Differential gene expression and immune cell infiltration in maedi-visna virus-infected lung tissues.

BACKGROUND: Maedi-visna virus (MVV) is a lentivirus that infects monocyte/macrophage lineage cells in sheep, goats, and wild ruminants and causes pneumonia, mastitis, arthritis, and encephalitis. The immune response to MVV infection is complex, and a complete understanding of its infection and pathogenesis is lacking. This study investigated the in vivo transcriptomic patterns of lung tissues in sheep exposed to MVV using the RNA sequencing technology. RESULT: The results indicated that 2,739 genes were significantly differentially expressed, with 1,643 downregulated genes and 1,096 upregulated genes. Many variables that could be unique to MVV infections were discovered. Gene Ontology analysis revealed that a significant proportion of genes was enriched in terms directly related to the immune system and biological responses to viral infections. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the most enriched pathways were related to virus-host cell interactions and inflammatory responses. Numerous immune-related genes, including those encoding several cytokines and interferon regulatory factors, were identified in the protein-protein interaction network of differentially expressed genes (DEGs). The expression of DEGs was evaluated using real-time polymerase chain reaction and western blot analysis. CXCL13, CXCL6, CXCL11, CCR1, CXCL8, CXCL9, CXCL10, TNFSF8, TNFRSF8, IL7R, IFN-γ, CCL2, and MMP9 were upregulated. Immunohistochemical analysis was performed to identify the types of immune cells that infiltrated MVV-infected tissues. B cells, CD4+ and CD8+ T cells, and macrophages were the most prevalent immune cells correlated with MVV infection in the lungs. CONCLUSION: Overall, the findings of this study provide a comprehensive understanding of the in vivo host response to MVV infection and offer new perspectives on the gene regulatory networks that underlie pathogenesis in natural hosts.

326K at E Protein Is Critical for Mammalian Adaption of TMUV.

Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses to the poultry industry in China since 2010. However, the potential threat of TMUV to mammals has not been well studied. In this study, a TMUV HB strain isolated from diseased ducks showed high virulence in BALB/c mice inoculated intranasally compared with the reference duck TMUV strain. Further studies revealed that the olfactory epithelium is one pathway for the TMUV HB strain to invade the central nervous system of mice. Genetic analysis revealed that the TMUV HB virus contains two unique residues in E and NS3 proteins (326K and 519T) compared with duck TMUV reference strains. K326E substitution weakens the neuroinvasiveness and neurovirulence of TMUV HB in mice. Remarkably, the TMUV HB strain induced significantly higher levels of IL-1ß, IL-6, IL-8, and interferon (IFN)-α/ß than mutant virus with K326E substitution in the brain tissue of the infected mice, which suggested that TMUV HB caused more severe inflammation in the mouse brains. Moreover, application of IFN-ß to infected mouse brain exacerbated the disease, indicating that overstimulated IFN response in the brain is harmful to mice upon TMUV infection. Further studies showed that TMUV HB upregulated RIG-I and IRF7 more significantly than mutant virus containing the K326E mutation in mouse brain, which suggested that HB stimulated the IFN response through the RIG-I-IRF7 pathway. Our findings provide insights into the pathogenesis and potential risk of TMUV to mammals.

Epidemiology of Cardiopulmonary Arrest and Outcome of Resuscitation in PICU Across China: A Prospective Multicenter Cohort Study.

Objective: To investigate the epidemiology and the effectiveness of resuscitation from cardiopulmonary arrest (CPA) among critically ill children and adolescents during pediatric intensive care unit (PICU) stay across China. Methods: A prospective multicenter study was conducted in 11 PICUs in tertiary hospitals. Consecutively hospitalized critically ill children, from 29-day old to 18-year old, who had suffered from CPA and required cardiopulmonary resuscitation (CPR) in the PICU were enrolled (December 2017-October 2018). Data were collected and analyzed using the "in-hospital Utstein style." Neurological outcome was assessed with the Pediatric Cerebral Performance Category (PCPC) scale among children who had survived. Factors associated with the return of spontaneous circulation (ROSC) and survival at discharge were evaluated using multivariate logistic regression. Results: Among 11,599 admissions to PICU, 372 children (3.2%) had CPA during their stay; 281 (75.5%) received CPR, and 91 (24.5%) did not (due to an order of "Do Not Resuscitate" requested by their guardians). Cardiopulmonary disease was the most common reason for CPA (28.1% respiratory and 19.6% circulatory). The most frequent initial dysrhythmia was bradycardia (79%). In total, 170 (60.3%) of the total children had an ROSC, 91 had (37.4%) survived till hospital discharge, 28 (11.5%) had survived 6 months, and 19 (7.8%) had survived for 1 year after discharge. Among the 91 children who were viable at discharge, 47.2% (43/91) received a good PCPC score (1-3). The regression analysis results revealed that the duration of CPR and the dose of epinephrine were significantly associated with ROSC, while the duration of CPR, number of CPR attempts, ventricular tachycardia/ventricular fibrillation (VT/VF), and the dose of epinephrine were significantly associated with survival at discharge. Conclusion: The prevalence of CPA in critically ill children and adolescents is relatively high in China. The duration of CPR and the dose of epinephrine are associated with ROSC. The long-term prognosis of children who had survived after CPR needs further improvement.

A programmable high-expression yeast platform responsive to user-defined signals.

Rapidly growing yeasts with appropriate posttranslational modifications are favored hosts for protein production in the biopharmaceutical industry. However, limited production capacity and intricate transcription regulation restrict their application and adaptability. Here, we describe a programmable high-expression yeast platform, SynPic-X, which responds to defined signals and is broadly applicable. We demonstrated that a synthetic improved transcriptional signal amplification device (iTSAD) with a bacterial-yeast transactivator and bacterial-yeast promoter markedly increased expression capacity in Pichia pastoris. CRISPR activation and interference devices were designed to strictly regulate iTSAD in response to defined signals. Engineered switches were then constructed to exemplify the response of SynPic-X to exogenous signals. Expression of α-amylase by SynPic-R, a specific SynPic-X, in a bioreactor proved a methanol-free high-production process of recombinant protein. Our SynPic-X platform provides opportunities for protein production in customizable yeast hosts with high expression and regulatory flexibility.

Study on Preparation and Grinding Performance of Vitrified Bond CBN Grinding Wheel with Controllable Porosity.

Vitrified bond cubic boron nitride (CBN) grinding wheel specimens with controllable porosity were prepared by regulating the pore former dextrin content and varying the forming pressure, and the performance of the grinding camshaft was studied. The porosity of the specimens increases with the increase in dextrin content, and decreases first and then increases with the increase in the forming pressure. The grinding experiments show that the dextrin content is negatively correlated with the grinding force and grinding temperature, while the grinding force and grinding temperature of the specimens increase and then decrease with the increase in the forming pressure. When we observe and measure the grinding surface of the specimen and workpiece, we see that the surface roughness of the specimen after grinding is smaller than that before grinding. In addition, the greater the porosity of the specimen, the rougher the surface of the workpiece after grinding.

Next-generation sequencing for the genetic characterization of Maedi/Visna virus isolated from the northwest of China.

BACKGROUND: Maedi/Visna virus (MVV) is a contagious viral pathogen that causes considerable economic losses to the sheep industry worldwide. OBJECTIVES: In China, MVV has been detected in several regions, but its molecular characteristics and genetic variations were not thoroughly investigated. METHODS: Therefore, in this study, we conducted next-generation sequencing on an MVV strain obtained from northwest China to reveal its genetic evolution via phylogenetic analysis. RESULTS: A MVV strain obtained from Inner Mongolia (NM) of China was identified. Sequence analysis indicated that its whole-genome length is 9193 bp. Homology comparison of nucleotides between the NM strain and reference strains showed that the sequence homology of gag and env were 77.1%-86.8% and 67.7%-75.5%, respectively. Phylogenetic analysis revealed that the NM strain was closely related to the reference strains isolated from America, which belong to the A2 type. Notably, there were 5 amino acid insertions in variable region 4 and a highly variable motif at the C-terminal of the surface glycoprotein (SU5). CONCLUSIONS: The present study is the first to show the whole-genome sequence of an MVV obtained from China. The detailed analyses provide essential information for understanding the genetic characteristics of MVV, and the results enrich the MVV library.

[Association of non-thyroidal illness syndrome with interleukin-6 and interleukin-10 in critically ill children with sepsis].

OBJECTIVE: To study the incidence rate of non-thyroidal illness syndrome (NTIS) in critically ill children with or without sepsis and the association of NTIS with interleukin-6 (IL-6) and interleukin-10 (IL-10). METHODS: A retrospective analysis was performed on the medical data of 97 children with sepsis (sepsis group) and 80 non-sepsis children with bacterial infection (non-sepsis group). The correlations of IL-6 and IL-10 with the thyroid function parameters triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) were analyzed. RESULTS: There were no significant differences in age and sex between the sepsis and non-sepsis groups (P>0.05). Compared with the non-sepsis group, the sepsis group had a significantly higher Sequential Organ Failure Assessment score, a significantly longer length of hospital stay, and a significantly higher rate of use of ventilator (P<0.05). As for inflammation markers, the sepsis group had significantly higher levels of C-reactive protein, procalcitonin, and IL-6 than the non-sepsis group (P<0.05). As for thyroid function parameters, the sepsis group had significantly lower levels of T3, T4, free T3, free T4, and TSH than the non-sepsis group (P<0.05). Compared with the non-sepsis group, the sepsis group had significantly higher incidence rates of NTIS, low T3 and T4, and low TSH (P<0.001). The correlation analysis revealed that IL-6 level was not correlated with T3, T4, and TSH levels in children with or without sepsis (P>0.05), but the pooled analysis of the two groups showed that IL-6 level was negatively correlated with T3 and T4 levels (P<0.001). CONCLUSIONS: Children with sepsis have a higher incidence rate of NTIS than those without sepsis. The high level of IL-6 may be associated with the development of NTIS.

Mycoplasma ovipneumoniae-derived lipid-associated membrane proteins induce cytokine secretion in mouse peritoneal macrophages through TLR2 signalling.

BACKGROUND: Mycoplasma ovipneumoniae (M. ovi) is the causative agent of chronic non-progressive pneumonia in sheep, goats, bighorn, and wild small ruminants. However, the mechanism of infection and immune response to M. ovi remain unclear. Invading microbes express lipid-associated membrane proteins (LAMPs) on the cell surface that interact with host cells to facilitate infection, and are thus the major molecules recognised by the host immune system. Upon LAMP recognition, Toll-like receptor 2 (TLR2) and NLRP3 inflammasome sense the pathogens and signalling pathways for cytokine secretion. In this study, we investigated whether M. ovi and M. ovi-derived LAMPs are immuno-biologically active compounds capable of activating mouse peritoneal macrophages and explored the underlying mechanism. RESULTS: After infection of wild-type mice with M. ovi, the expression of TLR2 and NLRP3 at the transcriptional and translational levels was determined with reverse transcription-polymerase chain reaction and flow cytometry. In addition, the cytokine levels and associated pathways were detected in infected wild-type, Tlr2-/-, and Nlrp3-/- mice via enzyme-linked immunosorbent assays and western blotting. The nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signalling pathways were found to mediate the expression of inflammatory cytokines in M. ovi or M. ovi-derived LAMP-infected peritoneal macrophages, and cytokines were not induced in Tlr2-/- and/or Nlrp3-/- macrophages. CONCLUSION: Host cytokine production is activated in response to M. ovi-derived LAMPs through the NF-κB and MAPK signalling pathway via TLR2.

iTRAQ-based proteomic analysis of Mycoplasma bovis NM-28 strain from two generations for vaccine screening.

Mycoplasma bovis is an important pathogenic bacterium affecting cows and cattle. Clinically, an inactivated vaccine of M. bovis is mainly used to prevent infection by this bacterium. The changes that occur in the antigen when M. bovis is continuously passaged in vitro remain unknown. Therefore, we performed an in vitro serial passage of the M. bovis NM-28 strain, which was isolated and identified in our laboratory. An isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics method was used to analyse the differences between generations 3 and 60. Many major membrane proteins or protective antigens reported in the literature did not exhibit changes between these generations. We found an imbalance between growth rate and nutrition in the 60th generation. The proteomics results were verified by western blotting and real-time PCR. Growth curves were also prepared based on colony-forming units (CFUs) between the 3rd and 60th generations. The number of colonies in the 60th generation in the stationary phase was 5 × 109 CFU mL-1, which was 10-fold higher than that in the 3rd generation. The 60th generation of the NM-28 strain can be used as an inactivated vaccine strain of M. bovis to lower production costs compared to use of the 3rd generation.

Unraveling the action mechanism of polygonum cuspidatum by a network pharmacology approach.

As a popular Chinese herbal medicine (CHM), polygonum cuspidatum is widely used to treat various diseases in China. However, its biological function and action mechanism have yet to be systematically explored. In the present study, we first identified 14 potential active ingredients of polygonum cuspidatum using the TCMSP server and then conducted an in silico target prediction for these ingredients using PharmMapper. The subsequent KEGG pathway enrichment analysis of the 57 identified potential targets revealed that they were closely associated with cancer and gynecological disorders. Furthermore, a protein-protein interaction network of these targets was constructed using STRING and Cytoscape, through which 11 core targets were excavated according to degree, a key topological parameter. Meanwhile, we developed a novel formula, in which the "R value" is determined by average shortest path length and closeness centrality, two other key topological parameters, to evaluate the reliability of these predicted core targets. Intriguingly, among the top 10 core targets excavated using this new formula, 7 overlapped with the former 11 core targets, showing a good consistency in these core targets between the different prediction algorithms. Next, 7 ingredients were identified/validated from the crude extract of polygonum cuspidatum using UPLC-MS/MS. Noteworthy, 6 potential targets predicted for these 7 ingredients overlapped with the 7 core targets excavated from the previous in silico analyses. Further molecular docking and druggability analyses suggested that polydatin may play a pivotal role in manifesting the therapeutic effects of polygonum cuspidatum. Finally, we carried out a series of cell functional assays, which validated the anti-proliferative effects of polygonum cuspidatum on gynecological cancer cells, thus demonstrating our network pharmacology approach is reliable and powerful enough to guide the CHM mechanism study.

CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris.

BACKGROUND: Pichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris. RESULTS: A non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (PAOX1UP-g2, PTEF1UP-g1, and PFLD1UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR-Cas9-mediated gene co-integration method in P. pastoris. CONCLUSIONS: A CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

Genome-wide SNP analysis using 2b-RAD sequencing identifies the candidate genes putatively associated with resistance to ivermectin in Haemonchus contortus.

BACKGROUND: The excessive and uncontrolled use of anthelmintics, e.g. ivermectin (IVM) for the treatment of livestock parasites has led to widespread resistance in gastrointestinal nematodes, such as Haemonchus contortus. There is an urgent need for better management of drug-use in nematode control and development of novel anthelmintics. Discovery and identification of anthelmintic resistance-associate molecules/markers can provide a basis for rational anthelmintics-use and development of novel drugs. Recent studies have shown that ivermectin resistance in H. contortus is likely to be multi-genic in nature except for several genes coding for IVM target and efflux pump. However, no other IVM resistance-associated genes were characterized by conventional methods or strategies. In the present study we adopted a new strategy, i.e. using genome-wide single nucleotide polymorphism (SNP) analysis based on 2b-RAD sequencing, for discovering SNPs markers across the genomes in both IVM susceptible and resistant isolates of H. contortus and identifying potential IVM resistance-associated genes. RESULTS: We discovered 2962 and 2667 SNPs within both susceptible and resistant strains of H. contortus, respectively. A relative lower and similar genetic variations were observed within both resistant and susceptible strains (average π values were equal to 0.1883 and 0.1953, respectively); whereas a high genetic variation was found across both strains (average π value was equal to 0.3899). A significant differentiation across 2b-RAD tags nucleotide sites was also observed between the two strains (average FST value was equal to 0.3076); the larger differences in average FST were observed at SNPs loci between coding and noncoding (including intronic) regions. Comparison between resistant and susceptible strains revealed that 208 SNPs loci exhibited significantly elevated FST values, 24 SNPs of those loci were located in the CDS regions of the nine genes and were likely to have signature of IVM directional selection. Seven of the nine candidate genes were predicted to code for some functional proteins such as potential IVM target and/or efflux pump proteins, component proteins of receptor complex in membrane on neuromuscular cells, and transcriptional regulation proteins. Those genes might be involved in resistance to IVM. CONCLUSIONS: Our data suggest that candidate genes putatively associated with resistance to IVM in H. contortus may be identified by genome-wide SNP analysis using 2b-RAD sequencing.

[Effect of continuous hemofiltration on inflammatory mediators and hemodynamics in children with severe hand, foot and mouth disease].

OBJECTIVE: To investigate the effect of continuous veno-venous hemofiltration (CVVH) on inflammatory mediators in children with severe hand, foot and mouth disease (HFMD), and to investigate its clinical efficacy. METHODS: A total of 36 children with stage IV HFMD were enrolled and randomly divided into conventional treatment group and CVVH group (n=18 each). The children in the CVVH group were given CVVH for 48 hours in addition to the conventional treatment. The levels of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and lactic acid in peripheral venous blood, heart rate, blood pressure, and left ventricular ejection fraction were measured before treatment and after 24 and 48 hours of treatment. RESULTS: After 24 hours of treatment, the conventional treatment group had a significantly reduced serum IL-2 level (P<0.01), and the CVVH treatment group had significantly reduced serum levels of IL-2, IL-6, IL-10, and TNF-α (P<0.05). After 48 hours of treatment, both groups had significantly reduced serum levels of IL-2, IL-6, IL-10, and TNF-α (P<0.01), and the CVVH group had significantly lower levels of these inflammatory factors than the conventional treatment group (P<0.01). After 48 hours of treatment, heart rate, systolic pressure, and blood lactic acid level were significantly reduced, and left ventricular ejection fraction was significantly increased in both groups, and the CVVH group had significantly greater changes in these indices except systolic pressure than the conventional treatment group (P<0.01). CONCLUSIONS: CVVH can effectively eliminate inflammatory factors, reduce heart rate and venous blood lactic acid, and improve heart function in children with severe HFMD.

[Clinical research of timing of application of antibiotics in septic shock of pediatric patients].

OBJECTIVE: To investigate the value of timing of antibiotics in pediatric septic shock. METHODS: Eighty children with septic shock treated with bundle treatment in Department of Critical Care Medicine were retrospectively analyzed. Eighty children with septic shock were divided into observation group (n=40, anti-infection therapy within 1 hour after admission) and control group (n=40, anti-infection therapy 1-6 hours after admission). The contents of lactate, C-reaction protein (CRP) and procalcitonin (PCT) were compared between two groups at admission and 24 hours and 72 hours after admission. RESULTS: Lactate in the observation group was significantly lower than that of the control group within the first 24 hours after admission (8.65 ± 2.84 mmol/L vs. 11.75 ± 3.20 mmol/L, P<0.01). CRP in the observation group was significantly lower than that of the control group 24 hours and 72 hours after admission (66.25 ± 8.55 mg/L vs. 91.77 ± 7.71 mg/L, 22.03 ± 7.46 mg/L vs. 50.11 ± 7.30 mg/L, both P<0.01). PCT in the observation group was significantly lower than that of the control group 72 hours after admission (0.67 ± 0.31 µg/L vs. 1.16 ± 0.25 µg/L, P<0.01). Time for shock recovery in the observation group was significantly shorter than that of the control group (6.80 ± 3.70 hours vs. 12.80 ± 3.63 hours, P<0.05), but no statistical difference in mortality rate between groups was found [5% (2/40) vs. 10% (4/40), P>0.05]. CONCLUSION: With the early empirical anti-infection treatment in pediatric septic shocked patients, time for recovery from shock can be shortened and successful rate of resuscitation can be improved.