Abstract:Microsporidia is a type of obligate intracellular parasitic single-cell eukaryote. It is a fungal pathogen causing by microsporidiosis. More than 1500 microsporidians have been identified. Among them, 17 species in 9 genera can infect humans. Human microsporidia can infect the intestine, liver, lungs, brain and other parts, causing chronic diarrhea, hepatitis, keratitis, encephalitis and systemic infections. Exploration and development of rapid and efficient human microsporidia diagnostic methods are thus important for pathogenic microorganism detection. Conventional detection methods include transmission Hematoxylin-eosin stain (EM), Hematoxylin-eosin stain, Methylene blue, Giemsa, Gram stain, Weber's Chromotrope-based staining, Calcofluor White staining, microsporidian antigen, antibody detections, quantitative real-time PCR, loop-mediated isothermal amplification and DNA dot hybridization model. The development of detection methods would greatly aid the research of microsporidia infection and control.

Abstract:Surveying diversity of microbial communities has great significance for exploiting microbial resource, exploring functions of microbial communities, elucidating relations between microbial communities and their habitat. With the proposal of the concept of "metagenomics" and the development of sequencing technology, 16S rRNA gene profiling has been widely applied in the survey of microbial diversity. This review introduces four important stages of 16S rRNA gene sequencing analysis, including selection of sequencing platforms and hypervariable regions, sequencing data preprocess and methods of diversity analysis. Furthermore, the current challenges and future prospects to 16S rRNA gene profiling are discussed. This review aims to provide a reference for microbial diversity researches.

Abstract:Coccidiosis brings great economic loss to the poultry industry. The urgent need of green and healthy feed makes the prevention and control of coccidiosis face new challenges. In recent years, a safe and effective new anticoccidial method is needed by the poultry farming, owing to increasing bans of anticoccidial drugs. Recent researches have demonstrated probiotics can prevent coccidiosis-induced secondary infection by competitively excluding colonization of pathogens bacteria and stimulate the secretion of host antibacterial peptides, mucins and tight junction proteins to resist coccidiosis. In addition, it can enhance the anti-coccidial ability by activating the immune response. This review summarizes what is currently known on mechanisms about how probiotics prevent and control coccidiosis by modulating the gut microbiota, ameliorating mucus barrier, affecting function of immune system, to provide a reference for the development of probiotic products to control coccidiosis.

Abstract:Neurodegenerative diseases are generally characterized by loss of synapses and death of neurons, leading to decreased cognitive function, dementia and loss of motor function. Increasing epidemiological and experimental evidence indicates that chronic bacterial, viral and fungal infections may cause neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis and Multiple sclerosis. Chronic infections in the central nervous system could lead to a series of cellular disfunction such as protein misfolding and aggregation, increased oxidative stress, abnormal autophagy, neuronal apoptosis or necrosis. Pathogen infection may also trigger the release of inflammatory mediators and the activation of host immune responses. Furthermore, infection could lead to chronic nerve inflammation and metabolic dysfunction. Here we review recent progress in the research of the regulatory roles and mechanisms of infections in the pathogenesis of neurodegenerative diseases. A better understanding of the relationship between pathogenic infections and neurodegenerative diseases will promote the development of new drugs and therapies.

Abstract:Oral cavity harbors human oral microecosystem, one of the largest microbial reservoirs in the human body. The oral microorganisms usually stably adhere to the surfaces of oral mucosa and teeth in the form of biofilm, and the composition and interaction of biofilms are complicated. Under pathological conditions, microbes can penetrate the pulp cavity and alveolar bone to cause common oral infectious diseases such as dental caries, periapical periodontitis, periodontitis. The infection caused by oral biofilms is even closely related to many systemic diseases. Currently, it is still challenging to study the pathogenic mechanism of the oral biofilm. Moreover, under the influence of the host immunity and various physical and chemical factors inside or outside the oral cavity, the phenotypes and biological characteristics of biofilms remain unclear due to lag of technological advances to mimic and study biofilms in vitro. Microfluidics is a multi-disciplinary field that involves flexible control of micro-scale fluids. It can not only accurately simulate the physical and chemical microenvironment but also be used to conduct real-time observation, high-throughput testing, and single-cell analysis. In this article, we will review the recent research progress of microfluidic technologies in biofilm study, and elaborate on the application prospects of microfluidics for the study of microbial biofilms in the oral microecosystem.

Abstract:Pseudomonas aeruginosa, one of the three leading pathogens of human opportunistic infection, is a Gram-negative bacterium that can cause severe and persistent infections in patients with immunocompromised and cystic fibrosis. The reason for this persistent infection is mainly due to the synergy of bacteria with their own regulatory network after receiving external signals. These bacteria attach to solid surfaces and produce macromolecular substances such as extracellular polysaccharides, matrix proteins, and extracellular DNA. These macromolecules can form highly structured membranous complexes that wrap themselves to form biofilm morphotype and population structure. Biofilms can effectively facilitate bacterial colonization, resistance to antimicrobial substances and host immune responses, and cell-to-cell signal communication within a bacterial community. Thus, it is one of the most important causes of chronic infection and repeated infection of pathogens in clinical treatment. This review focuses on the recent processes in study of the P. aeruginosa biofilm components and their roles in biofilm formation, as well as the function of related regulatory systems, including the quorum sensing system (las, rhl, pqs and iqs) and the c-di-GMP system. Through this review, we provide a more systematic understanding the development of P. aeruginosa biofilm, which will reinforce the treatment of P. aeruginosa infection.

Abstract:Sophorolipids are glycolipids biosurfactants largely produced by various yeasts. They have excellent surface/interfacial activities and chemical stability even at extreme pH, temperature and salinity. More important, sophorolipids have many advantages over the chemical surfactants including non-toxic, non-irritating and easy-to-biodegrade, with many potential applications in many fields. Through the efforts made in the past five decades, the fermentation level of sophorolipids has been highly improved, but the downstream processing that counts for 60% to 80% of the total production cost, is still a challenge. This review highlights the properties and structures of sophorolipids, the biosynthesis pathway of sophorolipids, and the progress in fermentation and separation of sophorolipids in recent years. In addition, the prospects doe sophorolipids fermentation and separation towards their industrialization and commercialization are also addressed.

Abstract:As the most abundant phytoplankton in the marine environment, Prochlorococcus plays a vital role in driving element cycle and energy flow of marine ecosystem. Numerous studies indicate that the growth and photosynthetic activity of Prochlorococcus are vulnerable to environmental stresses, thus resulting in the damage to marine ecosystem stability. Therefore, it is crucial to study the responses of Prochlorococcus to environmental stresses for maintaining the marine ecological stability. Generally, Prochlorococcus could adapt to different light and nutrient conditions in the ocean by differentiating into different ecotypes. However, they cannot cope with sudden changes in the marine environment. Here, we review the progress on the responses of Prochlorococcus at physiological and molecular level to environmental stresses, which includes the important role of cyclic electron transport around photosystem I in light changes, RNA-regulated gene expression, and the protection Prochlorococcus against oxidative stress by heterotrophic bacteria. Moreover, we discuss the future directions for studies on Prochlorococcus responding to environmental stresses, aiming to provide fundamental bases to further explore anti-stress mechanisms of Prochlorococcus.

Abstract:Chronic hepatitis B virus (HBV) infection remains a significant worldwide medical problem. Despite the availability of an effective prophylactic vaccine, an estimated 250 million individuals worldwide are chronically infected. Chronic infection leads to over 1 million deaths annually. Currently, interferon-alpha (IFN-α) and nucleoside/nucleotide analogues drugs are available and reduce both new infection rates and the development of liver disease in HBV-positive persons, but it is difficult to achieve the ideal clinical treatment endpoint. Immune checkpoint inhibitors are an important strategy for reversing T cell exhaustion, that aims at reinvigorating dysfunctional T cells represents a promising approach to induce a functional cure of a chronic infection. In this review, we summarize the recent advances in immune checkpoint inhibitors of programmed death receptor 1/cell programmed death ligand 1 (PD-1/PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and ITIM domin (TIGIT), T-cell immunoglobulin and mucin domin protein-3 (Tim-3), and lymphocyte activation gene-3 (Lag-3) in chronic hepatitis B.

Abstract:[Objective] (a) To determine the virulence of Metarhizium anisopliae to the second instar larvae of Spodoptera litura, (b) To investigate the response of the intestinal bacterial community and antioxidant enzyme to the infection of M. anisopliae, and (c) to explore the defense mechanisms of S. litura against M. anisopliae. [Methods] The toxicity of different concentration of M. anisopliae against 2^nd instar larvae of S. litura was tested by Dipping method. The intestinal bacterial community was determined by Illumina MiSeq high-throughput sequencing of 16S rDNA amplicons. [Results] Different concentrations of spore suspension had virulence to the 2^nd instar larvae of S. litura. After 7 days of treatment, the half lethal concentration (LC₅₀) was 3.944×10⁷ spores/mL. The 1.0×10⁹ spores/mL treatment had the fastest half lethal time (LT₅₀) of 4.6 d, with a corrected lethal rate of 81.03%. Antioxidant enzyme activities were significantly higher in the treated alive larvae compared with that in the control group. The intestinal bacterial community diversity of lethal S. litura larvae was significantly higher than that of the control group, and the bacterial community composition differed significantly between the lethal and control group. [Conclusion] The mortality rate and lethal efficiency of M. anisopliae against S. litura larvae were positively correlated with the concentration of M. anisopliae. The antioxidant enzyme in S. litura larvae may play important roles in resisting the infection of M. anisopliae. The infection of M. anisopliae leads to increase of diversity and change of composition of the intestinal bacterial community of S. litura larvae. Genera, such as Enterococcus, Escherichia and Pseudomonas, may be important factors affecting the mortality of S. litura larvae against infection of M. anisopliae.

Abstract:[Objective] Coprisidins (A and B) are structural unique naphthoquinone-oxindole alkaloids from an insect gut-associated Streptomyces with bioactivities for cancer prevention. As the first example of natural alkaloids with naphthoquinone-oxindole skeleton, the biosynthesis of coprisidins remains unclear. Exploration of the coprisidins biosynthetic mechanism would gain new insights into the biosynthesis of type II polyketide natural products. [Methods] The genome of coprisidin-producing strain Streptomyces sp. SNU607 was sequenced and in silico analysis revealed the biosynthetic potential. The coprisidins biosynthetic cluster was cloned and identified by the gene inactivation and heterologous expression experiments. A biosynthetic pathway for coprisidins was proposed according to the in vivo results and bioinformatics analysis. [Results] Genome analysis of Streptomyces sp. SNU607 revealed 23 gene clusters potentially for secondary metabolites with four of them related to polyketide synthases (PKSs). Gene inactivation and heterologous expression confirmed that a type II PKS gene cluster, which contains 30 putative open reading frames, is responsible for coprisidins biosynthesis. The copH/I/M/N/O genes could constitute a five gene cassette for the biosynthesis of butyryl start unit of coprisidins. Sequence alignment of KS_β (CopB) suggested that coprisidins are biosynthesized by a type II system with a nascent chain length of C20, which is afterward modified by a variety of accessory enzymes. [Conclusion] The rare naphthoquinone-oxindole backbone of coprisidins is assembled by the minimal type II PKS (CopB-C-A) utilizing a butyryl starter unit, along with ketoreductases and cyclases to form the anthracycline-like four ring system. Subsequently, tailoring enzymes and oxidative arrangement are proposed to shape the final naphthoquinone-oxindole skeleton. Considering the structure feature of coprisidins, this study would set the stage for biosynthetic mechanism studies of naphthoquinone-oxindole alkaloids and expand the structural diversity of products synthesized by type II PKSs.

Abstract:[Objective] To isolate, screen and identify strains with rice protein degradation activity and their key proteases, so as to provide ideal enzymes for the preparation of rice oligopeptides. [Methods] Strains with the ability to degrade rice protein were screened from the soil near grain warehouse by "hydrolysis circle", and identified by 16S rRNA analysis. Single factor experiment was done to obtain optimal nitrogen source. Enzymes were then characterized. The yield of rice oligopeptides was detected by HPLC, and the preparation conditions were further optimized. [Results] The best strain was identified as Serratia sp. JWG-D15. The highest protease SoPRO production was achieved by using rice protein as nitrogen source with optimal temperature at 40℃ and optimal pH of 8.0. The highest yield of rice oligopeptides was 72.38%, with 20 U/mg protease SoPRO, and 40 mg/mL rice protein at 40℃ for 4 hours. [Conclusion] Strain JWG-D15 had highest protease SoPRO production by using rice protein as nitrogen source, and the yield of rice oligopeptides was the highest. This study enriched enzyme sources for the preparation of rice oligopeptide.

Abstract:[Objective] To explore the effects of spontaneous and inoculated wine fermentations on the yeast diversity and genetic diversity of Saccharomyces cerevisiae. [Methods] Spontaneous and inoculated fermentations were conducted with Chardonnay using different S. cerevisiae strains of NXU17-26, UCD522 and UCD2610. We used 26S rDNA D1/D2 sequence analysis and Interdelta fingerprint to distinguish the yeasts at interspecific and intraspecific levels, respectively. The diversity of S. cerevisiae strains was analysed by cluster analysis and diversity index. [Results] The fermentation curve of spontaneous fermentation was relatively smooth, and the fermentation speed of inoculated fermentations was significantly faster than spontaneous fermentation. The isolated yeasts were identified as 6 genera and 11 species with 6 species of 5 genera of non-Saccharomyces in spontaneous fermentation. The yeast diversity in inoculated fermentation was far lower than that in spontaneous fermentation, which was composed of Saccharomyces cerevisiae and two non-Saccharomyces. UCD2610 dominated the corresponding fermentation and its genotype accounted for 48.78%; while in the fermentation inoculated with NXU17-26 and UCD522, we didn't find the same genotype as them. Genetic differences between Saccharomyces cerevisiae strains isolated from UCD522 fermentation were small, while obvious in NXU17-26 and UCD2610 fermentations. Diversity index results showed that UCD2610 showed a more prominent position in the fermentation; Saccharomyces cerevisiae strains isolated in UCD522 fermentation showed a higher diversity, and there were more unknown factors affecting the population diversity and the concentration of different genotypes of Saccharomyces cerevisiae. [Conclusion] Fermentation methods have a significant effect on the yeast diversity and the genetic diversity of Saccharomyces cerevisiae strains. This study provides reference for the control of microorganisms during wine fermentation.

Abstract:[Objective] rocE encodes arginine permease in arginine degradation pathway. To determine the mechanism of transcriptional regulation of rocE, we analyzed the transcriptional activity of rocE in Bacillus thuringiensis (Bt). [Methods] Transcriptional units of rocE gene cluster were analyzed by RT-PCR. Transcriptional activity of rocE promoter (ProcE) was analyzed by β-galactosidase assay. rocE mutant of Bt HD73 strain was constructed by homologous recombination. The HTH domain of RocR with His fusion protein was purified by HiTrap chelating column. The binding of rocE promoter with RocR-HTH protein was verified by electrophoresis mobility shift assays. [Results] Transcriptional activity of ProcE was induced by arginine in M9 medium. The transcriptional activity of ProcE was sharply decreased in sigL (encodes Sigma 54) and rocR mutants compared with that in HD73 wild-type in Schaeffer's sporulation medium (SSM) and arginine induced medium. ProcE could bind to RocR-HTH protein. Mutation of rocE had no significant differences on growth and Cry1Ac protein production. However, the sporulation efficiency of the rocE mutant was 65.5%, and that of the HD73 strain was 85.7%. The results of significance analysis show that the difference was significant (P<0.05). [Conclusion] Transcriptional activation of rocE is controlled by Sigma 54, and positive regulated by RocR. rocE gene is related to sporulation efficiency.

Abstract:[Objective] We detected and analyzed β-lactam resistance in E. coli in broiler chickens in Shandong province. [Methods] The viscera samples were obtained from broiler chickens in the slaughterhouse. We screened β-lactam resistant E. coli, used the disk diffusion assay to determine antimicrobial-resistance, extracted bacterial DNA, conducted phylogenetic and biochemical assays, identified β-lactam-resistance genes and the structure of integrons and performed conjugation assays. [Results] More than 80% isolates were resistant to 3 or more β-lactams. The bla_TEM and bla_CTX-M resistance genes encoding class A β-lactamases have a higher rate, 86.7% and 81%, respectively, but only bla_CTX-M and β-lactam resistance showed a significant correlation. The rate of β-lactam resistance genes in B₁ and D₂ subtypes of E. coli was higher, and the resistance was significantly enhanced, while the A₀ and A₁ subtypes were more sensitive. Although integrons were commonly detected in E. coli, their correlation with β-lactamase genes is low. [Conclusion] The results show that broiler-derived E. coli has highly resistant to β-lactams, and multi-drug resistance is widespread. The relationship between the resistance of β-lactams and the phylogeny in E. coli is clarified, and it provides a reference for the epidemiological surveillance of β-lactam resistance in E. coli from broilers.

Abstract:[Objective] To label the nuclei and peroxisomes of Botrytis cinerea with fluorescent proteins, as a tool for further investigation on biogenesis and dynamics of the cellular structures of the fungus. [Methods] The green fluorescent protein (GFP) and the red fluorescent proteins (DsRed and mCherry) were used as reporter proteins to label the nuclei and peroxisomes in B. cinerea. Three fluorescent labeling vectors were separately introduced into the B. cinerea strain B05.10 via Agrobacterium tumefaciens-mediated transformation. The resulting transformants were selected and confirmed by PCR, and then analyzed with the confocal fluorescent microscope. [Results] The single-spore purified recombinant strains expressing red or green fluorescence were obtained. The PCR amplification and fluorescence detection indicated that the fluorescent genes were integrated into the genome of the transformants. Round fluorescent spots were detected in mycelia and conidia of the strains with nuclear labeling, overlapping well with DAPI staining. In the strains with peroxisomes labeling, small green or red fluorescent dots were present in mycelia and conidia. Upon induction on lipids, the number of the dots was significantly increased, corresponding well with the described distribution profile of peroxisomes. In addition, the blue fluorescence produced by Calcofluor white staining did not interfere with the fluorescence of red or green fluorescent proteins, capable of forming well-integrated multi-fluorescent images. [Conclusion] We obtained the B. cinerea strains with fluorescent labeled nuclei or peroxisomes, which are maybe ideal tools for studying the biogenesis and dynamics of cellular organelles, the developments and even the pathogenesis of the fungus.

Abstract:[Objective] Farnesol (FOH, C₁₅H₂₆O) is a natural acyclic sesquiterpene alcohol with an aromatic odor. It is widely used in the industrial production of cosmetics and pharmaceuticals and also considered as an aviation jet-fuel substitute. Although food-grade S. cerevisiae can synthesize endogenous FOH, the yield is too low to meet the requirements for industrial production. Therefore, in this work, the FOH synthesis pathway in S. cerevisiae was metabolically engineered to overproduce FOH. [Methods] To improve the intracellular level of farnesyl pyrophosphate (FPP), the direct precursor for FOH synthesis, the mevalonate pathway in S. cerevisiae CEN.PK2-1D was enhanced via constitutive overexpression of genes encoding the two key enzymes whereas the ergosterol pathway was weakened by replacing the EGR9 promoter with the HXT1 promoter. In addition, five genes encoding endogenous phosphatases and two heterologous synthases that could catalyze FOH synthesis, were individually overexpressed to determine the enzyme with optimum performance. [Results] Shake flask culture experiments showed that constitutive overexpression of genes encoding the truncated HMG-CoA reductase (tHMGR1) and the FPP synthase ERG20 in S. cerevisiae CEN.PK2-1D increased the production of FOH by 50.8 fold to reach 5.08 mg/L. Down-regulation of the squalene synthase encoding gene ERG9, by replacing its promoter with the HXT1 promoter, further increased the FOH titer to 239.17 mg/L. On this basis, a maximum yield of FOH (393.13 mg/L) was obtained when the endogenous phosphatase PAH1 was overexpressed. [Conclusion] In this study, metabolic engineering of S. cerevisiae was used to increase the production titer of FOH to 393.13 mg/L. This is the highest reported yield of FOH from S. cerevisiae under shake flask culture conditions.

Abstract:[Objective] To investigate culturable yeast diversity in Yamzhog Yumco Lake and analyze the influence of environmental factors on yeast diversity. [Methods] Yeasts were isolated by membrane filtration flat culture. Identification was based on sequence analysis of ITS domains of rRNA gene, combining with traditional classification method. SPSS 20.0 and CANOCO 5 were used to examine correlations between culturable yeast diversity and environmental factors. [Results] In total of 297 yeast strains were isolated, these strains were identified as 25 species in 16 genera. Vishniacozyma was the dominating genera in the Lake. Vishniacozyma victoriae was the dominating species. Statistical analysis indicated that pH, electric conductivity (EC), total dissolved salt (TDS) and salt were significantly positive related to yeast species number and genera number among different sites, total phosphorus (TP) is significantly negative related to yeast genera number among different sites and different regions, TP is significantly negative related to yeast species number among different regions, additionally, pH and TP were important factors influencing the community structure of yeast in the Yamzhog Yumco Lake. [Conclusion] The yeast community from Yamzhog Yumco Lake showed high species richness, the yeast community structure was different at different sites, and anthropogenical influenced is an important factor for the yeast community. The relationship between yeast community and human activities deserves further study.

Abstract:[Objective] Pine wilt disease caused by the pine wood nematode Bursaphelenchus xylophilus is a destructive disease of pines and one of the most dangerous forestry diseases in China. Bacillus velezensis strains are widely used in agriculture as microbial agents for promoting plant growth and the major bacterial resources in China's microbial fertilizer industry. In this study we evaluated the inhibition of Bu. xylophilus by Ba. velezensis. [Methods] Ba. velezensis FZB42 was used as a representative strain in the study. The inhibition was determined using supernatants of FZB42 cultures under different conditions, the supernatants of different mutants, the extract of plantazolicin, and the direct FZB42 contact on the mortality/survival rate of Bu. xylophilus. An FZB42 mutant deficient in biofilm formation was constructed and its effect on the survival rate of Bu. xylophilus was also determined.[Results] Compared with Landy medium, the supernatant of FZB42 cultured in LB medium had a significant inhibitory effect on Bu. xylophilus. The supernatant collected at the 48^th hour after inoculation was more inhibitory than that collected at the 24^th hour. The inhibitory efficacy improved with the increase of supernatant concentration and treatment time. Using LB culture supernatant collected at the 48^th hour to treat nematodes for 48 h could result in a mortality rate of the nematodes as high as about 50%. The tests with the supernatants of different mutants and the culture extracts showed that plantazolicin, a bacteriocin reported to have a nematocidal activity against Caenorhabditis elegans, had no inhibitory effect on Bu. xylophilus. Direct contact experiments also showed that FZB42 significantly decreased the survival rate of Bu. xylophilus, although the bacterial biofilm formation could enhance Bu. xylophilus resistance to stress. [Conclusion] We demonstrated with laborious but strict assays that Ba. velezensis FZB42 had an inhibitory effect on the pine wood nematode Bu. xylophilus. The molecular basis and mechanism of the inhibitory effect need to be further explored, but it has proved to be not related with plantazolicin. As a group of biocontrol strains with good safety, profound basis research and high degree of development, the potential application value of Ba. velezensis in the control of the pine wood nematode deserves our attention.

Abstract:[Objective] The objective of this study is to clarify the differences of number, type and expression profile of circRNAs between Ascosphaera apis mycelium and spore, and discuss the potential role of common circRNAs, specific circRNAs and differentially expressed circRNAs (DEcircRNAs). [Methods] Based on previously gained high-quality RNA-seq data of A. apis mycelium (AaM) and spore (AaS), circRNAs were predicted using find_circ software. Common circRNAs and specific circRNAs were filtered via Venn analysis. DEcircRNAs of AaM vs AaS were screened following the standard of P ≤ 0.05 and|log₂ fold change|≥ 1. Function and pathway annotation of source genes of circRNAs was done via alignment against GO and KEGG databases. CircRNA-targeted miRNAs and miRNA-targeted mRNAs were predicted using TargetFinder software. Cytoscape software was used to visualize competing endogenous RNA (ceRNA) regulation network. DEcircRNAs were verified by RT-qPCR. [Results] There were 13210156 and 19011000 anchors reads in AaM and AaS, respectively, among them 6124922 and 11392886 ones can be mapped to the reference genome of A. apis. In AaM and AaS groups, 1868 and 2225 circRNA were respectively identified, and the numbers of common circRNAs, specific circRNAs in AaM and specific circRNAs in AaS were 1098, 770 and 1127, respectively. In addition, the length of circRNAs in AaM and AaS was mainly distributed among 1000-2000 nt, and the most abundant circularization type was intergenic circRNA. Further, 456 upregulated circRNAs and 97 downregulated circRNAs were contained in AaM vs AaS comparison group. Source genes of common circRNAs were annotated to 29 functional terms and 14 pathway classifications; source genes of specific circRNAs in AaM were annotated to 31 functional terms and 17 pathway classifications; source genes of specific circRNAs in AaS were annotated to 34 functional terms and 16 pathway classifications; source genes of DEcircRNAs were annotated to 29 functional terms and 40 pathways. ceRNA regulation network analysis showed that 36 common circRNAs can target four miRNAs, further regulating six mRNAs related to endocytosis. Additionally, four (255) specific circRNAs in AaM (AaS) can target two (two) miRNAs, further regulating eight (two) mRNAs related to biosynthesis of secondary metabolites; nine DEcircRNAs can target two DEmiRNAs, further regulating three DEmRNAs associated with MAPK signal pathway. The result of RT-qPCR displayed that the expression trend of 10 DEcircRNAs was consistent with that in the sequencing result, verifying the reliability of our sequencing data. [Conclusion] Common circRNAs, specific circRNAs and DEcircRNAs in A. apis mycelium and spore may regulate material and energy metabolisms, endocytosis, biosynthesis of secondary metabolites, and MAPK signaling pathway through controlling the expression of source genes and serving as ceRNAs, further affecting the mycelium growth, spore germination, and pathogenicity of A. apis.

Abstract:[Objective] To understand the basic characteristics of the genome sequence of the representative strain of Staphylococcus aureus from dairy cows with mastitis in Ningxia, and to further explore its drug-resistant genotype, virulence and evolutionary relationship, to serve as a theoretical basis for veterinary clinical prevention and treatment. [Methods] In total 97 clinical isolates of S. aureus were tested for antimicrobial susceptibility by the paper disk method. Staphylococcal protein A typing and multi-site sequence typing were done at the same time. According to the typing results, 16 representative strains were sequenced and the obtained sequences were analyzed by network database. [Results] Drug susceptibility test shows that 97 strains were resistant to 18 antibacterial drugs, of which 9 strains of methicillin-resistant Staphylococcus aureus (MRSA) were completely resistant to 8 antibacterial drugs, such as penicillin, ampicillin, oxacillin, ceftiofur, sulfisoxazole, erythromycin, gentamicin, and clindamycin. Methicillin-sensitive Staphylococcus aureus (MSSA) strains were highly resistant to penicillin, ampicillin, and sulfisoxazole. Antibiotic resistance genes database (ARDB) annotation analysis shows that 16 representative strains carried 21 drug resistance genes, among which the carrying rates of norA, tet38, bacA, and mepA were higher, correlating with to the results of drug sensitivity test. Virulence Factors Database (VFDB) annotation analysis shows that all strains carried a variety of virulence genes related to diseases such as adhesion, host immune escape, secretion, extracellular enzyme encoding, and iron uptake. MRSA strains all carried more virulence factors, MSSA strains carried different number of virulence factors. The results of gene island prediction show that 16 representative strains had different number of gene islands, and MRSA strains carried more gene islands and virulence gene islands, but the number of drug resistance gene islands was not significantly different from that of MSSA. The results of SNP analysis show that some isolates had higher homology, and the two MRSA with higher homology had little differences in the basic sequence characteristics of the whole genome, both carried similar resistance and virulence genes. [Conclusion] The drug resistance of S. aureus isolates from cattle in Ningxia is serious and highly virulent. Our findings provide a reference for the comparative analysis of genome sequence information of livestock-associated MRSA (LA-MRSA) and MSSA, and the clinical prevention and control of S. aureus infection in Ningxia.

Abstract:Kinamycin gene cluster contains two ketosynthase (KS_α and KS_β) coding genes, namely alpA, alpB and alpR, alpQ, the first two are necessary for the synthesis of kinamycin, and alpR and alpQ are thought to be involved in the synthesis of other compounds.[Objective] Since alpR and alpQ are adjacent to the essential gene alpS in the kinamycin synthesis gene cluster, this study aims to identify their correlation with kinamycin biosynthesis.[Methods] The PCR-targeting multigene knockout was done on the bacterial artificial chromosome library plasmid, and the constructed library plasmid was introduced into Streptomyces albus J1074, a general Streptomyces host. The fermentation products of the mutant and the wild-type strains were detected by high performance liquid chromatography. [Results] AlpR and AlpQ had no direct effect on the synthesis of kinamycin, but the yield of kinamycin increased significantly in ΔalpRQ mutant.[Conclusion] AlpR and AlpQ are likely KS_α and KS_β for the biosynthesis of another type II polyketide compound, competing for common synthetic precursors with AlpA and AlpB. This study also demonstrates that alpR and alpQ cannot complement the function of alpA and alpB.

Abstract:[Objective] This study aims to investigate the differential expression pattern of long non-coding RNAs (lncRNAs) and their regulatory function involved in Apis cerana cerana 6-day-old larval gut response to Ascosphaera apis infection. [Methods] Un-infected and Ascosphaera apis-infected 6-day-old larval guts of Apis cerana cerana (AcCK and AcT) were sequenced using strand-specific cDNA library-based RNA-seq technology. Structural characteristics and expression pattern of lncRNAs were analyzed using related bioinformatic softwares. DElncRNAs were screened followed by investigation of their cis-acting role and competitive endogenous RNA (ceRNA) network. RT-qPCR was conducted to verify the sequencing data and expression pattern of DElncRNAs. [Results] Here, 642 known lncRNAs and 487 novel lncRNAs were identified. Compared with mRNAs, these Apis cerana cerana lncRNAs were shorter in exon and intron length, fewer in exon number and lower in expression level. Additionally, 43 antisense lncRNAs were discovered to have complementary relationship with 40 sense-strand mRNAs. In AcCK vs. AcT comparison group, 367 up-regulated lncRNAs and 268 down-regulated ones were identified. In total, 194 DElncRNAs were found to potentially regulate 461 upstream and downstream genes, which were annotated to 38 functional terms such as cellular process, metabolic process and catalytic activity, as well as 191 pathways including amino acid metabolism, endocytosis and MAPK signaling pathways. Moreover, 180 DElncRNAs can target 50 DEmiRNAs, further regulating 6365 mRNAs; additionally, complex regulatory networks existed among them. [Conclusion] These results demonstrated that partial antisense lncRNAs may participate in host response to Ascosphaera apis infection; some DElncRNAs may regulate upstream and downstream genes relative to material metabolism and immune-associated pathways, thus mediating host Ascosphaera apis-response; a portion of DElncRNAs including TCONS_00010661 and TCONS_00003104 were likely to regulate Jak-STAT signaling pathway and oxidative phosphorylation and corresponding enriched genes via ceRNA networks, further participating in the response of host to Ascosphaera apis invasion.

Abstract:[Objective] To investigate the effect of mediator subunit Med15B mediated fatty acid metabolism on Candida glabrata low pH tolerance. [Methods] Overexpression of genes elo1 and ole1, encoding fatty acid elongase and Δ9 desaturase, in strain Δmed15B we obtained an overexpressed strain Δmed15B/elo1-ole1. The expression level of genes elo1 and ole1, the membrane fatty acid composition, the membrane integrity and stress tolerance of parent strain ATCC55, strain Δmed15B and overexpression strain Δmed15B/elo1-ole1 were determined and compared at pH 2.0 and pH 6.0. [Results] At pH 2.0, the expression level of genes elo1 and ole1 in overexpression strain Δmed15B/elo1-ole1 was increased by 4.1-fold and 3.3-fold compared to those of strain Δmed15B, and increased by 2.5-fold and 2.2-fold compared to those of parent strain ATCC55, respectively. The average acyl-chain length of overexpression strain Δmed15B/elo1-ole1 reached 17.4 longer than that of the deletion strain (16.8) and parent strain (17.1), and the ratio of unsaturated fatty acid to saturated fatty acid (UFA/SFA ratio) increased by 53.1% and 41.5%, respectively. Furthermore, the percentage of propidium iodide (PI)-stained cell in overexpression strain Δmed15B/elo1-ole1 was decreased by 60.8% and 37.7%, respectively, compared to that of strain Δmed15B and parent strain ATCC55. Similarly, overexpression strain Δmed15B/elo1-ole1 exhibited more tolerance to low pH and the half-maximal inhibition (IC₅₀) values for pH stress was reached pH 2.3, which was pH 3.7 of strain Δmed15B and pH 3.1 of parent strain ATCC55. [Conclusion] These results indicated that overexpression genes elo1 and ole1 in strain Δmed15B can enhance cell tolerance to low pH by increasing the content of long chain and unsaturated fatty acids.