Virus-host coevolutionary analyses of an Alphabaculovirus with a wide host range.

Baculoviruses are highly host specific, and their host range is usually restricted to a single or a few closely related insect species, except for few virus species, e.g. Alphabaculovirus aucalifonicae and Alphabaculovirus mabrassicae. In this study, two new alphabaculovirus isolates were isolated from the larvae of Mamestra brassicae and Mythimna separata, which were named as Mamestra brassicae multiple nucleopolyhedrovirus isolate QD (MbMNPV-QD) and Mythimna separata multiple nucleopolyhedrovirus isolate Hb (MyseMNPV-Hb), respectively. The Kimura two-parameter values based on the concatenated 38 core genes of baculovirus revealed that MbMNPV (isolates QD/CHb1/K1/CTa), MyseMNPV-Hb, Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) and Mamestra configurata nucleopolyhedrovirus B (MacoNPV-B) were different isolates of a same virus species. A phylogenetic tree of baculoviruses and nudiviruses constructed from their 20 homologous gene sequences, and that of their isolated hosts constructed from 13 protein-coding genes of the insect mitochondrial genomes, were used to analyse the coevolution of baculoviruses with their isolated hosts. The results showed that M. brassicae was the most likely ancestral host of these virus isolates, included MbMNPV isolates, MyseMNPV-Hb, HearMNPV, and MacoNPV-B. Therefore, we concluded that these virus isolates belong to the existing virus species - Alphabaculovirus mabrassicae with M. brassicae as their ancestral host.

MUC4 O-GlcNAcylation Regulates the Epithelial Phenotype in Conjunctival Epithelial Cells.

In the present study, our aim was to explore the role of MUC4 in IL-4-stimulated conjunctival epithelial cells and the underlying mechanisms. Human recombinant IL-4 was employed in human conjunctival epithelial cells (HConEpic) cells, and MUC4 shRNA (sh-MUC4) was constructed to explore the functional role of MUC4. The protein level of MUC4, O-GlcNAc transferase (OGT), O-GlcNAc hydrolase (OGA), zonula occludens 1 (ZO-1), gap junction protein beta 2 (GJB2), claudin-8 (CLDN8), and E-cadherin were detected by Western blot in HConEpic cells, the interaction between MUC4 and OGT/OGA was assessed by co-immunoprecipitation (IP) and Western blot in 293T cells. Our results showed that IL-4 significantly up-regulated MUC4 and OGT protein levels in HConEpic cells, while down-regulated OGA protein level. Also, IL-4 down-regulated ZO-1, GJB2, CLDN8, and E-cadherin protein levels in HConEpic cells, while which was markedly reversed by sh-MUC4. Additionally, OGT inhibitor significantly reduced MUC4 protein level, and elevated ZO-1, GJB2, CLDN8, and E-cadherin protein levels in HConEpic cells, while OGA inhibitor resulted in the opposite results. Furthermore, in addition to the interaction between OGT/OGA and MUC4, Co-IP and Western blot also revealed the alteration of MUC4 O-GlcNAcylation in 293T cells treated with OGT/OGA inhibitor. Above findings suggested that OGT/OGA inhibitor regulated MUC4 protein level by affecting MUC4 O-GlcNAcylation to regulate ZO-1, GJB2, CLDN8, and E-cadherin protein levels in HConEpic cells, which was achieved via inhibiting the interaction between OGT/OGA and MUC4. This study may provide a better understanding of the pathogenesis of allergic conjunctivitis (AC).

Transcriptome and the gut microbiome analysis of the impacts of Brucella abortus oral infection in BALB/c mice.

Brucellosis is a zoonotic infectious disease caused by Brucella spp, which could cause serious economic losses to animal husbandry and threaten human public health. Ingestion of contaminated animal products is a common way to acquire Brucella infection in humans, while research on effect of oral Brucella infection on host gut microbiota and the gene expression in intestinal tissues is limited. In the present study, 16S rRNA sequencing and RNA sequencing were conducted to explore gut microbiota and expression profiles of mRNAs in the colon of BALB/c mice, which were infected by Brucella abortus 2308. The fecal samples were collected at 7 and 28 days post infection to observe changes in the gut microbiota during Brucella infection. In the alpha diversity analysis, significantly increased Chao 1 index was observed at 28 days after Brucella infection. The Bray-Curtis distancebased principal coordinate analysis indicated that the WT group showed a separation from the Brucella infection groups. In addition, analysis of composition of microbes revealed that Prevotellaceae_NK3B31_group were more abundant in 1 week and 4 week infection groups, while Turicibacter was only more abundant in 4 week infection group. Based on the RNA-seq assay, a total of 45 differentially expressed genes were detected between Brucella abortus infection group and control group. Furthermore, KEGG pathway enrichment analysis showed that protein processing in endoplasmic reticulum, Legionellosis, Spliceosome, Hippo signaling pathway and Influenza A were significantly enriched in response to Brucella abortus infection. Our finding will help to improve the knowledge of the mechanisms underlying Brucella infection and may provide novel targets for future treatment of this pathogen infection.

From methane to value-added bioproducts: microbial metabolism, enzymes, and metabolic engineering.

Methane is abundant in nature, and excessive emissions will cause the greenhouse effect. Methane is also an ideal carbon and energy feedstock for biosynthesis. In the review, the microorganisms, metabolism, and enzymes for methane utilization, and the advances of conversion to value-added bioproducts were summarized. First, the physiological characteristics, classification, and methane oxidation process of methanotrophs were introduced. The metabolic pathways for methane utilization and key intermediate metabolites of native and synthetic methanotrophs were summarized. Second, the enzymatic properties, crystal structures, and catalytic mechanisms of methane-oxidizing and metabolizing enzymes in methanotrophs were described. Third, challenges and prospects in metabolic pathways and enzymatic catalysis for methane utilization and conversion to value-added bioproducts were discussed. Finally, metabolic engineering of microorganisms for methane biooxidation and bioproducts synthesis based on different pathways were summarized. Understanding the metabolism and challenges of microbial methane utilization will provide insights into possible strategies for efficient methane-based synthesis.

ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus.

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

Comparative study of different sewage sludge incineration treatments based on environmental and economic life cycle assessment.

Incineration is one of the most widely used treatments in the field of sewage sludge disposal. However, the choice of sewage sludge incineration process is still controversial. In this study, the comparative life cycle assessment of sewage sludge incineration processes, including the mono-incineration, co-incineration in coal-fired power plants and co-incineration in municipal solid waste (MSW) incineration plants, was carried out from the perspective of environment, carbon footprint and economy. The environmental assessment results show that terrestrial ecotoxicity, freshwater ecotoxicity, marine ecotoxicity, human carcinogenic toxicity and human non-carcinogenic toxicity are the most significant environmental impacts. And the environmental performance of co-incineration in coal-fired power plants is the best. Moreover, the environmental impact is most sensitive to the dehydrant, electricity and fly ash chelating agent. Co-incineration in MSW incineration plants has the lowest carbon emissions, with only 70.50% and 82% of the carbon emissions from mono-incineration and co-incineration in coal-fired power plants, respectively. Furthermore, sewage sludge mono-incineration has the highest disposal costs because of the higher depreciation and solid waste disposal costs. The comprehensive evaluation results reveal that the optimization should focus on the selection of dehydrant and fly ash chelating agent, as well as the improvement of the equipment efficiency.

Genomic analysis of two Chinese isolates of hyphantria cunea nucleopolyhedrovirus reveals a novel species of alphabaculovirus that infects hyphantria cunea drury (lepidoptera: arctiidae).

BACKGROUND: Baculoviruses act as effective biological control agents against the invasive pest Hyphantria cunea Drury. In this study, two Chinese Hyphantria cunea nucleopolyhedrovirus (HycuNPV) isolates, HycuNPV-BJ and HycuNPV-HB, were deep sequenced and compared with the Japanese isolate, HycuNPV-N9, to determine whole-genome level diversity and evolutionary history. RESULTS: The divergence of the phylogenetic tree and the K2P distances based on 38 core-gene concatenated alignment revealed that two Chinese HycuNPV isolates were a novel species of Alphabaculovirus that infected Hyphantria cunea in China. The gene contents indicated significant differences in the HycuNPV genomes between the Chinese and Japanese isolates. The differences included gene deletions, acquisitions and structural transversions, but the main difference was the high number of single nucleotide polymorphisms (SNPs). In total, 10,393 SNPs, corresponding to approximately 8% of the entire HycuNPV-N9 genome sequence, were detected in the aligned reads. By analyzing non-synonymous variants, we found that hotspot mutation-containing genes had mainly unknown functions and most were early expressing genes. We found that the hycu78 gene which had early and late promoter was under positive selection. Biological activity assays revealed that the infectivity of HycuNPV-HB was greater than that of HycuNPV-BJ, and the killing speed of HycuNPV-HB was faster than that of HycuNPV-BJ. A comparison of molecular genetic characteristics indicated that the virulence differences between the two isolates were affected by SNP and structural variants, especially the homologous repeat regions. CONCLUSIONS: The genomes of the two Chinese HycuNPV isolates were characterized, they belonged to a novel species of Alphabaculovirus that infected Hyphantria cunea in China. We inferred that the loss or gain of genetic material in the HycuNPV-HB and HycuNPV-BJ genomes resulted in new important adaptive capabilities to the H. cunea host. These results extend the current understanding of the genetic diversity of HycuNPV and will be useful for improving the applicability of this virus as a biological control agent.

The Overexpression of Phasin and Regulator Genes Promoting the Synthesis of Polyhydroxybutyrate in Cupriavidus necator H16 under Nonstress Conditions.

Cupriavidus necator H16 is an ideal strain for polyhydroxybutyrate (PHB) production from CO2. Low-oxygen stress can induce PHB synthesis in C. necator H16 while reducing bacterial growth under chemoautotrophic culture. The optimum growth and PHB synthesis of C. necator H16 cannot be achieved simultaneously, which restricts PHB production. The present study was initiated to address the issue through comparative transcriptome and gene function analysis. First, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen stress and nonstress conditions was studied. Three types of genes were discovered to have differential levels of transcription: those involving PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen stress conditions, acetoacetyl-coenzyme A (CoA) reductase gene phaB2, PHB synthase gene phaC2, phasins genes phaP1 and phaP2, and regulator genes uspA and rpoN were upregulated 3.0-, 2.5-, 1.8-, 2.7-, 3.5-, and 1.6-fold, respectively. Second, the functions of upregulated genes and their applications in PHB synthesis were further studied. It was found that the overexpression of phaP1, phaP2, uspA, and rpoN can induce PHB synthesis under nonstress conditions, while phaB2 and phaC2 have no significant effect. Under the optimum conditions, the PHB percentage content in C. necator H16 was increased by 37.2%, 28.4%, 15.8%, and 41.0%, respectively, with overexpression of phaP1, phaP2, uspA, and rpoN, and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5%, respectively, under nonstress chemoautotrophic conditions. Similar promotion by phaP1, phaP2, uspA, and rpoN was observed in heterotrophically cultured C. necator H16. The PHB percentage content and PHB production were increased by 54.4% and 103.1%, respectively, with the overexpression of rpoN under nonstress heterotrophic conditions. IMPORTANCE Microbial fixation of CO2 is an effective way to reduce greenhouse gases. Some microbes, such as C. necator H16, usually accumulate PHB when they grow under stress. Low-oxygen stress can induce PHB synthesis when C. necator H16 is autotrophically cultured with CO2, H2, and O2, while under stress, growth is restricted, and total PHB yield is reduced. Achieving the optimal bacterial growth and PHB synthesis at the same time is an ideal condition for transforming CO2 into PHB by C. necator H16. The present study was initiated to clarify the molecular basis of low-oxygen stress promoting PHB accumulation and to realize the optimal PHB production by C. necator H16. Genes upregulated under nonstress conditions were identified through comparative transcriptome analysis and overexpression of phasin, and regulator genes were demonstrated to promote PHB synthesis in C. necator H16.

Pal Affects the Proliferation in Macrophages and Virulence of Brucella, and as Mucosal Adjuvants, Provides an Effective Protection to Mice Against Salmonella Enteritidis.

The purpose of this study was to elucidate the roles of peptidoglycan-associated lipoprotein (Pal protein) in the proliferation of Brucella in macrophage and bacterial virulence, and to evaluate the immune effect of Pal protein to Salmonella enteritidis. Murine macrophage-like cell line Raw264.7 was stimulated by recombinant Pal protein, and the expression of TNF-α and IFN-γ were up-regulated, but not it of IL-1ß and IL-6. The macrophages infection and in vitro simulated stress assays showed that deletion of pal gene reduced the proliferation of Brucella in macrophages, the survival in acidic, oxidative and polymyxin B-contained environment. The mice infection assay showed that mice challenged with the pal mutant strain were found to have more severe splenomegaly, but less bacterial load. After oral immunization of mice, Pal protein induced a higher titer of mucosal and humoral antibody (IgA and IgG) against heat-killed Salmonella enteritidis, and a stronger Th1 cellular immune response. The challengte experiments showed Pal protein elevated the survival rate and reduced the bacterial load of spleens in immunized mice. In conclusion, our results revealed the important roles of pal gene in Brucella virulence, and Pal protein was a potentially valuable adjuvant against mucosal pathogens, such as Salmonella enteritidis.

The Pseudogene BMEA_B0173 Deficiency in Brucella melitensis Contributes to M-epitope Formation and Potentiates Virulence in a Mice Infection Model.

It is widely acknowledged that pseudogenes play important roles in bacterial diversification and evolution and participate in gene regulation and RNA interference (RNAi). However, the function of most pseudogenes in Brucella spp remains poorly understood, warranting further studies.To comprehensively analyze the function of the pseudogenes BMEA_B0173 in Brucella melitensis strain 63/9, a BMEA_B0173 in-frame deleted mutant strain was constructed. Then, the phenotypes of the mutant strain, such as growth characteristics and bacterial virulence, were assessed in mice infection models. Finally, iTRAQ analysis was performed to investigate the gene expression profile affected by the pseudogenes BMEA_B0173. In this study, we found that BMEA_B0173 deletion exhibited increased agglutination with M monospecific sera. In a mouse model of chronic infection, the BMEA_B0173 deletion strain displayed increased colonization in the spleen compared to the wild-type pathogen. The iTRAQ assay revealed that 252 proteins were differentially expressed between the BMEA_B0173 deletion and the wild-type strains. In addition, deletion of BMEA_B0173 significantly increased the expression of proteins involved in the denitrification pathway, iron metabolism, and several transcriptional regulators, which might cause increased virulence of the mutant strain. In conclusion, this study preliminary uncovered the function of the pseudogene BMEA_B0173 in Brucella melitensis 63/9 and provided novel insights for studying the pathogenesis of Brucella strains.

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

Identification of four genes and biological characteristics of esophageal squamous cell carcinoma by integrated bioinformatics analysis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) has become one of the most serious diseases affecting populations worldwide and is the primary subtype of esophageal cancer (EC). However, the molecular mechanisms governing the development of ESCC have not been fully elucidated. METHODS: The robust rank aggregation method was performed to identify the differentially expressed genes (DEGs) in six datasets (GSE17351, GSE20347, GSE23400, GSE26886, GSE38129 and GSE77861) from the Gene Expression Omnibus (GEO). The Search Tool for the Retrieval of Interacting Genes (STRING) database was utilized to extract four hub genes from the protein-protein interaction (PPI) network. Module analysis and disease free survival analysis of the four hub genes were performed by Cytoscape and GEPIA. The expression of hub genes was analyzed by GEPIA and the Oncomine database and verified by real-time quantitative PCR (qRT-PCR). RESULTS: In total, 720 DEGs were identified in the present study; these genes consisted of 302 upregulated genes and 418 downregulated genes that were significantly enriched in the cellular component of the extracellular matrix part followed by the biological process of the cell cycle phase and nuclear division. The primary enriched pathways were hsa04110:Cell cycle and hsa03030:DNA replication. Four hub genes were screened out, namely, SPP1, MMP12, COL10A1 and COL5A2. These hub genes all exhibited notably increased expression in ESCC samples compared with normal samples, and ESCC patients with upregulation of all four hub genes exhibited worse disease free survival. CONCLUSIONS: SPP1, MMP12, COL10A1 and COL5A2 may participate in the tumorigenesis of ESCC and demonstrate the potential to serve as molecular biomarkers in the early diagnosis of ESCC. This study may help to elucidate the molecular mechanisms governing ESCC and facilitate the selection of targets for early treatment and diagnosis.

Wide-bound salt tolerance of the inocula from marine sediment and their specific microbial community.

The microorganisms in marine sediment are promising candidates for the treatment of the saline wastes due to their property of salt tolerance. However, the knowledge about the microbial community and property of the marine sediments is still limited. In the present study, the salt tolerance of the microorganisms in the marine sediment that was collected from a marine fish farm was investigated by being used as inoculum for anaerobic digestion. The microbial communities were analyzed by high-throughput sequencing. The inoculum from the wastewater plant (IWTP) was taken as a control. The inoculum from the marine sediment (IMS) showed excellent capacity for anaerobic digestion at salinities of 0.3%-6%. Even at a salinity of 9%, the methane yield remained 60% of the highest yield. IMS provides promising microbial resources for the treatment of both fresh-water and saliferous organic wastes. While the IWTP was sensitive to salt, the methane yield decreased to 56% of the highest yield at the salinity of 3%. The bacterial taxonomic richness of IMS was about half of that in IWTP. Eighty-one genera were identified only in IWTP but not in IMS. The IMS possessed fewer bacterial members related to the nitrogen cycle than IWTP, but more members related to the sulfur cycle. The members of animal parasites or symbionts in IMS were significantly fewer than those in IWTP. The archaeal compositions of IMS and IWTP were different. The relative abundance of the unidentified archaea in IMS was much higher than that in IWTP with 12.52% vs 0.06% at phylum level. The findings of this work expand our understanding of the microorganisms in marine sediments and will promote the application of them in waste treatment.

Postoperative drain after transoral endoscopic thyroidectomy vestibular approach (TOETVA) with single incision.

OBJECTIVE: To find the optimal size of a drain for the reliable drainage and the best cosmetic result in TOETVA. To explore the normal drainage flow rate after TOETVA. METHODS: A prospective randomized controlled trial was performed in a single center from December 2016 to December 2018. One hundred and fifty-three (153) patients had TOETVA with a single incision and were randomly divided into two groups. Self-made drainage tubes with a small diameter (outer diameter 2.0 mm, inner diameter 1.0 mm) were used in 80 patients (experimental group). No. 8 tubes were used in 73 patients (control group). The clinical characteristics and results between both groups were compared by t test or chi-square test, and the results of normal drainage flow rate were calculated. RESULTS: The experimental group had a longer intraoperative tube-inserting time, compared with the control group (9.5 ± 2.5 min vs. 5.6 ± 1.4 min, p = 0.001), a smaller scar six months after the operation (1.8 ± 2.3 mm vs. 3.1 ± 2.6 mm, p = 0.002), and a lower Vancouver Scar Scale score at both one month (3.20 ± 1.44 vs. 4.19 ± 1.92, p = 0.001) and six months after the operation(1.43 ± 1.84 vs. 2.40 ± 2.37, p = 0.006). The drainage volume, pain score on the first day, postoperative complications (tube blockage, air leakage, subcutaneous hydrops, hematoma, regional infection), and the extubation time were not significantly different. The average drainage of 148 patients without postoperative complications was 78.3 ± 10.9 ml. The cumulative drainage within 8 h, and 32 h after the operation accounted for 53.2% and 91.9% of the total drainage, respectively. The residual drainage at 32 h was estimated to be 6.5 ± 2.9 ml (P95 = 11.0 ml). A linear regression equation between total volume (Vt) and the size of resected tissue (S) was established: Vt = 1.625 S + 56.604 (p = 0.0001). CONCLUSION: In TOETVA, a small drain can provide a good cosmetic appearance and reliable drainage. The main exudation period of the wound is within 8 h after the operation. If a residual volume of less than 11 ml is considered to be self-absorbable, the shortest safe extubation point for 95% of patients should be 32 h after the operation.

Identification and validation of immune-related lncRNA prognostic signature for breast cancer.

The prognosis of patients with breast cancer is closely related to both the infiltration of immune cells and the expression of lncRNAs. In this study, we evaluated the infiltration of immune cells in 1109 breast cancer samples obtained from TCGA by applying the ssGSEA to the transcriptome of these samples, thereby generating high immune cell infiltration group and low immune cell infiltration group. On the basis of these groupings, we found 696 differentially expressed lncRNAs which were sequentially subjected to univariate Cox regression and stepwise multiple Cox regression analysis. 11 lncRNAs were identified as prognostic signature for breast cancer. Kaplan-Meier analysis, univariate Cox regression, multivariate Cox regression, and ROC analyses further revealed that this 11-lncRNA signature was a novel and important prognostic factor independent of multiple clinicopathological parameters. The TIMER database showed that this 11-lncRNA prognostic signature for breast cancer was associated with the infiltration of immune cell subtypes.

Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice.

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health. However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging. A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA. Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.

Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions.

The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) in C. necator H16, while the growth of which is restricted. Under autotrophic culture using carbon dioxide, hydrogen, and oxygen as substrates, the oxygen concentration below 6.9% (v/v) in the mixture is considered as a safe condition. It also expected to achieve cell rapid growth and large accumulation of PHB simultaneously under the oxygen-limiting condition in C. necator H16. In this study, a metabolically engineered strain capable of both rapid growth and large accumulation of PHB under oxygen-limiting conditions was constructed based on the transcriptomic analysis. In the comparative transcriptomic analysis, the genes related to energy-generating of C. necator H16 at autotrophic culture were downregulated under oxygen-limiting conditions (3%, v/v). Besides, the genes related to the key intermediates (pyruvate and acetyl-CoA) metabolism in PHB biosynthetic pathway were analyzed. Most of which were downregulated, except the genes ldh, iclA, and ackA2 respectively encoding L-lactate dehydrogenase, isocitrate lyase, and acetate kinase were upregulated under oxygen-limiting conditions (3%, v/v). The Vitreoscilla hemoglobin (VHb) has the ability to promote aerobic metabolism and energy generation. To promote the bacterium growth and improve the energy generation in C. necator H16 under oxygen-limiting conditions, the VHb gene was introduced into C. necator H16 with the optimized promoter PphaC1-j5. Moreover, VHb was localized to the periplasmic space of the bacterium by the traction of membrane-bound hydrogenase (MBH) signal peptide. By optimizing the knockout of different genes, it was found that knockout of ldh can improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression of vgb and ldh knockout in C. necator H16. Compared with the control (Reh (p2)) under oxygen-limiting conditions (3%, v/v), the dry cell weight (DCW), PHB content, and PHB production of Reh01 (p2M-pj-v) increased by 31.0%, 30.9%, and 71.5%, respectively. From the perspectives of transcriptome and metabolic engineering, the work provides new ideas to achieve rapid cell growth and large PHB accumulation in C. necator under oxygen-limiting and autotrophic conditions.

Intestinal Flora Dysbiosis Aggravates Cognitive Dysfunction Associated With Neuroinflammation in Heart Failure.

BACKGROUND: Cognitive dysfunction after heart failure (HF) is characterized by neuroinflammation, which plays an important role in the occurrence and development of cognitive dysfunction. Recent studies have shown that an intestinal flora imbalance may also trigger neuroinflammation in Alzheimer's disease. The present study was designed to reveal that intestinal flora dysbiosis caused by HF aggravates neuroinflammation-associated cognitive impairment. METHODS AND RESULTS: Adult male Sprague-Dawley rats were fed daily for 2 weeks with probiotics or placebo until the day of surgery. HF was then triggered by 8 weeks of sustained coronary artery occlusion. 16S rDNA sequencing was used to confirm intestinal flora dysbiosis after HF and demonstrate that the changes paralleled intestinal pathology scores. The permeability of the blood-brain barrier was increased after HF, and such an increase in permeability may increase the levels of inflammatory cytokines caused by intestinal flora disorders. The changes in the intestinal flora caused by probiotics significantly reduced the level of neuroinflammation. In addition, probiotic administration considerably improved the impaired spatial memory in HF rats. CONCLUSIONS: We conclude that intestinal flora dysbiosis plays a potential role in aggravating the impaired cognition associated with neuroinflammation and that these effects may be attenuated by probiotics.

Deletion of the LuxR-type regulator VjbR in Brucella canis affects expression of type IV secretion system and bacterial virulence, and the mutant strain confers protection against Brucella canis challenge in mice.

Brucella spp. are facultative intracellular pathogens and zoonotic agents which pose a huge threat to human health and animal husbandry. The B. melitensis, B. abortus, and B. suis cause undulant fever and influenza-like symptoms in humans. However, the effects of B. canis have not been extensively studied. The quorum sensing-dependent transcriptional regulator VjbR influences the Brucella virulence in smooth type Brucella strains, such as B. melitensis, B. abortus and rough type Brucella ovis. However, the function of VjbR in the rough-type B. canis is unknown. In the present study, we discovered that deletion of this regulator significantly affected Brucella virulence in macrophage and mice infection models. The expression levels of virB operon and the ftcR gene were significantly altered in the vjbR mutant strain. We further investigated the protective effect of different doses of the vjbR mutant in mice and the results indicated that VjbR conferred protection against the virulent B. canis strain. This study presents the first evidence that the transcriptional regulator VjbR has important function in B. canis. In addition, according to its reduced virulence and the protective immunity it induces in mice, it can be a potential live attenuated vaccine against B. canis.

The role of Caspase-1/GSDMD-mediated pyroptosis in Taxol-induced cell death and a Taxol-resistant phenotype in nasopharyngeal carcinoma regulated by autophagy.

Taxol has been widely used as a first-line chemotherapeutic agent for the treatment of advanced nasopharyngeal carcinoma (NPC). However, acquired drug resistance has caused great difficulties in clinical treatment. Pyroptosis is a newly discovered programmed cell death pathway, and Caspase-1 and gasdermin D (GSDMD) play key roles in driving canonical pyroptosis. Increasing evidence suggests that pyroptosis is associated with the development of cancer; however, the function and mechanism of pyroptosis in NPC remain obscure. In this study, we observed that Taxol treatment caused pyroptotic cell death, along with activation of Caspase-1 and maturation of IL-1ß, as well as cleavage of GSDMD, which is the canonical pyroptosis executor. Furthermore, Taxol-induced pyroptotic cell death could be suppressed by Caspase-1 inhibitor (Z-YVAD-FMK) and GSDMD knockout. Moreover, NPC parental cells demonstrated higher levels of pyroptosis than Taxol-resistant cells, and pyroptosis mediated by Caspase-1/GSDMD suppression induced by a Caspase-1 inhibitor and GSDMD knockout could induce a Taxol-resistant phenotype in vitro and in vivo. By transfecting an siRNA targeting Beclin-1 into NPC Taxol-resistant cells, we discovered that autophagy could negatively regulate pyroptosis by inhibiting Caspase-1/GSDMD activation. Taken together, our results indicated that Caspase-1/GSDMD mediated Taxol-induced pyroptosis and a Taxol-resistant phenotype in NPC cell lines, which may be regulated by autophagy. Thus, we provide novel insight into the mechanisms of Taxol-induced cell death and a promising approach to improve the therapeutic outcomes of patients with advanced NPC.