Molecular epidemiology of hepatitis C virus genotypes in different geographical regions of Chinese mainland and a phylogenetic analysis.

BACKGROUND: Hepatitis C virus (HCV) infection remains a major public health problem in Chinese mainland. Investigation of the distribution of genotypes contributed to the prevention, diagnosis and treatment of HCV infection. Therefore, we conducted a study on the distribution of HCV genotypes and phylogenetic analysis to provide an up-to-date understanding of the molecular epidemiology of genotypes in Chinese mainland. METHODS: Our retrospective multicenter study enrolled 11,008 samples collected between August 2018 and July 2019 from 29 provinces/municipalities (Beijing, Hebei, Inner Mongolia, Shanxi, Tianjin, Gansu, Ningxia, Shaanxi, Xinjiang, Heilongjiang, Jilin Liaoning, Henan, Hubei Hunan, Anhui, Fujian, Jiangsu, Jiangxi, Shandong, Shanghai Zhejiang, Guangdong, Guangxi, Hainan, Chongqing, Guizhou, Sichuan and Yunnan). Phylogenetic analysis of each subtype was performed to infer the evolutionary relationship of sequences from diverse regions. Two independent samples t tests were used for the comparison of continuous variables, and chi-square tests were used for the comparison of categorical variables. RESULTS: Four genotypes (1, 2, 3 and 6) were found, including 14 subtypes. HCV genotype 1 was dominant, accounting for 49.2%, followed by genotypes 2, 3 and 6, accounting for 22.4%, 16.4%, and 11.9%, respectively. Additionally, the top five subtypes were 1b, 2a, 3b, 6a and 3a. Proportions of genotypes 1 and 2 decreased while genotypes 3 and 6 increased over past years (P < 0.001). Genotypes 3 and 6 were concentrated in the population aged 30 to 50 years, and male carriers had lower proportions of subtypes 1b and 2a than female carriers (P < 0.01). Genotypes 3 and 6 were more prevalent in southern parts of Chinese mainland. Nationwide spreads of subtypes 1b and 2a were associated with sequences from northern parts of Chinese mainland, while subtypes 3a, 3b and 6a were associated with sequences from southern parts of Chinese mainland. CONCLUSIONS: HCV subtypes 1b and 2a remained the most common subtypes in Chinese mainland, and their proportions decreased over the past years, while the proportions of genotypes 3 and 6 increased. Our investigation provided an accurate epidemiological picture of the circulating viral strains in Chinese mainland, contributing to the prevention, diagnosis and treatment of HCV infection. TRIAL REGISTRATION: Not applicable.

Efficient electrotransformation of Rhodococcus ruber YYL with abundant extracellular polymeric substances via a cell wall-weakening strategy.

Rhodococcus spp. have broad potential applications related to the degradation of organic contaminants and the transformation or synthesis of useful compounds. However, some Gram-positive bacteria are difficult to manipulate genetically due to low transformation efficiency. In this study, we investigated the effects of chemicals including glycine, isonicotinic acid hydrazide (INH), Tween 80 and penicillin G, as well as cell growth status, competent cell concentration, electroporation field strength, electroporation time and heat shock time, on the electrotransformation efficiency of the tetrahydrofuran-degrading bacterium Rhodococcus ruber YYL with low transformation efficiency. The highest electrotransformation efficiency was 1.60 × 106 CFU/µg DNA after parameter optimization. GmhD (D-glycero-D-manno-heptose 1-phosphate guanosyltransferase) gene, which is important in the biosynthesis of lipopolysaccharide, was deleted via the optimized electrotransformation method. Compared with wild-type strain, YYL ΔgmhD showed extremely high electrotransformation efficiency because the surface of it had no mushroom-like extracellular polymeric substances (EPS). In addition, the results showed that cell wall-weakening reagents might cause some translucent substances like EPS, to detach from the cells, increasing the electrotransformation efficiency of strain YYL. We propose that these results could provide a new strategy for unique bacteria that are rich in EPS, for which genetic manipulation systems are difficult to establish.

Enrichment and characterization of a highly efficient tetrahydrofuran-degrading bacterial culture.

Tetrahydrofuran (THF) is a ubiquitous toxic and carcinogenic pollutant. Screening for pure or mixed-culture microorganisms that can efficiently degrade THF is difficult due to its chemical stability. In this study, an enrichment culture, H-1, with a stable THF-degrading ability and microbial community structure was enriched from activated sludge and could efficiently degrade 95% of 40 mM THF within 6 days. The optimal THF degradation conditions for H-1 were an initial pH of 7.0-8.0 and a temperature of 30 °C. The substrate tolerance concentration of H-1 reached 200 mM. Heavy metals tolerance concentrations of Cu2+, Cd2+ and Pb2+ of H-1 was 0.5 mM, 0.4 mM and 0.03 mM, and 4 mM Mn2+ did not significantly influence the THF degradation ratio or biomass of H-1. H-1 might be a good material for actual wastewater treatment because of its efficient THF degradation performance and ability to resist various stressful conditions. In addition, the THF-degrading efficiency of H-1 was enhanced by the addition of moderate carbon sources. High-throughput sequencing of the 16S rRNA gene showed that Rhodococcus sp. (a potential THF-degrading strain) and Hydrogenophaga sp. (a potential non-THF-degrading strain) were the dominant microorganisms in the H-1 culture. These results indicate the potential coexistence of cooperation and competition between THF-degrading bacteria and nondegrading bacteria in this enrichment culture.

Metabolite Cross-Feeding between Rhodococcus ruber YYL and Bacillus cereus MLY1 in the Biodegradation of Tetrahydrofuran under pH Stress.

Bacterial consortia are among the most basic units in the biodegradation of environmental pollutants. Pollutant-degrading strains frequently encounter different types of environmental stresses and must be able to survive with other bacteria present in the polluted environments. In this study, we proposed a noncontact interaction mode between a tetrahydrofuran (THF)-degrading strain, Rhodococcus ruber YYL, and a non-THF-degrading strain, Bacillus cereus MLY1. The metabolic interaction mechanism between strains YYL and MLY1 was explored through physiological and molecular studies and was further supported by the metabolic response profile of strain YYL, both monocultured and cocultured with strain MLY1 at the optimal pH (pH 8.3) and under pH stress (pH 7.0), through a liquid chromatography-mass spectrometry-based metabolomic analysis. The results suggested that the coculture system resists pH stress in three ways: (i) strain MLY1 utilized acid metabolites and impacted the proportion of glutamine, resulting in an elevated intracellular pH of the system; (ii) strain MLY1 had the ability to degrade intermediates, thus alleviating the product inhibition of strain YYL; and (iii) strain MLY1 produced some essential micronutrients for strain YYL to aid the growth of this strain under pH stress, while strain YYL produced THF degradation intermediates for strain MLY1 as major nutrients. In addition, a metabolite cross-feeding interaction with respect to pollutant biodegradation is discussed.IMPORTANCERhodococcus species have been discovered in diverse environmental niches and can degrade numerous recalcitrant toxic pollutants. However, the pollutant degradation efficiency of these strains is severely reduced due to the complexity of environmental conditions and limitations in the growth of the pollutant-degrading microorganism. In our study, Bacillus cereus strain MLY1 exhibited strong stress resistance to adapt to various environments and improved the THF degradation efficiency of Rhodococcus ruber YYL by a metabolic cross-feeding interaction style to relieve the pH stress. These findings suggest that metabolite cross-feeding occurred in a complementary manner, allowing a pollutant-degrading strain to collaborate with a nondegrading strain in the biodegradation of various recalcitrant compounds. The study of metabolic exchanges is crucial to elucidate mechanisms by which degrading and symbiotic bacteria interact to survive environmental stress.

pH Stress-Induced Cooperation between Rhodococcus ruber YYL and Bacillus cereus MLY1 in Biodegradation of Tetrahydrofuran.

Microbial consortia consisting of cooperational strains exhibit biodegradation performance superior to that of single microbial strains and improved remediation efficiency by relieving the environmental stress. Tetrahydrofuran (THF), a universal solvent widely used in chemical and pharmaceutical synthesis, significantly affects the environment. As a refractory pollutant, THF can be degraded by some microbial strains under suitable conditions. There are often a variety of stresses, especially pH stress, that inhibit the THF-degradation efficiency of microbial consortia. Therefore, it is necessary to study the molecular mechanisms of microbial cooperational degradation of THF. In this study, under conditions of low pH (initial pH = 7.0) stress, a synergistic promotion of the THF degradation capability of the strain Rhodococcus ruber YYL was found in the presence of a non-THF degrading strain Bacillus cereus MLY1. Metatranscriptome analysis revealed that the low pH stress induced the strain YYL to up-regulate the genes involved in anti-oxidation, mutation, steroid and bile acid metabolism, and translation, while simultaneously down-regulating the genes involved in ATP production. In the co-culture system, strain MLY1 provides fatty acids, ATP, and amino acids for strain YYL in response to low pH stress during THF degradation. In return, YYL shares the metabolic intermediates of THF with MLY1 as carbon sources. This study provides the preliminary mechanism to understand how microbial consortia improve the degradation efficiency of refractory furan pollutants under environmental stress conditions.

Selection and evaluation of reference genes for expression analysis of Cassi.

Cassia obtusifolia, belonging to legume family, is important in many fields with high pharmaceutical, economic, and ecological values. These interests of C. obtusifolia triggered in-depth and fundamental genetic and molecular research. Therefore, the stable reference gene is necessary for normalization of the gene expression studies. In this study, 10 candidate reference genes were subjected to expression analysis in 12 different tissues and under different stresses by qRT-PCR. The expression stability was evaluated using geNorm, NormFinder, and BestKeeper software. In conclusion, different suitable reference genes were selected in different tissues and under different stress. CYP1, EF1α2, ACT2, UBQ1 were the most stable reference genes in all samples. The relative expression levels of WRKY gene were detected to confirm the reliability of the selected reference genes. These results provided suitable reference genes that could be used for normalization in C. obtusifolia tissues and under different stress.

Isolation, structure modeling and function characterization of a trypsin inhibitor from Cassia obtusifolia.

A trypsin inhibitor gene (CoTI1) from Cassia obtusifolia was isolated and the deduced amino acid sequence was attributed to the Kunitz-type trypsin inhibitor. The recombined CoTI1, expressed in E. coli, exhibited strong inhibitory effect on bovine trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura. CoTI1 thus presents insecticidal properties that may be useful for the genetic engineering of plants. Leu84, Arg86 and Thr88 were predicted as three key residues by molecular modeling in which Arg86, inserted into the substrate pocket of trypsin, interacted directly with residue Asp189 of trypsin causing the specific inhibition against trypsin. The predicted results were confirmed by site-directed mutagenesis with L84A, R86A and T88A, respectively. The substantial changing expression level of CoTI1 under salt, drought and abscisic acid treatment suggested that CoTI1 might play important role in the resistance against abiotic stress.

De novo assembly and analysis of Cassia obtusifolia seed transcriptome to identify genes involved in the biosynthesis of active metabolites.

A cDNA library generated from seeds of Cassia obtusifolia was sequenced using Illumina/Solexa platform. More than 12,968,231 high quality reads were generated, and have been deposited in NCBI SRA (SRR 1012912). A total of 40,102 unigenes (>200 bp) were obtained with an average sequence length of 681 bp by de novo assembly. About 34,089 (85%) unique sequences were annotated and 8694 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Among them, 131 unigenes, which are involved in the biosynthesis and (or) regulation of anthraquinone, carotenoid, flavonoid, and lipid, the 4 best known active metabolites, were identified from cDNA library. In addition, three lipid transfer proteins were obtained, which may contribute to the lipid molecules transporting between biological membranes. Meanwhile, 30 cytochrome P450, 12 SAM-dependent methyltransferases, and 12 UDP-glucosyltransferase unigenes were identified, which could also be responsible for the biosynthesis of active metabolites.

In silico analysis on structure and DNA binding mode of AtNAC1, a NAC transcription factor from Arabidopsis thaliana.

NAC (NAM/ATAF/CUC) transcription factors regulate the expression of the target genes by formation of NAC-DNA complex, which are involved in development, stress responses and nutrient distribution in many metaphyta plants. AtNAC1, a NAC transcription factor from Arabidopsis thaliana, plays an important role in auxin signaling and root development. In order to understand the structure and DNA binding model of AtNAC1, the 3D structure model of AtNAC1 was constructed and docked with its target DNA. The structure of AtNAC1 monomer contained four α-helices and eight ß-sheets. Two homo monomers of AtNAC1 formed a homo-dimer. The N-terminal sheet S1, Arg24 and Glu31 played an important role in forming AtNAC1 homo-dimer. AtNAC1 dimer interacted with DNA via its core ß-sheet (S5) which contained WKATGKD motif inserting into the major groove of DNA and formed a tight AtNAC1-DNA complex. The DNA sites for AtNAC1 binding were 5'-CTGACGTA-3' and 5'-GATGACGC-3'. Lys102, Ala103, Thr104, Gly105, Lys106, and Asp107 interacting with sugars/bases of DNA were probably responsible for specific recognition of DNA sites. Meanwhile, Arg91, Lys135, and Lys171 binding with phosphate groups of DNA backbone might be the key residues for affinity with DNA. The study provided the in silico framework to understand the interactions of AtNAC1 with DNA at the molecular level.