to cytokines, potentially providing protection against cytokine toxicity.

From Hyper- to Hypoinsulinemia and Diabetes: Effect of KCNH6 on Insulin Secretion.

Glucose-stimulated insulin secretion from islet ß cells is mediated by KATP channels. However, the role of non-KATP K+ channels in insulin secretion is largely unknown. Here, we show that a non-KATP K+ channel, KCNH6, plays a key role in insulin secretion and glucose hemostasis in humans and mice. KCNH6 p.P235L heterozygous mutation co-separated with diabetes in a four-generation pedigree. Kcnh6 knockout (KO) or Kcnh6 p.P235L knockin (KI) mice had a phenotype characterized by changing from hypoglycemia with hyperinsulinemia to hyperglycemia with insulin deficiency. Islets from the young KO mice had increased intracellular calcium concentration and increased insulin secretion. However, islets from the adult KO mice not only had increased intracellular calcium levels but also had remarkable ER stress and apoptosis, associated with loss of ß cell mass and decreased insulin secretion. Therefore, dysfunction of KCNH6 causes overstimulation of insulin secretion in the short term and ß cell failure in the long term.

Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells.

AIM: To study the capacity of bone marrow mesenchymal stem cells (BM-MSCs) trans-differentiating into islet-like cells and to observe the effect of portal vein transplantation of islet-like cells in the treatment of streptozotocin-induced diabetic rat. METHODS: BM-MSCs were isolated from SD rats and induced to differentiate into islet-like cells under defined conditions. Differentiation was evaluated with electron microscopy, RT-PCR, immunofluorescence and flow cytometry. Insulin release after glucose challenge was tested with ELISA. Then allogeneic islet-like cells were transplanted into diabetic rats via portal vein. Blood glucose levels were monitored and islet hormones were detected in the liver and pancreas of the recipient by immunohistochemistry. RESULTS: BM-MSCs were spheroid adherent monolayers with high CD90, CD29 and very low CD45 expression. Typical islet-like cells clusters were formed after induction. Electron microscopy revealed that secretory granules were densely packed within the cytoplasm of the differentiated cells. The spheroid cells expressed islet related genes and hormones. The insulin-positive cells accounted for 19.8% and mean fluorescence intensity increased by 2.6 fold after induction. The cells secreted a small amount of insulin that was increased 1.5 fold after glucose challenge. After transplantation, islet-like cells could locate in the liver expressing islet hormones and lower the glucose levels of diabetic rats during d 6 to d 20. CONCLUSION: Rat BM-MSCs could be transdifferentiated into islet-like cells in vitro. Portal vein transplantation of islet-like cells could alleviate the hyperglycemia of diabetic rats.

Preliminary research on DNA methylation changes during murine palatogenesis induced by TCDD.

2,3,7,8-Tetrachlrodibenzo-p-dioxin (TCDD) has been shown to induce cleft palate through growth factor and receptor expression changes during palatogenesis. DNA methylation is an important epigenetic modification that can regulate gene expressions and may be involved in TCDD-induced cleft palate. In this study, we investigated the effects of TCDD on the global and CpG DNA methylation status and the expression levels of DNA methyltransferases (Dnmts) in palate tissue of fetal mice. Pregnant C57BL/6J mice were administered with corn oil or TCDD 28 µg/kg at gestation day 10.5(GD10.5), and sacrificed at GD13.5, 14.5, 15.5. Fetal palates were collected for molecular analysis. Global DNA methylation status was detected by Methylamp™ Global DNA Methylation Quantification Ultra Kit. The expression of DNA methyltransferases were examined by quantitative real-time PCR(q-PCR). Methylation Specific PCR (MSP) was performed to analyze CpG methylation status of Dnmts. We found that the global DNA methylation level and the expression of Dnmt3a were higher at GD13.5 in the TCDD group. The methylation level of CpG site 2 in the promoter region of Dnmt3a in the control group was higher than that of the TCDD group at GD13.5. The low CpG methylation level of Dnmt3a at GD13.5 which causes the up-expression of Dnmt3a may induce global hypermethylation in fetal palate tissue. The aberrant global methylation status at GD13.5 may be the cause of palate malformation in fetal mice induced by TCDD.

[Transfection of pemt-2-cDNA inhibits the expression of cell cycle related proteins in rat CBRH-7919 hepatoma cells].

OBJECTIVES: To unravel the molecular mechanism of proliferation inhibition induced by transfection of pemt2-cDNA into rat CBRH-7919 hepatoma cells. METHODS: We started with the highly expressed PEMT2 clone. Cell culture and Western blotting techniques were used to examine the expression of cyclinD1/CDK4, cyclinE/CDK2, phospho-Rb, caspase-3, c-jun and caveolins. RESULTS: Our results showed that CDK4, CDK2, phospho-Rb and c-jun were down regulated in the pemt2 highly expressed cell clone. The high expression clone of pemt2-transfected cells also showed over expression of caspase-3. CONCLUSION: The reductions of proliferation and apoptosis of pemt2 transfected cells could be related to the G1 phase arrest induced by down-regulation of the cell cycle-associated proteins.

Diagnostic Value of Common Inflammatory Markers on Fever of Unknown Origin.

This study was designed to evaluate the diagnostic value of common inflammatory markers with regard to fever of unknown origin (FUO). We investigated 383 patients who were hospitalized with FUO at the Henan Province People's hospital between January 2009 and June 2015. Of all the cases, infectious diseases accounted for 33.9%, neoplasms for 21.1%, collagen vascular diseases for 25.1%, miscellaneous diseases for 4.7%, and no diagnosis for 15.1%. Patients in the neoplasm group were older than those in the infectious disease, collagen vascular disease, and miscellaneous disease groups (p = 0.006, p < 0.0001, and p = 0.001, respectively). The duration of fever before admission of patients in the neoplasm and collagen vascular disease group was longer than that of patients in the infectious disease group (p = 0.002 and p = 0.007, respectively). The diagnostic time after admission of patients from the neoplasm and collagen vascular disease groups was longer than that for patients from the infectious disease group (both p < 0.0001). Serum ferritin levels of patients in the infectious disease group were lower than those of patients in the neoplasm and collagen vascular disease groups (p = 0.029 and p = 0.032, respectively), while serum procalcitonin (PCT) levels in the infectious disease group was higher than that in the neoplasm and collagen vascular disease groups (p = 0.016 and p = 0.007, respectively). Therefore, FUO remains a clinical problem in China and serum ferritin and PCT may be useful in discriminating infectious from non-infectious causes (neoplasms and collagen vascular diseases) in patients with FUO.