The Transcription Factor SsZNC1 Mediates Virulence, Sclerotial Development, and Osmotic Stress Response in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a fungal pathogen with a broad range of hosts, which can cause diseases and pose a great threat to many crops. Fungal-specific Zn2Cys6 transcription factors (TFs) constitute a large family prevalent among plant pathogens. However, the function of Zn2Cys6 TFs remains largely unknown. In this study, we identified and characterized SsZNC1, a Zn2Cys6 TF in S. sclerotiorum, which is involved in virulence, sclerotial development, and osmotic stress response. The expression of SsZNC1 was significantly up-regulated in the early stages of S. sclerotiorum infection on Arabidopsis leaves. The target deletion of SsZNC1 resulted in reduced virulence on Arabidopsis and oilseed rape. In addition, sclerotial development ability and growth ability under hyperosmotic conditions of SsZNC1 knockout transformants were reduced. A transcriptomic analysis unveiled its regulatory role in key cellular functions, including cellulose catabolic process, methyltransferase activity, and virulence, etc. Together, our results indicated that SsZNC1, a core regulatory gene involved in virulence, sclerotial development and stress response, provides new insight into the transcription regulation and pathogenesis of S. sclerotiorum.

Development of gut microbiota along with its metabolites of preschool children.

BACKGROUND: To reveal the changes of intestinal microbial abundance and composition, as well as the microbiota metabolic levels of bile acids and short chain fatty acids of healthy preschool children during their growth. METHODS: Feces of 120 healthy newborns and 150 healthy children aged 6 months to 6 years were collected. Then the composition of intestinal flora was analyzed by 16S rRNA, and the contents of bile acids and short chain fatty acids in feces were detected by LC-MS and GS methods, respectively. RESULTS: The composition and function of intestinal microflora were not stable in neonatal period but significantly improved at 6 months after birth, and gradually stabilized and tended to adult-like formation after 2-3 years old. The levels of short chain fatty acids and secondary bile acids were consistent with the development of gut microbiota. CONCLUSION: The age of 6 months may be a critical period for the development of intestinal microflora in children.

Metagenomic profiling reveals dominance of gram-positive bacteria in the gut microbiome shifts associated with immunoglobulin A vasculitis (Henoch-Schönlein Purpura).

OBJECTIVES: Immunoglobulin A vasculitis (IgAV), previously known as Henoch-Schönlein purpura, is the most common vasculitis that has a classical skin manifestation of palpable purpuric rash. Factors pertinent to IgAV remain inadequately understood. Here, we aimed to examine the gut microbiome shifts associated with IgAV and its recovery. METHODS: Stool samples were collected from 10 children with IgAV (6-14 years old) before and after a multi-drug therapy, along with 9 age-matched healthy children. The samples were subjected to metagenomic analyses to investigate the taxonomic and functional shifts of the gut microbiome. RESULTS: The analyses revealed that compared with healthy controls, treatment-naïve patients exhibited substantial taxonomic and functional alterations of gut microbiota, including 104 IgAV-depleted species and 7 IgAV-elevated species (FDR < 0.05). After treatment, the IgAV patients displayed a partial restoration of the microbiota shifts, as the relative abundances of some biomarkers (e.g. 9 genera and 22 species) became comparable (FDR > 0.1) between the patients and healthy controls. The treatment-responsive features included Weissella, Faecalibacterium prausnitzii and Bifidobacterium pseudocatenulatum and three components of a putative glutamine transport system. Importantly, gram-positive bacteria accounted for over 85% of the numbers and total relative abundance of the species that were associated with IgAV and responsive to the treatment. In addition, of the 122 IgAV-depleted bacterial genes, 82 were mainly contributed by gram-positive bacteria and 12 by gram-negative bacteria. CONCLUSIONS: Gram-positive bacteria are the main drivers underlying the gut microbiome shifts of IgAV, which may assist rational management of the disease.

Developmental Patterns of Fecal Bile Acids in Healthy Neonates and Children.

BACKGROUND Normal profiles of FBAs in healthy neonates and children in Kunming city and surrounding areas in China have not been previously determined. The objective of this study was to determine a developmental pattern of fecal bile acids (FBAs) in healthy neonates and children. MATERIAL AND METHODS A cross-sectional study was performed on 238 healthy neonates and children recruited in the First Affiliated Hospital of Kunming Medical University, China from October 2015 to September 2016. Secreted primary and secondary FBAs in fresh feces were quantitated by liquid chromatography mass spectrometry (LC-MS). Amounts of FBAs in feces were compared among various age groups. RESULTS Trace amounts of cholic acid and chenodiol acid of primary FBAs were detectable at day 3 after birth, with a significant increase from day 3 to day 7. The primary FBAs gradually decreased from day 25 to the age of 6 years old. In contrast, a significant amount of glycochenodeoxycholic acid was detected on day 3 but decreased to a trace amount by day 7 and onwards. Primary FBAs appeared to maintain a high level, accounting for 98% of total FBAs, with no significant changes from day 7 to day 25 after birth. They gradually decreased from 90% to 10% from age 6 months to 6 years old. While the secondary FBAs were barely detected in neonates, only accounting for 2% of total FBAs, they were gradually elevated to 90% of total FBAs from age 6 months to 6 years old. CONCLUSIONS The liver can effectively synthesize primary bile acids 7 days after birth, and fecal primary bile acids tend to be stable after the neonate stage. Secondary bile acids continuously increase along with the maturation of intestinal flora, which reaches a relatively stable level at around 3 years old.

Gut Microbiota Dysbiosis Associated with Bile Acid Metabolism in Neonatal Cholestasis Disease.

Neonatal cholestasis disease (NCD) is a complex and easily mis-diagnosed condition. We analyzed microbiota community structure in feces and measured short-chain fatty acids, bile acids (BAs) and liver function of 12 healthy, 13 NCD, and 13 treated infants after diagnosis. Based on 16S rRNA gene amplicon sequencing and gas-chromatographic-mass-spectrometric analysis of secondary BAs, we identified microbial genera and metabolites that associate with abnormal bile secretion. Streptococcus gallolyticus and Parabacteroides distasonis, and Lactobacillus gasseri had higher relative abundance in healthy and NCD infants respectively. Compared to NCD patients, healthy infants had higher LCA, CDCA and GCDCA fecal concentrations. The three microbial species and three secondary bile acids were selected as potential non-invasive combined biomarkers to diagnose NCD. We propose that microbiota-metabolite combined biomarkers could be used for diagnosis of NCD, and this may contribute to improved early clinical diagnosis of NCD in the future.

[Levels of short-chain fatty acids in enterobacteria-related metabolites in the feces of infants with cholestatic hepatopathy].

OBJECTIVE: To compare the levels of short-chain fatty acids in enterobacteria-related metabolites in feces between infants with cholestatic hepatopathy and healthy infants. METHODS: Thirty infants with cholestatic hepatopathy were enrolled in this study as the disease group, while 30 healthy infants were enrolled as the control group. Fecal specimens were collected from the disease group before and after treatment and from the control group. Gas chromatography was used to quantitatively determine the content of short-chain fatty acids in the feces of both groups including acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid. RESULTS: There were no significant differences in the concentrations of acetic acid and propionic acid between the control and disease groups before and after treatment, as well as no significant changes in the two markers in the disease group after treatment (P>0.05). The disease group had a significantly increased concentration of butyric acid after treatment (P<0.05). The concentrations of isobutyric acid and isovaleric acid in the control group were significantly higher than those in the disease group before and after treatment (P<0.05). CONCLUSIONS: Intestinal protein metabolites in infants with cholestatic hepatopathy are significantly different from those in healthy infants, whereas there is no significant difference with respect to carbohydrate metabolites.

[Significance of detection of biomarker fecal bile acids in the diagnosis and treatment of childhood Henoch-Schönlein purpura].

OBJECTIVE: To investigate the changes and clinical significance of biomarker fecal bile acids (BA) in children with Henoch-Schönlein purpura (HSP). METHODS: Nineteen children with HSP and twenty-seven healthy children were enrolled in this study. The stool samples were obtained at the acute and remission phases. Fecal BA levels were measured by high performance liquid chromatography mass spectrometry (HPLC-MS). RESULTS: The fecal cholic acid level in the HSP remission group was significantly higher than in the HSP acute group and the healthy control group (P<0.016). The fecal chenodeoxycholic acid level in the HSP remission group was significantly higher than in the healthy control group (P<0.016). The levels of fecal secondary colonic bile acids, deoxycholic acid and lithocholic acid, in the HSP acute and remission groups were significantly lower than in the healthy control group(P<0.05, P<0.016 respectively). No significant differences were found in the levels of fecal urosodeoxycholic acid among the three groups (P>0.05). CONCLUSIONS: Fecal secondary colonic bile acids, deoxycholic acid and lithocholic acid, are in decrease in children with HSP at the acute stage, which may be involved in the pathogenesis and treatment outcomes of HSP.

[Effects of hyperglycaemia in pregnancy and relevant factors on neonatal body composition].

OBJECTIVE: To compare body composition at birth in the appropriate-for-gestational-age infants of women with gestational diabetes mellitus (GDM) and normal glucose tolerance and determine the influencing factors of body composition in infants of women with GDM and normal glucose tolerance. METHODS: A study was conducted on 160 appropriate-for-gestational-age infants (90 males and 70 females) of women with gestational diabetes mellitus (GDM group) and 284 appropriate-for-gestational-age infants (139 males and 145 females) of women with normal glucose tolerance (control group). Anthropometric measurements were obtained within 24 to 48 hours of birth. Multiple stepwise regression was used to determine the correlating factors of fat mass, percent of body fat and fat free mass mass. RESULTS: There were no significant difference in gestational age, birth weight, length, body mass index, circumferences of head, chest and upper arm, biceps, abdominal superficial skin fold between two groups (all P > 0.05), but GDM group was characterized by higher skin folds of triceps and subscapular and flank versus control group(all P = 0.000). GDM group had greater fat mass but decreased fat free mass versus control group ((585 ± 59) vs (480 ± 74) g, 17.8% ± 0.8% vs 14.7% ± 1.9%, (2685 ± 127) vs (2784 ± 109) g, all P = 0.000). Stepwise regression showed that maternal fasting glucose level of oral glucose tolerance test and pre-gravid body mass index correlated with fat mass and percent of body fat. Fasting glucose level had the strongest correlation with fat mass and percent of body fat (P = 0.004, 0.006). Gestational age and maternal height correlated with fat free mass in GDM group (P = 0.040, 0.013). On the other hand, maternal weight gain correlated with fat mass (P = 0.015), fasting glucose level and maternal prepartal weight were correlated with percent of body fat (P = 0.002, 0.043) and pre-gravid body mass index had correlation with fat free mass in control group (P = 0.004). CONCLUSIONS: The appropriate-for-gestational-age infants of women with GDM have increased fat mass and percent of body fat, but decreased fat free mass. Maternal fasting glucose level of oral glucose tolerance test, pre-gravid body mass index, weight gain and maternal prepartal weight are influencing factors of body composition in neonates.

[Effect of breastfeeding on childhood overweight in the offspring of mothers with gestational diabetes mellitus].

OBJECTIVE: To investigate whether breastfeeding can reduce the risk of childhood overweight in the offspring of mothers with gestational diabetes mellitus (GDM). METHODS: Follow-up was performed on 1189 offspring of mothers with GDM between January 2003 and December 2009. The influence of the manner and duration of breastfeeding between 0 to 3 months after birth on the risk of childhood overweight in the offspring of mothers with GDM was analyzed by logistic regression. RESULTS: After correcting confounding factors such as pre-pregnancy BMI, gestational weight gain, gestational blood sugar, sex, birth weight, age and farther's body weight, it was found that the risk of childhood overweight in the offspring who received exclusive breastfeeding during the first 3 months after birth was lower than in the artificial feeding group (OR: 0.479, 95%CI: 0.256-0.897). Offspring who were breastfed for 0 to 3 months, 4 to 6 months and over 6 months had a lower risk of childhood overweight than the artificial feeding group (OR: 0.456, 95%CI: 0.233-0.827; OR: 0.29, 95%CI: 0.103-0.817; OR: 0.534, 95%CI: 0.280-0.970), offspring who were breastfed for 4 to 6 months had a lower risk of childhood overweight than those who were breastfed for 0 to 3 months (OR: 0.372, 95%CI: 0.129-0.874), and offspring who were breastfed for more than 6 months did not show significantly lower risk of overweight than those who were breastfed for less than 6 months (OR: 0.769, 95%CI: 0.470-1.258). CONCLUSIONS: Within 3 months of birth, breastfeeding, especially exclusively, may reduce the risk of childhood overweight in the offspring of mothers with GDM. Within 6 months of birth, the risk of childhood overweight decreases as the duration of breastfeeding increases, but prolonging the duration of breastfeeding cannot necessarily reduce the risk of childhood overweight after postnatal six months.

Effect of high-dose methotrexate chemotherapy on intestinal Bifidobacteria, Lactobacillus and Escherichia coli in children with acute lymphoblastic leukemia.

High-dose methotrexate (HDMTX) chemotherapy is generally accepted as an effective method for the treatment and prevention of extramedullary leukemia in children. However, it is unknown whether HDMTX chemotherapy kills intestinal bacteria on a large scale, thus causing dysbacteriosis, which may in turn influence the progress or prognosis of leukemia. The aim of this study was to examine changes in intestinal flora in children with acute lymphoblastic leukemia (ALL) treated with HDMTX chemotherapy. Bacterial DNA in stool from 36 healthy children and 36 ALL children were tested at A(260) with a spectrophotometer before and after HDMTX chemotherapy. The primers of Bifidobacteria, Lactobacillus and Escherichia coli were designed according to the 16SrRNA/DNA bacterial sequences. Bacteria were qualitatively and quantitatively confirmed by routine polymerase chain reaction (PCR) and fluorescent quantitative PCR, respectively. Our data showed that the total amount of flora in the stools of children with ALL was decreased by 29.6% compared with healthy children (P < 0.01). The total amount of flora in the stools of children with ALL on the third and seventh days after chemotherapy were 1496.5 ± 577.1 and 1966.6 ± 598.3 ng/µL, respectively, which was notably less than before chemotherapy (2436.3 ± 768.6 ng/µL). The amount of Bifidobacteria, Lactobacillus and E. coli in the intestinal tract in the ALL group after chemotherapy had an apparent change, which decreased most clearly on the third day, and partially recovered on the seventh day after chemotherapy. HDMTX chemotherapy can cause intestinal dysbacteriosis in children with ALL. The amount of Bifidobacteria, Lactobacillus and E. coli decreased significantly compared with the control group.

[Effects of high-dose methotrexate therapy on intestinal bacterial flora in children with acute lymphoblastic leukemia].

OBJECTIVE: To investigate the effects of high-dose methotrexate (HDMTX) therapy on intestinal bacterial flora in children with acute lymphoblastic leukemia (ALL). METHODS: Thirty-six children with ALL of pre-and post-HDMTX therapy and 36 control children were enrolled. The bacterial DNA in stool was extracted. The primers for Bacillus bifidus and Escherichia coli with the 16SrRNA/DNA sequence of bacteria were designed. The bacteria were identified through general PCR. The standard curve of both bacterial DNA was produced using a series of dilution of accurately quantified bacterial DNA. The unknown samples were measured by 16SrRNA/DNA-targeted PCR. The amount of stool Bacillus bifidus and Escherichia coli among 36 control children and 36 children with ALL of pre- and post-HDMTX therapy were obtained by using the standard curves. RESULTS: Bacillus bifidus logarithmic absolute value of the first day before treatment, of third day after treatment, of seventh day after treatment in patients with ALL and the control was 7.24 +/- 0.33, 6.00 +/- 0.27, 6.59 +/- 0.33, and 9.49 +/- 0.41, respectively (P < 0.01). Escherichia coli logarithmic absolute value of the first day before treatment, of third day after treatment, of seventh day after treatment in patients with ALL and the control was 6.62 +/- 0.42, 5.96 +/- 0.42, 7.02 +/- 0.41, and 7.52 +/- 0.43, respectively (P < 0.01). The logarithm of the amount of stool Bacillus bifidus and Escherichia coli in control group was higher in ALL group (F = 739.61, 88.67, P < 0.01). There were significant difference (P < 0.01) in the logarithm of the amount of stool Bacillus bifidus and Escherichia coli between pre-therapy and post-therapy group. The logarithm of the bacterium was very low on third day after treatment, but gradually increased on the seventh day after treatment. CONCLUSIONS: (1) HDMTX therapy has great effects on intestinal flora of Bacillus bifidus and Escherichia coli and significantly reduced the bacteria in children with ALL. (2) Probiotics had significantly decreased in ALL group than in control group.

Anti-NSP4 antibody can block rotavirus-induced diarrhea in mice.

BACKGROUND: Rotavirus infection is the most common cause of infectious diarrhea and gastroenteritis among children worldwide. The viral proteins (VP), especially VP4- and VP7-induced neutralizing antibodies, were considered to be critical in protective immunity to rotavirus disease. However, whether the antibody to rotavirus nonstructural protein 4 (NSP4) protects against rotavirus-induced diarrhea directly is not completely clear, especially for the protective time course. MATERIALS AND METHODS: To obtain direct evidence, 12-day-old ICR mice were treated with NSP4 and entire rotavirus to induce diarrhea. RESULTS: Both NSP4 and rotavirus-treated mice developed diarrhea, which was accompanied by histological changes in the small intestine compared to age-matched control mice. Anti-NSP4 antibody demonstrated protection against both entire rotavirus-induced diarrhea and NSP4-induced diarrhea. The histological changes in the small intestinal were reversible. These data show that early intervention with anti-NSP4 antibody can prevent rotavirus-induced diarrhea in mice; late intervention with anti-NSP4 antibody could halt diarrhea progression in mice. CONCLUSIONS: Our findings demonstrate for the first time that administration of anti-NSP4 antibody is effective both prior to and during the time course of rotavirus infection. These observations extend our knowledge of rotavirus infection and its therapeutic options.

[Immunogenetic analysis of human leukocyte antigen DRB1, DQB1 locus among Han ethnic children with Helicobacter pylori infection in Kunming].

OBJECTIVE: To explore the immunogenetic features of human leukocyte antigen DRB1, DQB1 locus and children with Helicobacter pylori (H. pylori) infection in Han ethnic population in Kunming and its association with digestive diseases and H. pylori to better understand the immunogenetic features of the H. pylori infection. METHODS: Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used to study the HLA-DRB1, DQB1 allelic frequency distribution on 35 children with H. pylori infection and 37 healthy controls in Han ethnic population in Kunming. RESULTS: Allelic frequencies of HLA-DRB1 * 0901, DQB1 * 03032 in the H. pylori infection group were lower than those of the healthy control group (7.14% vs. 31.08%, chi(2) = 13.16, Pc < 0.012; 5.71% vs. 25.68%, chi(2) = 10.68, Pc = 0.007) but the rest alleles' frequencies did not show significant differences. CONCLUSION: These result suggested that HLA-DRB1 * 0901, DQB1 * 03032 might protect the H. pylori infection in Han ethnic population in Kunming.

[Possible association between HLA-HRB1 and DQB1 genes frequency and susceptibility or resistance to Helicobacter pylori infection in Kunming Yi ethnic group children].

OBJECTIVE: To study if there is any association between frequency of HLA-DRB1 and DQB1 genes and susceptibility or resistance to Helicobacter pylori (Hp) infection among children of Yi ethnic group in Kunming for understanding the immunogenetic features of the digestive diseases associated with Hp infection. METHODS: Peripherial blood samples were collected from 156 children of Yi ethnic group in a primary school in Kunming city by cluster sampling and the blood Hp-IgG tests (ELISA) were performed. The samples were divided into two groups (Hp-IgG-positive group and Hp-IgG-negative group) according to the blood Hp-IgG test results. There were 61 children in Hp-IgG-positive group and 95 children in Hp-IgG-negative group. Forty children who were chosen from each group by simple random sampling underwent (13)carbon-urea breath test ((13)C-UBT). Thirty-three children who were Hp-IgG-positive and (13)C-UBT-positive were defined as currently Hp- infected group; 39 children who were Hp-IgG-negative and (13)C-UBT-negative were defined as Hp-non-infected group. DNA specimens were extracted from the lymphocytes of their peripheral blood samples. HLA-DRB1 and DQB1 DNA typing was performed by using polymerase chain reaction with sequence specific primers (PCR-SSP). HLA-DRB1, DQB1 allelic frequency distribution among currently Hp infected and non-infected children was compared. RESULTS: HLA-DRB1 * 12 gene frequency among children in Hp non-infected group was higher than that in the currently Hp-infected group (42.31% vs. 14.52%, P < 0.001, Pc < 0.012); however, HLA-DRB1 * 11 gene frequency in the Hp-non-infected group was lower than that in the currently Hp-infected group (3.85% vs. 12.9%, P < 0.05, Pc > 0.05). HLA-DQB1 * 0301 gene frequency in the Hp non-infected group was higher than that in the currently Hp-infected group (55.13% vs. 32.26%, P < 0.007, Pc < 0.05); however, HLA-DQB1 * 04 gene frequency in the Hp non-infected group was lower than that in currently Hp infected group (2.56% vs. 11.29%, P < 0.05, Pc > 0.05). CONCLUSIONS: HLA-DRB1 * 12 and HLA-DQB1 * 0301 gene may be associated with protection against Hp infection in Kunming Yi ethnic group children. Further studies with larger sample size are needed to clarify if HLA-DRB1 * 11 and HLA-DQB1 * 04 are associated with susceptible gene to Hp infection.

[Analysis on molecular characteristic of VP7 and NSP4].

OBJECTIVE: To explore the molecular characteristics and molecular variation of human rotavirus (HRV) strains and to understand the relationship between clinical characteristics and epidemiology of different HRV-VP7 and NSP4. METHODS: Double-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 were amplified with RT-PCR. Then cDNAs were sequenced and compared with 4 human rotavirus NSP4 (Wa, KUN, AU-1, Hochi)) and 3 animal rotavirus NSP4 (EW, OSU, SA11) available in the GenBank while the epidemic strains of human rotavirus isolated in different areas of China were compared, using the Clustal-mp, DNAssist, MEGA2 software. The G serotype of VP7 was analysed by PCR. RESULTS: Serotype G1 was prevalent in 2002 while serotype G3 was the prevalent in Kumming in 2003. The NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 belonged to Wa with highly conservative amino acid. Samples isolated in the same years but not in the same area shared higher homology. Symptoms associated with heavy diarrhea did not seem to be associated with NSP4 molecular variation (P > 0.05). CONCLUSION: Obvious variations of VP7 typing were seen in the same season, as well as in different areas and years. Due to the stable nature of NSP4, it seem to be a better candidate for vaccine production, than VP7.

[Analysis of HLA-DRB1,DQB1 allele polymorphism in the Kunming Yi nationality population].

OBJECTIVE: To investigate the HLA-DRB1, DQB1 allele polymorphism in Kunming Yi nationality population. METHODS: HLA-DRB1, DQB1 DNA types in 70 healthy children of Yi nationality in Kunming were analyzed by polymerase chain reaction with sequence specific primer (PCR-SSP). RESULTS: Twelve alleles at HLA-DRB1 locus were observed in the 70 children: the alleles with gene frequencies higher than 10% were HLA-DRB1*12(33.57%), DRB1*0901(11.43%), DRB1*04(11.43%); the alleles with gene frequencies between 10% and 5% were HLA-DRB1*01(8.57%), DRB1*11(7.86%), DRB1*14(7.14%), DRB1*15(7.14%), DRB1*08(5%); the alleles with gene frequencies lower than 5% were HLA-DRB1*03(2.86%), DRB1*13(2.14%), DRB1*07(1.43%), DRB1*16(1.43%). Seven alleles at HLA-DQB1 locus were observed in the 70 children: the alleles with gene frequencies higher than 10% were HLA-DQB1*0301(45%), DQB1*05(22.14%), DQB1*0303(12.14%); the alleles with gene frequencies between 10% and 5% were HLA-DQB1*04(6.43%), DQB1*06(6.43%); the alleles with gene frequencies lower than 5% were HLA-DQB1*0201(4.29%) and DQB1*0302(3.57%). CONCLUSION: The distribution of HLA-DRB1, DQB1 allele polymorphism in the Kunming Yi nationality population is distinctive. It is neither like that in the South Han population nor like that in the North Han population.