Coaggregated E. faecalis with F. nucleatum regulated environmental stress responses and inflammatory effects.

To investigate the cell-cell interactions of intergeneric bacterial species, the study detected the survival of Enterococcus faecalis (Ef) under monospecies or coaggregation state with Fusobacterium nucleatum subsp. polymorphum (Fnp) in environmental stress. Ef and Fnp infected the human macrophages with different forms (Ef and Fnp monospecies, Ef-Fnp coaggregates, Ef + Fnp cocultures) for exploring the immunoregulatory effects and the relevant molecular mechanisms. Meanwhile, the transcriptomic profiles of coaggregated Ef and Fnp were analyzed. Ef was shown to coaggregate with Fnp strongly in CAB within 90 min by forming multiplexes clumps. Coaggregation with Fnp reinforced Ef resistance against unfavorable conditions including alkaline, hypertonic, nutrient-starvation, and antibiotic challenges. Compared with monospecies and coculture species, the coaggregation of Ef and Fnp significantly facilitates both species to invade dTHP-1 cells and aid Ef to survive within the cells. Compared with coculture species, dual-species interaction of Ef and Fnp significantly decreased the levels of pro-inflammatory cytokines IL-6, TNF-α, and chemokines MCP-1 secreted by dTHP-1 cells and lessened the phosphorylation of p38, JNK, and p65 signaling pathways. The transcriptome sequencing results showed that 111 genes were differentially expressed or Ef-Fnp coaggregated species compared to Ef monospecies; 651 genes were differentially expressed for Fnp when coaggregation with Ef. The analysis of KEGG pathway showed that Ef differentially expressed genes (DEGs) were enriched in quorum sensing and arginine biosynthesis pathway; Fnp DEGs were differentially concentrated in lipopolysaccharide (LPS) biosynthesis, biofilm formation, and lysine degradation pathway compared to monospecies. KEY POINTS: • Coaggregated with Fnp aids Ef's survival in environmental stress, especially in root canals after endodontic treatment. • The coaggregation of Ef and Fnp may weaken the pro-inflammatory response and facilitate Ef to evade killed by macrophages. • The coaggregation between Ef and Fnp altered interspecies transcriptional profiles.

Circular RNA circFNDC3AL Upregulates BCL9 Expression to Promote Chicken Skeletal Muscle Satellite Cells Proliferation and Differentiation by Binding to miR-204.

Skeletal muscle is a heterogeneous tissue that is essential for initiating movement and maintaining homeostasis. The genesis of skeletal muscle is an integrative process that lasts from embryonic development to postnatal stages, which is carried out under the modulation of many factors. Recent studies have shown that circular RNAs (circRNAs), a class of non-coding RNAs, are involved in myogenesis. However, more circRNAs and their mechanisms that may regulate skeletal muscle development remain to be explored. Through in-depth analysis of our previous RNA-Seq data, circFNDC3AL was found to be a potentially functional circRNA highly expressed during embryonic development of chicken skeletal muscle. Therefore, in this study, we investigated the effect of circFNDC3AL on skeletal muscle development in chickens and found that circFNDC3AL promoted chicken skeletal muscle satellite cell (SMSC) proliferation and differentiation. To gain a thorough understanding of the exact modulatory mechanisms of circFNDC3AL in chicken skeletal muscle development, we performed target miRNA analysis of circFNDC3AL and found that circFNDC3AL has a binding site for miR-204. Subsequently, we demonstrated that miR-204 inhibited chicken SMSC proliferation and differentiation, which showed the opposite functions of circFNDC3AL. Furthermore, we identified the miR-204 target gene B-cell CLL/lymphoma 9 (BCL9) and validated that miR-204 had an inhibitory effect on BCL9, while the negative effect could be relieved by circFNDC3AL. In addition, we verified that BCL9 performed the same positive functions on chicken SMSC proliferation and differentiation as circFNDC3AL, as opposed to miR-204. In conclusion, our study identified a circRNA circFNDC3AL that upregulates BCL9 expression to promote the proliferation and differentiation of chicken SMSCs by binding to miR-204.

ß-Sitosterol Ameliorates Endometrium Receptivity in PCOS-Like Mice: The Mediation of Gut Microbiota.

Background: Polycystic ovary syndrome (PCOS), one of the most common endocrine diseases in women of childbearing age, has been found to be accompanied by changes in the gut microbiota. The Bu Shen Yang Xue formula (BSYXF) is a traditional Chinese medicine widely used for the treatment of PCOS. This study aimed to investigate whether the protective effects of ß-sitosterol, the main active ingredient of BSYXF, on PCOS was mediated by regulating gut microbiota. Methods: The presence of ß-sitosterol in BSYXF was detected by liquid chromatography-mass spectrometry. The PCOS-like mouse model was induced by dehydroepiandrosterone. The fecal supernatant of ß-sitosterol-treated mice was prepared for fecal microbiota transplantation (FMT). Body weight and wet weight of the uterus and ovary of the mice were recorded for organ index calculation. Hematoxylin and eosin stain was used to assess the endometrial morphology and microenvironment changes. Expression of endometrial receptivity markers cyclooxygenase-2 (COX-2), Integrin ανß3, leukemia inhibitory factor (LIF), and homeobox A10 (HOXA10) in the endometrium were determined by immunohistochemistry and western blot analysis. Enzyme-linked immunosorbent assay was employed to detect the expression of follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), and testosterone (T) in the serum. The diversity of gut microbiota was examined by 16S rDNA gene sequencing. Results: With the treatment of ß-sitosterol and ß-sitosterol-FMT, the uterine index of PCOS-like mice increased, the ovarian index decreased, levels of COX-2, LH and T decreased, and levels of Integrin ανß3, LIF, HOXA10, FSH, and P increased. Under ß-sitosterol treatment, the structure of the gut microbiota in PCOS-like mice was also changed. Conclusion: ß-sitosterol regulates the endometrial receptivity of PCOS and harmonizes the sex hormone balance, which may be related to the changes in the structure and composition of gut microbiota, thus affecting the pathological process of PCOS.

Characteristics and function of cathepsin L3 from Schistosoma japonicum.

Schistosomiasis is still prevalent and seriously endangering the health of people and livestock in many countries. There have been great efforts to develop vaccines against schistosomiasis for prolonged protection in epidemic areas. Molecules from lung-stage schistosomula have been regarded as potential vaccine candidates against schistosomiasis. Our previous work has shown that cathepsin L3 from Schistosoma japonicum (SjCL3) is expressed in lung-stage schistosomula, but its role is not well known. In the present study, we characterized SjCL3 and detected its effect as a possible vaccine in vivo and in vitro. From the results of quantitative PCR (qPCR) and western blot, SjCL3 was present throughout the lifecycle of the worm, and its relative expressed level was higher in the liver eggs and adult worms than other stages. Additionally, immunofluorescence assay showed that SjCL3 was mainly concentrated in the eggshell, alimentary canal, and musculature of worms. Compared with the adjuvant group, the immunization of SjCL3 in mice resulted in a 28.9% decrease in worm burden and a 29.2% reduction in egg number in the host liver. In antibody-dependent cell-mediated cytotoxicity (ADCC) insecticidal experiments in vitro, the existence of SjCL3 could in part suppress adherence between macrophages and worm. The above results indicated that the immunization of SjCL3 could induce limited immune protection against S. japonicum infection in mice, and this protease played a role in breaking the process of ADCC, which was beneficial to the survival of worms.

Severe viral gastroenteritis in children after suboptimal rotavirus immunization in Taiwan.

BACKGROUND: The study aimed to investigate the molecular epidemiology of severe viral gastroenteritis (AGE) in children in Taiwan after the implementation of the rotavirus vaccine in the private sector. METHODS: Fecal samples from hospitalized children with severe AGE from April 2004 to March 2011 were examined by reverse transcription-polymerase chain reaction or polymerase chain reaction to identify enteric viral pathogens. The study period was divided to prevaccine (before September 2006) and postvaccine (after October 2006) periods. The prevalence of enteric viruses between the 2 periods was analyzed. The disease burdens of rotavirus- and norovirus-associated diseases were assessed according to vaccine implementation status and were adjusted for age. RESULTS: A total of 755 stool samples were collected from hospitalized patients with AGE; enteric viruses were identified in 586 patients (77.6%), including 44 with concomitant bacterial infection. Viral enteric infection by rotavirus, norovirus, astrovirus, sapovirus, enteric adenovirus, multiple viruses and bacterial coinfections were found in 216 (28.6%), 128 (17.0%), 24 (3.2%), 6 (0.8%), 69 (9.1 %), 99 (13.1%) and 44 (5.8%) patients, respectively. A significant increase of norovirus infection was found in the postvaccine period (P < 0.001); on the other hand, rotavirus infection in infants has been reduced substainally (P = 0.056) and the annual peak of rotavirus infection has gradually become less prominent, with a significant decline of coinfection of rotavirus with other pathogens. CONCLUSIONS: Suboptimal use of rotavirus vaccines in the private sector caused a slow but modest impact on severe rotavirus AGE, whereas norovirus infection became more common.

Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma.

BACKGROUND: Pre-S deletion mutant of hepatitis B virus (HBV) affects the expression of middle and small surface proteins, resulting in intracellular accumulation of large surface protein. The correlation between pre-S deletion mutant and risk of hepatocellular carcinoma (HCC) in hepatitis B virus carriers remains unclear. METHODS: Using molecular assays, pre-S deletion mutant of HBV were determined in 266 patients with chronic HBV genotype B or C infection. They included 202 asymptomatic carriers and 64 HCC patients. RESULTS: The overall prevalence of pre-S deletion mutant was 16.5%. Hepatocellular carcinoma (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.23-8.48, P = 0.02) and genotype C (OR, 3.19; 95%CI, 1.54-6.62, P = 0.002) were independently associated with the presence of pre-S deletion mutant. The prevalence of pre-S deletion mutant was comparable between HCC patients with genotype B and C infection. Nevertheless, in asymptomatic carriers, patients with genotype C infection were significantly associated with the presence of pre-S deletion mutant compared to those with genotype B infection (20.8% vs 7.2%, P = 0.007). Compared with age- and genotype B-matched asymptomatic carriers, young HCC patients (<50 years of age) had a significantly higher frequency of pre-S deletion (3.4% vs 20%, P = 0.04). CONCLUSIONS: Pre-S deletion mutant is more frequent in HBV carriers with genotype C infection, and those with pre-S deletion mutant may be associated with the development of HCC, irrespective of HBV genotype.

Role of hepatitis B virus precore/core promoter mutations and serum viral load on noncirrhotic hepatocellular carcinoma: a case-control study.

BACKGROUND: Apart from the presence of liver cirrhosis, hepatitis B virus (HBV) factors have also been shown to play a role in the development of hepatocellular carcinoma (HCC). Studying HBV-related noncirrhotic HCC may help clarify the effect of viral factors. METHODS: In a hospital-based, age- and genotype-matched study, we aimed to determine the role played by basal core promoter (BCP) T1762/A1764 mutation, precore A1896 mutation, and serum viral load in noncirrhotic hepatocarcinogenesis by comparing 44 patients with HBV-related noncirrhotic HCC, 45 patients with chronic hepatitis B, and 42 patients with HBV-related cirrhotic HCC. HBV genotype, precore and BCP mutations, and viral load were determined by molecular assays. RESULTS: In univariate analysis, statistically significant odds ratios were obtained for male sex (P=.005) and BCP T1762/A1764 mutation (P=.0003) in patients with noncirrhotic HCC, compared with patients with chronic hepatitis B. By multiple logistic regression analysis, male sex, BCP T1762/A1764 mutation, and viral load >or=10(5) copies/mL were independently associated with the risk of noncirrhotic HCC. The virologic characteristics were similar between patients with cirrhotic HCC and those with noncirrhotic HCC. CONCLUSIONS: Our results suggest that BCP T1762/A1764 mutation and higher viral load may be involved in the carcinogenesis of cirrhotic and noncirrhotic HCC.

Effect of plasma homocysteine level and urinary monomethylarsonic acid on the risk of arsenic-associated carotid atherosclerosis.

Arsenic-contaminated well water has been shown to increase the risk of atherosclerosis. Because of involving S-adenosylmethionine, homocysteine may modify the risk by interfering with the biomethylation of ingested arsenic. In this study, we assessed the effect of plasma homocysteine level and urinary monomethylarsonic acid (MMA(V)) on the risk of atherosclerosis associated with arsenic. In total, 163 patients with carotid atherosclerosis and 163 controls were studied. Lifetime cumulative arsenic exposure from well water for study subjects was measured as index of arsenic exposure. Homocysteine level was determined by high-performance liquid chromatography (HPLC). Proportion of MMA(V) (MMA%) was calculated by dividing with total arsenic species in urine, including arsenite, arsenate, MMA(V), and dimethylarsinic acid (DMA(V)). Results of multiple linear regression analysis show a positive correlation of plasma homocysteine levels to the cumulative arsenic exposure after controlling for atherosclerosis status and nutritional factors (P < 0.05). This correlation, however, did not change substantially the effect of arsenic exposure on the risk of atherosclerosis as analyzed in a subsequent logistic regression model. Logistic regression analyses also show that elevated plasma homocysteine levels did not confer an independent risk for developing atherosclerosis in the study population. However, the risk of having atherosclerosis was increased to 5.4-fold (95% CI, 2.0-15.0) for the study subjects with high MMA% (> or =16.5%) and high homocysteine levels (> or =12.7 micromol/l) as compared to those with low MMA% (<9.9%) and low homocysteine levels (<12.7 micromol/l). Elevated homocysteinemia may exacerbate the formation of atherosclerosis related to arsenic exposure in individuals with high levels of MMA% in urine.

Role of hepatitis B viral load and basal core promoter mutation in hepatocellular carcinoma in hepatitis B carriers.

BACKGROUND: Several hepatitis B viral factors correlate with the progression of chronic liver disease. However, the independent and interactive effects of each known viral factor on the development of hepatocellular carcinoma (HCC) remain largely unknown. METHODS: In a cross-sectional, retrospective, hospital-based setting, we comprehensively compared viral factors in 160 chronic hepatitis B virus (HBV) carriers and 200 patients with HCC, to clarify the independent and joint effect of each factor. RESULTS: In univariate analysis, statistically significant odds ratios (ORs) were obtained for male sex (P < .001), advanced age (P < .001), HBV genotype C infection (P = .005), the precore A1896 mutation (P < .001), and the basal core promoter (BCP) T1762/A1764 mutation (P < .001). According to the results of multiple logistic-regression analysis, advanced age, male sex, the precore A1896 mutation, the BCP T1762/A1764 mutation, and an HBV load > or = 10(5) copies/mL were independently associated with the development of HCC. Compared with patients with an HBV load < 10(5) copies/mL and the BCP A1762/G1764 wild-type strain, the adjusted OR of developing HCC was > or = 30 in patients with an HBV load > or = 10(5) copies/mL and the BCP T1762/A1764 mutant, irrespective of the presence of the precore A1896 mutation and viral genotype. CONCLUSIONS: HBV load and the BCP T1762/A1764 mutation are important in hepatocarcinogenesis.

Lack of interferon sensitivity-determining region in the genome of hepatitis B virus genotype Ba.

BACKGROUND/AIMS: In chronic hepatitis B, both host and viral factors may predict the response to interferon (IFN) treatment. Whether IFN sensitivity-determining regions exist within the hepatitis B virus (HBV) genomic background remains largely unknown. We therefore performed full-length viral genomic comparison between HBVs obtained from IFN responders and non-responders. METHODS: We enrolled 18 HBV genotype Ba patients who had received 24-week IFN 5 MU three times weekly and were followed monthly for 12 months post-treatment. There were 10 responders and eight non-responders. Pretreatment full-length viral nucleotide consensus sequence was obtained. In six non-responders and four responders, post-treatment viral nucleotide sequence was further compared with their corresponding pre-treatment specimens. In addition, the average number of nucleotide substitutions of the HBV quasispecies was compared between three responders and three non-responders. RESULTS: HBV nucleotide consensus sequence was identical between responders and non-responders. We found 0-15 (mean 7.7) nucleotide substitutions in the post-treatment HBV genome in the six non-responders and 0-14 (mean 3.8) nucleotide substitutions in the four responders, respectively. Genetic complexity of HBV quasispecies was comparable between responders and non-responders. CONCLUSIONS: Our results suggest that an IFN sensitivity-determining region might not exist within the genome of HBV genotype Ba. Host factors and virus-host interactions may be more important in determining the response to IFN treatment.