Identification a novel Ganoderma FIP gene from Ganoderma capense and its functional expression in Pichia pastoris.

Ganoderma capense is a precious medicinal fungus in China. In this study, a novel fungal immunomodulatory protein gene, named as FIP-gca, was cloned from G. capense by homologous cloning. Sequencing analysis indicated that FIP-gca was composed of 336 bp, which encoded a polypeptide of 110 amino acids. Protein sequence blasting and phylogenetic analysis showed that FIP-gca shared homology with other Ganoderma FIPs. FIP-gca was effectively expressed in Pichia pastoris GS115 at an expression level of 166.8 mg/L and purified using HisTrap™ fast-flow prepack columns. The immunomodulation capacity of rFIP-gca was demonstrated by that rFIP-gca could obviously stimulate cell proliferation and increase IL-2 secretion of murine spleen lymphocytes. Besides, antitumor activity of rFIP-gca towards human stomach cancer AGS cell line was evaluated in vitro. Cell wound scratch assay proved that rFIP-gca could inhibit migration of AGS cells. And flow cytometry assay revealed that rFIP-gca could significantly induce apoptosis of AGS cells. rFIP-gca was able to induce 18.12% and 22.29% cell apoptosis at 0.3 µM and 0.6 µM, respectively. Conclusively, the novel FIP-gca gene from G. capense has been functionally expressed in Pichia and rFIP-gca exhibited ideal immunomodulation and anti-tumour activities, which implies its potential application and study in future.

Characterization of changes in the intestinal microbiome following combination therapy with zinc preparation and conventional treatment for children with rotavirus enteritis.

Background: Rotavirus (RV) is one of the most common pathogens causing diarrhea in infants and young children worldwide. Routinely, antiviral therapy, intestinal mucosa protection, and fluid supplementation are used in clinic, however this is not efficacious in some severe cases. Zinc supplementation has previously been shown to improve resolution of symptoms from infectious diarrhea. Methods: In this study differences in response rate, duration of hyperthermia, vomiting, and diarrhea, and the persistence time of cough and lung rales in groups were compared. 16SrDNA gene sequencing technology was used to analyze and compare changes in the intestinal microflora of children with RV enteritis who received the conventional treatment with or without the zinc preparation. In addition, the correlations between the differential bacterial species and the related inflammatory factors were determined. Results: Conventional therapy combined with the zinc preparation significantly shortened the duration of hyperthermia, vomiting, and diarrhea compared with the conventional treatment alone. In addition, the time to symptom relief showed that the absorption time of cough and lung rales was significantly shorter in the combination treatment group than that in the conventional treatment group in the children with pneumonia. Further, compared with the conventional treatment, the combined treatment significantly increased the diversity and abundances of florae as compared with the conventional treatment. This combination therapy containing zinc preparation markedly increased the abundances of Faecalibacterium, Bacteroidales, Ruminoccoccoccus, and Lachnospiraceae at the genus level. The LEfSe analysis suggested that Clostridiumbolteae were most significantly altered after the combination therapy. In addition, a correlation analysis revealed significantly negative correlations between the inflammatory factors especially IL-6, TNF-a, CRP and some intestinal florae such as Bacteroides, Faecalibacterium, Blautia, Parabacteroides, Subdoligranulum, and Flavonifractor. Conclusion: Compared with the conventional therapy alone, the combined therapy with the zinc preparation significantly improves symptoms caused by RV. The combination therapy containing the zinc preparation significantly increases the diversity and abundances of some beneficial groups of bacteria. Further, The presence of these groups was further negatively correlated with relevant inflammatory factors. More importantly, this combination therapy containing the zinc preparation provides a reference for the clinical management of children with RV enteritis.

A Case of Fungemia Caused by Postoperative Chronic Lumbar Intervertebral Disc Infection.

Postoperative surgical site infection is one of the serious postoperative complications of spine surgery, especially fungal infections. Late-stage surgical site fungal infections often lack typical clinical symptoms and have a variable clinical presentation. In this case, the patient was a senior patient with usually only tolerable pain and discomfort, which was detected 2 years after the first surgery. Such cases are even rarer for fungal bloodstream infections caused by delayed postoperative chronic fungal osteomyelitis and deserve further study for early identification and intervention to minimize harm.

Analysis of Intestinal Short-Chain Fatty Acid Metabolism Profile After Probiotics and GLP-1 Treatment for Type 2 Diabetes Mellitus.

Type 2 diabetes accounts for about 90% of diabetes patients, and the incidence of diabetes is on the rise as people's lifestyles change. Compared with GLP-1 treatment, probiotic treatment can directly regulate homeostasis of the host gut microbe, and thus homeostasis of its metabolites. Currently, the regulatory role of probiotics on intestinal metabolites after treatment of type 2 diabetes mellitus remains unclear. The purpose of this study was to investigate the therapeutic effect of probiotics on type 2 diabetes mellitus and its regulatory effect on short-chain fatty acids, which are metabolites of intestinal microorganisms. I collected feces from 15 patients with diabetes before treatment and 15 patients with type 2 diabetes after treatment with GLP-1 and probiotics. The abundance of short-chain fatty acids in feces was determined by GC-MS. Results Both GLP-1 and probiotics could improve the levels of blood glucose, urine glucose and BMI in patients with type 2 diabetes. After glP-1 treatment, two short-chain fatty acids (butyric acid and valerate acid) in intestine were significantly changed. Propionic acid and isovalerate were significantly changed after probiotic treatment. At the same time, KEGG signal pathway enrichment results showed that probiotics intervention mainly achieved the purpose of treating type 2 diabetes through regulating protein and carbohydrate metabolism. Taken together, our study shows changes in intestinal short-chain fatty acids after probiotics or GLP-1 treatment of type 2 diabetes, which will provide us with new insights into the mechanism of probiotics treatment of type 2 diabetes, as well as potential intervention targets for diabetes treatment.

ROS1-mediated decrease in DNA methylation and increase in expression of defense genes and stress response genes in Arabidopsis thaliana due to abiotic stresses.

BACKGROUND: Small interfering RNAs (siRNAs) target homologous genomic DNA sequences for cytosine methylation, known as RNA-directed DNA methylation (RdDM), plays an important role in transposon control and regulation of gene expression in plants. Repressor of silencing 1 (ROS1) can negatively regulate the RdDM pathway. RESULTS: In this paper, we investigated the molecular mechanisms by which an upstream regulator ACD6 in the salicylic acid (SA) defense pathway, an ABA pathway-related gene ACO3, and GSTF14, an endogenous gene of the glutathione S-transferase superfamily, were induced by various abiotic stresses. The results demonstrated that abiotic stresses, including water deficit, cold, and salt stresses, induced demethylation of the repeats in the promoters of ACD6, ACO3, and GSTF14 and transcriptionally activated their expression. Furthermore, our results revealed that ROS1-mediated DNA demethylation plays an important role in the process of transcriptional activation of ACD6 and GSTF14 when Arabidopsis plants are subjected to cold stress. CONCLUSIONS: This study revealed that ROS1 plays an important role in the molecular mechanisms associated with genes involved in defense pathways in response to abiotic stresses.

Clinical application, evaluation and analysis of influencing factors of on tuberculosis γ-Interferon enzyme-linked immunosorbent assay.

OBJECTIVE: To assess sensitivity and specificity of the interferon gamma release assay test, and to pinpoint the influencing factors that should be taken care of in clinical application. METHODS: This study was conducted at the First People's Hospital in the Yunnan Province of China from October 2018 to March 2019, and comprised samples collected from outpatient and inpatients. To detect mycobacterium tuberculosis, acid-fast staining on sputum smear was performed on relevant tissues suspected of extrapulmonary tuberculosis. Tuberculosis interferon gamma release assay test and pathology samples were examined. Tuberculosis-specific cell reaction assay kit was used for sampling. SysmexXN-2000 haematology analyser, VACUETTE SRS100/II and Beckman Coulter AU5800 were used to perform various analyses. Data was grouped and analysed using R statistical software. RESULTS: Of the 960 samples, 516(53.75%) cases tested positive for tuberculosis infection and 444(46.25%) tested negative. The sensitivity of the pathological results was 86% and the specificity was 96%. The sensitivity of the interferon gamma release assay test was 95% and specificity 82%. Interferon release test, pathological results and final diagnosis showed significant comparisons (p<0.05). Significant relationships were also established for factors, such as age, interferon release quantity, lymphocyte, C-reactive protein and counts of mono-nuclear cell (p<0.05). CONCLUSION: Interferon gamma release assay test was found to have high consistency with pathological results and final diagnosis and can be used as a subsidiary to traditional clinical imaging and pathological judgment.

The viral suppressor HCPro decreases DNA methylation and activates auxin biosynthesis genes.

Auxin has profound effects on plant growth and development. In addition to participating in plant growth and development, the auxin signaling pathway is involved in plant defense against pathogens. In this study, we investigated the molecular mechanism by which helper-component protease (HCPro) encoded by the Tobacco vein banding mosaic virus (TVBMV) activates auxin biosynthesis genes (YUCs) and interferes with the auxin signaling pathway. Our results demonstrated that the viral suppressor HCPro decreased the DNA methylation of dispersed repeats (DRs) within the promoters of YUC1, YUC5 and YUC10 and transcriptional activated these YUC genes targeted by RNA-directed DNA methylation (RdDM), leading to an increase in auxin accumulation in plants. Furthermore, we found that the induction of these YUCs by HCPro was attenuated in ros1 mutant plants, suggesting that HCPro-mediated transcriptional activation of the genes was partly dependent on ROS1-mediated DNA demethylation.

HEDD: the human epigenetic drug database.

Epigenetic drugs are chemical compounds that target disordered post-translational modification of histone proteins and DNA through enzymes, and the recognition of these changes by adaptor proteins. Epigenetic drug-related experimental data such as gene expression probed by high-throughput sequencing, co-crystal structure probed by X-RAY diffraction and binding constants probed by bio-assay have become widely available. The mining and integration of multiple kinds of data can be beneficial to drug discovery and drug repurposing. HEMD and other epigenetic databases store comprehensively epigenetic data where users can acquire segmental information of epigenetic drugs. However, some data types such as high-throughput datasets are not provide by these databases and they do not support flexible queries for epigenetic drug-related experimental data. Therefore, in reference to HEMD and other epigenetic databases, we developed a relatively comprehensive database for human epigenetic drugs. The human epigenetic drug database (HEDD) focuses on the storage and integration of epigenetic drug datasets obtained from laboratory experiments and manually curated information. The latest release of HEDD incorporates five kinds of datasets: (i) drug, (ii) target, (iii) disease, (vi) high-throughput and (v) complex. In order to facilitate data extraction, flexible search options were built in HEDD, which allowed an unlimited condition query for specific kinds of datasets using drug names, diseases and experiment types.Database URL: http://hedds.org/.

HC-Pro viral suppressor from tobacco vein banding mosaic virus interferes with DNA methylation and activates the salicylic acid pathway.

Salicylic acid (SA) is an important signalling molecule that is synthesized by plants and induces the expression of resistance genes. The SA pathway is typically activated by DNA viruses as well as RNA viruses. Here, we demonstrated that heper-component protease (HC-Pro) encoded by tobacco vein banding mosaic virus (TVBMV) decreases in DNA methylation at the promoters of the regulators ACD6 and NPR1 in the SA pathway. We found that the overexpression of HC-Pro increases the expression of components in the SA pathway in plants. The results revealed that HC-Pro interferes in DNA methylation and activates the SA pathway in the HC-Pro transgenic plants and TVBMV-infected plants. We further found that the accumulation of siRNAs derived from the promoter repeats of ACD6 and NPR1 is greatly reduced in the HC-Pro plants. Our results suggested that HC-Pro-mediated interference with DNA methylation is likely caused by a reduction in accumulation of siRNAs.

A new transient expression system for large-scale production of recombinant proteins in plants based on air-brushing an Agrobacterium suspension.

Plant transient expression using virus-based vectors is advantageous when high level of gene expression is desired within a short time. In this study, a new system, named "air-brush," has been developed to facilitate a scale-up production of recombinant proteins in plants. GFP was expressed successfully in Nicotiana benthamiana (Nb) plants by air-brushing an Agrobacterium suspension that contained the TMV-based vector p35S-30B-GFP. Key factors influencing the gene expression were optimized, including the Agrobacterium cell density, seedling age, and the growth temperature of plant materials. In addition, the pharmaceutical protein human acidic fibroblast growth factor (ha FGF) was also expressed in Nb plants by the air-brush system. The results demonstrated that using this system is highly advantageous; it is convenient, quick, easily scaled-up, and has a higher expression efficiency than leaf infiltration.

[A new agroinoculation technology for foreign gene expression in plants by means of transient expression].

Due to the laborious and scale-up limitation we have developed a simple method named "seed absorption" to express foreign proteins in plants by means of transient expression. It has been shown that the reporter gene GFP was expressed successfully in tomato (Solanum lycopersicum L.) plants by seed absorbing Agrobacterium suspension containing TMV-based p35S-30B-GFP vector. Various factors influencing the gene expression were optimized including Agrobacterium cell density and other inoculation conditions. This method has the special advantages as simple work process, ease to scale-up, and further expanding the host range of plant bioreactor than previous methods. We assume that the seed absorption method will facilitate the industrial scale production of the recombinant pharmaceutical proteins in plants.