[Study on mechanism of Chinese Yam polysaccharide synergizing nucleoside analogues against hepatitis B virus through p38 MAPK signaling pathway].

This study explore the molecular mechanism of the synergistic effect of Chinese Yam polysaccharides and nucleoside analogues(NAs) on hepatitis B virus(HBV) resistance. Different concentrations of Chinese Yam polysaccharide and entecavir were ad-ded to HepG2.2.15 cells. After the cytotoxicity was detected by cell counting kit-8(CCK-8), the optimal concentration and time of the two drugs to inhibit HepG2.2.15 cells were screened out. They were divided into control group, Chinese Yam polysaccharide group, entecavir group and combination drug group(Chinese Yam polysaccharide + entecavir). The drugs were added to HepG2.2.15 cells, ELISA was used to detect the effects of each group of drugs on the secretion of hepatitis B virus surface antigen(HBsAg) and hepatitis B virus e antigen(HBeAg) in cell supernatant, probe quantitative real-time PCR(probe qRT-PCR) was used to detect the effects of drugs on HBV-DNA in HepG2.2.15 cells, and Western blot was used to detect the effects of each group of drugs on the expression of p38 MAPK, p-p38 MAPK, NTCP proteins in HepG2.2.15 cells. The qRT-PCR was used to detect the effect of drugs on the expression of p38 MAPK and NTCP mRNA in HepG2.2.15 cells. The results showed that compared with control group, the concentrations of HBeAg and HBsAg in Chinese Yam polysaccharide group, entecavir group and combination group decreased(P<0.01 or P<0.001), and both of them inhibited HBV-DNA in HepG2.2.15 cells(P<0.01), and the HBV-DNA inhibition of HepG2.2.15 cells in the combination group was more obvious(P<0.001), and the protein expression levels of p-p38 MAPK and NTCP were significantly decreased(P<0.05 or P<0.01), the mRNA expression level of p38 MAPK increased, and the mRNA expression level of NTCP decreased(P<0.05 or P<0.01). To sum up, Chinese Yam polysaccharide can reduce the expression of NTCP protein and mRNA through p38 MAPK signaling pathway and cooperate with entecavir in anti-HBV.

CD146, a therapeutic target involved in cell plasticity.

Since its identification as a marker for advanced melanoma in the 1980s, CD146 has been found to have multiple functions in both physiological and pathological processes, including embryonic development, tissue repair and regeneration, tumor progression, fibrosis disease, and inflammations. Subsequent research has revealed that CD146 is involved in various signaling pathways as a receptor or co-receptor in these processes. This correlation between CD146 and multiple diseases has sparked interest in its potential applications in diagnosis, prognosis, and targeted therapy. To better comprehend the versatile roles of CD146, we have summarized its research history and synthesized findings from numerous reports, proposing that cell plasticity serves as the underlying mechanism through which CD146 contributes to development, regeneration, and various diseases. Targeting CD146 would consequently halt cell state shifting during the onset and progression of these related diseases. Therefore, the development of therapy targeting CD146 holds significant practical value.

Phosphorylated vimentin at Ser72 is associated with epithelial-mesenchymal transition in lupus nephritis.

OBJECTIVES: To measure the expression of vimentin and its phosphorylated forms in lupus nephritis (LN) and explore their potential role in LN development. METHODS: Lupus renal biopsies from LN patients and normal renal biopsies from kidney transplant donors were collected. The expression of vimentin and its phosphorylated forms (p-vimentin (Ser39, Ser56, Ser72, Ser83, and Tyr117)) were measured by Western blots and immunohistochemistry. To construct stable cell line that overexpress vimentin and its phosphorylated forms, an immortalized proximal tubule epithelial cell line (HK-2 cells) was utilized. The roles of vimentin and its phosphorylated forms on the migration of HK-2 cells were examined by transwell migration assay and wound healing analysis. RESULTS: We first observed a significant upregulation of vimentin protein in TGFß1-induced HK-2 cells. This finding was further confirmed in renal tissues obtained from LN patients and animal model. Interestingly, among the five phosphorylated forms of vimentin, only vimentin phosphorylated at Ser72 was upregulated in LN. Through the establishment of stable vimentin and its phosphorylated forms overexpression in HK-2 cells, we found that the overexpression of vimentin and its phosphorylated forms at Ser72 significantly enhances the cell migration. CONCLUSIONS: Vimentin phosphorylated on Ser72 is important for renal epithelial cell migration, which would enhance the progression of vimentin-induced epithelial-mesenchymal transition during LN development.

Rapid Genotyping of Campylobacter coli Strains from Poultry Meat by PFGE, Sau-PCR, and fla-DGGE.

The genotyping of Campylobacter coli was done using three methods, pulsed-field gel electrophoresis (PFGE), Sau-polymerase chain reaction (Sau-PCR), and denaturing gradient gel electrophoresis assay of flagellin gene (fla-DGGE) and the characteristics of these assays were compared. The results showed that a total of 53 strains of C. coli were isolated from chicken and duck samples in three markets. All isolates were clustered into 31, 33, and 15 different patterns with Simpson's index of diversity (SID) values of 0.972, 0.974, and 0.919, respectively. Sau-PCR assay was simpler, more rapid, and had higher discriminatory power than PFGE assay. Fla-DGGE assay could detect and illustrate the number of contamination types of C. jejuni and C. coli without cultivation, which saved more time and cost than Sau-PCR and PFGE assays. Therefore, Sau-PCR and fla-DGGE assays are both rapid, economical, and easy to perform, which have the potential to be promising and accessible for primary laboratories in genotyping C. coli strains.

[Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway].

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

Expression and Correlation of MIF and ERK1/2 in Liver Cirrhosis and Hepatocellular Carcinoma Induced by Hepatitis B.

Objective: To detect expression and phosphorylation level of macrophage migration inhibitor (MIF) and extracellular-regulated kinases 1 and 2 (ERK1/2) in hepatitis B-induced liver cirrhosis (HBILC) and hepatocellular carcinoma (HCC) with a background of HBILC and analyze the correlation of MIF and ERK1/2 with HBILC and HCC. Methods: Twenty cases of normal liver tissues were collected as a control group, and 48 specimens of HBILC tissues and 48 specimens of HCC tissues were collected as the experimental group, which were assigned as the HBILC group and HCC group, respectively. All tissue specimens were processed into tissue chips. The expressions of MIF, ERK1/2, and their phosphorylated proteins were detected via immunohistochemistry, and MIF and ERK1/2 nucleic acid expressions were detected by in situ hybridization. The results were statistically analyzed using the chi-square test. Results: Proteins and nucleic acids of MIF and ERK1/2 presented low expression in the control group and high expression in the HBILC group and HCC group. MIF expression in the three groups was 25.0%, 75.0%, and 79.17%, respectively, while that of the nucleic acids was 25.0%, 70.83%, and 68.75%, respectively. Expression of ERK1/2 in the three groups was 40.0%, 60.42%, and 81.25%, respectively, and that of nucleic acids was 40.0%, 79.17%, and 77.08%. Expression of pERK1/2 was low in the control and HBILC group and high in the HCC group. Expression of pERK1/2 in the three groups was 20%, 45.83%, and 75%, respectively. Expression of pERK1/2 in the HCC group was significantly different from that in the HBILC and control group (P<0.05), but the difference between the HBILC group and control group was not statistically significant (P>0.05). Conclusion: Occurrence and development of HBILC and HCC are not only related to the high expression of MIF but also closely related to the activation of the ERK1/2 signaling pathway.

Intergenic region polymorphism analysis: a novel genotyping method for Klebsiella pneumoniae.

AIMS: The ability to distinguish between Klebsiella pneumoniae strains is critical for outbreak investigations. A new typing method, intergenic region polymorphism analysis (IRPA), was developed, validated, and the discriminatory power was determined by comparison with multiple-locus variable-number tandem repeat analysis (MLVA) in this study. METHODS AND RESULTS: This method is based on the idea that every IRPA locus (polymorphic fragment of intergenic regions present in one strain but not in other strains or different fragment sizes in other strains) could divide strains into different genotypes. A 9-loci IRPA scheme was designed to type 64 K. pneumoniae isolates. Five IRPA loci were identified that conferred the same level of discrimination as the 9-loci initially examined. Among these K. pneumoniae isolates, 7.81% (5/64), 6.25% (4/64), 4.96% (3/64), 9.38% (6/64), and 1.56% (1/64) were capsular serotypes K1, K2, K5, K20, and K54, respectively. The discriminatory power of the IRPA method was better than that of MLVA expressed in Simpson's index of diversity (SI) at 0.997 and 0.988, respectively. The congruent analysis of the IRPA method and MLVA showed moderate congruence between the two methods (AR = 0.378). The AW indicated that if IRPA data are availabl, one can accurately predict the MLVA cluster. CONCLUSION: The IRPA method was found to have higher discriminatory power than MLVA and allowed for simpler band profile interpretation. The IRPA method is a rapid, simple, and high-resolution technique for molecular typing of K. pneumoniae.

Comparison of pulsed-field gel electrophoresis and a novel amplified intergenic locus polymorphism method for molecular typing of Campylobacter jejuni.

Campylobacter is regarded as the leading cause of zoonotic diseases and Campylobacter jejuni (C. jejuni) is one of the predominant pathogenic species. To track C. jejuni infections, various genotyping methods have been used. In this study, amplified intergenic locus polymorphism (AILP) was used to type C. jejuni for the first time. To confirm its feasibility, pulsed-field gel electrophoresis (PFGE) was performed as a control, and the results obtained by the AILP and PFGE methods were compared. Fifty-one isolates were resolved into 34 and 29 different genotypes with Simpson's indices of 0.976 and 0.967 using the AILP and PFGE methods, respectively. The adjusted Rand coefficient of the two approaches was as high as 0.845. In summary, the data showed that the two genotyping methods were similar for discriminating isolates and were both appropriate methods to distinguish whether two isolates were indistinguishable, but the AILP was faster and less costly than PFGE. Therefore, the AILP is a reliable, rapid, and highly discriminative method to genotype C. jejuni collected from poultry meat, which is helpful to effectively monitor C. jejuni.

Genome-wide association study analysis to resolve the key regulatory mechanism of biomineralization in Pinctada fucata martensii.

Biomineralization-controlled exo-/endoskeleton growth contributes to body growth and body size diversity. Molluscan shells undergo ectopic biomineralization to form the exoskeleton and biocalcified "pearl" involved in invading defence. Notably, exo-/endoskeletons have a common ancestral origin, but their regulation and body growth are largely unknown. This study employed the pearl oyster, Pinctada fucata marntensii, a widely used experimental model for biomineralization in invertebrates, to perform whole-genome resequencing of 878 individuals from wild and breeding populations. This study characterized the genetic architecture of biomineralization-controlled growth and ectopic biomineralization. The insulin-like growth factor (IGF) endocrine signal interacted with ancient single-copy transcription factors to form the regulatory network. Moreover, the "cross-phylum" regulation of key long noncoding RNA (lncRNA) in bivalves and mammals indicated the conserved genetic and epigenetic regulation in exo-/endoskeleton growth. Thyroid hormone signal and apoptosis regulation in pearl oysters affected ectopic biomineralization in pearl oyster. These findings provide insights into the mechanism underlying the evolution and regulation of biomineralization in exo-/endoskeleton animals and ectopic biomineralization.

Subtyping of Campylobacter coli isolated from raw poultry meat in retail markets using amplified intergenic locus polymorphism - A novel rapid subtyping method.

In order to provide more phylogenetic information of Campylobacter coli in large-scale epidemiological investigation, this work was undertaken to develop a novel genotyping method based on amplified intergenic locus polymorphism (AILP), by using pulsed-field gel electrophoresis (PFGE; using SmaI enzymes) as control. Eleven pairs of primers were selected to type C. coli strains for this purpose. A total of 68 C. coli isolates recovered from 51 retail raw chicken and 37 retail raw duck were subtyped. The Simpson's index of diversity (SID) of AILP and PFGE, as well as the adjusted Rand index (AR) and the adjusted Wallace coefficient (AW) between AILP and PFGE, were calculated. The new AILP method differentiated 68 C. coli isolates into 55 different subtypes (SID = 0.993), compared with 46 different profiles obtained from PFGE (SID = 0.980). The SID value of the AILP method was improved with the increasing number of primers, and a combination of 7 loci was selected as the optimal combination. The congruent analysis of the AILP method and PFGE showed moderate congruence between the two methods (AR = 0.462). The AW indicated that if AILP data is the available one can confidently predict the PFGE cluster. The results of this study showed that the AILP method had higher discrimination than PFGE and also allowed for significant reduction in time and cost.

Novel globular C1q domain-containing protein (PmC1qDC-1) participates in shell formation and responses to pathogen-associated molecular patterns stimulation in Pinctada fucata martensii.

The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.

Light-resetting impact on behavior and the central circadian clock in two vole species (genus: Lasiopodomys).

The behavioral circadian rhythms of subterranean rodents show intra- and interspecies diversity in terms of adaptation to dark underground environments, but the endogenous molecular mechanism of rhythm regulation in the suprachiasmatic nuclei (SCN) is stable to many species. In this study, we sought to determine the rhythms of behavior and central molecular regulatory mechanisms in the SCN of the subterranean Mandarin voles (Lasiopodomys mandarinus) compared with a related aboveground species, Brandt's voles (Lasiopodomys brandtii). Both species were reared under a 12 L:12 D cycle or in continuous darkness for 4 weeks. The pattern of wheel-running activity was similar in both species and had a periodicity of almost 24 h regardless of rearing conditions. However, the intensity of daily activity in Brandt's voles decreased markedly in darkness, while there was no significant difference in activity intensity in mandarin voles under different light regimes. In both vole species, all tested genes in the SCN showed significant time-dependent expression regardless of rearing conditions, and the expression levels of most genes did not differ significantly between different species and conditions. However, the peak phase shift in gene expression differed between the two species. In conclusion, behavioral patterns in mandarin and Brandt's voles were regulated by a stable molecular endogenous biological clock. The observed differences in activity intensity and phase shift suggest that different mechanisms regulate circadian rhythms in different living environments.

Matrine inhibits proliferation and migration of HepG2 cells by downregulating ERK1/2 signaling pathways.

OBJECTIVE: To research the effect of matrine on the proliferation and migration of HepG2 cells through extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. METHODS: HepG2 cell was selected and divided into blank control group, experimental group (matrine 1, 2, and 4 mg/mL), and positive control group (PD98059, ERK1/2 inhibitor). MTT measure was used to detect the effective time and concentration which matrine inhibits HepG2 cells. After 24 h, the effect of effective concentration of matrine on the of morphological changing HepG2 cells was observed. The invasion ability was assayed by transwell method, the expression of ERK1/2 and pERK1/2 were detected through Western blot, and reverse transcription polymerase chain reaction was used to test the expression level of ERK1/2 mRNA. RESULTS: With the increase of matrine concentration, the number of adherent HepG2 cells gradually decreased, the morphologic changes gradually became spherical, some cell morphology was incomplete, and even cell fragments appeared. The proliferation and invasion ability of HepG2 cells decreased. The expression of ERK1/2, pERK1/2, and ERK1/2 mRNA downregulated with the increase of matrine concentration (P < 0.05). CONCLUSION: Matrine inhibits the proliferation and migration of HepG2 cells by downregulating the ERK1/2 signaling pathway.