Establishment and application of a loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for detecting Clostridium piliforme.

BACKGROUND: Clostridium piliforme (causative agent of Tyzzer disease) infects various animals, including primates, and hence a threat to animal and human health worldwide. At present, it is detected using traditional methods, such as path morphology, polymerase chain reaction and enzyme-linked immunosorbent assay. Therefore, it is necessary to develop convenient, efficient visual molecular biological methods for detecting C. piliforme. OBJECTIVES: To establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme. METHODS: In this study, we designed internal and external primers based on the conserved 23S rRNA region of C. piliforme to develop a biotin-labelled diarrhoea-suffered loop-mediated isothermal amplification (LAMP) system for detecting of C. piliforme and assessed the specificity, sensitivity and repeatability of the LAMP system. RESULTS: The LAMP system did not exhibit cross-reactivity with 24 other common pathogenic species, indicating that it had good specificity. The minimum concentration of sensitivity was 1 × 10-7  ng/µL. Mouse models (Meriones unguiculatus) of Tyzzer disease were established and a LAMP-lateral flow dipstick (LAMP-LFD) was developed for detecting C. piliforme. The detection rate of C. piliforme was 5.08% in clean-grade animals and 9.96% in specific-pathogen-free-grade animals from Jiangsu, Zhejiang and Shanghai. In addition, the detection rates of C. piliforme were 10.1%, 8.6% and 20%, in animals from Hangzhou, Wenzhou and Shaoxing, respectively. The detection rate of C. piliforme was higher in experimental animals used in schools than in those used in companies and research institutes. CONCLUSIONS: The LAMP-LFD method established in this study can be used to detect C. piliforme in animals handled in laboratory facilities of universities, pharmaceutical enterprises and research and development institutions.

Mining host candidate regulators of schistosomiasis-induced liver fibrosis in response to artesunate therapy through transcriptomics approach.

BACKGROUND: Artesunate (ART) has been reported to have an antifibrotic effect in various organs. The underlying mechanism has not been systematically elucidated. We aimed to clarify the effect of ART on liver fibrosis induced by Schistosoma japonicum (S. japonicum) in an experimentally infected rodent model and the potential underlying mechanisms. METHODS: The effect of ART on hepatic stellate cells (HSCs) was assessed using CCK-8 and Annexin V-FITC/PI staining assays. The experimental model of liver fibrosis was established in the Mongolian gerbil model infected with S. japonicum cercariae and then treated with 20 mg/kg or 40 mg/kg ART. The hydroxyproline (Hyp) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in liver tissue were measured and histopathological changes of liver tissues were observed. Whole-transcriptome RNA sequencing (RNA-seq) of the liver tissues was performed. Differentially expressed genes (DEGs) were identified using bioinformatic analysis and verified by quantitative PCR (qPCR) and western blot assay. RESULTS: ART significantly inhibited the proliferation and induce the apoptosis of HSCs in a dose-dependent manner. In vivo, Hyp content decreased significantly in the ART-H group compared to the model (MOD) group and GPX activity was significantly higher in the ART-H group than in the MOD group. Besides, ART treatment significantly reduced collagen production (p <0.05). A total of 158 DEGs and 44 differentially expressed miRNAs related to ART-induced anti-schistosomiasis liver fibrosis were identified. The qPCR and western blot results of selected DEGs were consistent with the sequencing results. These DEGs were implicated in key pathways such as immune and inflammatory response, integrin-mediated signaling and toll-like receptor signaling pathways. CONCLUSION: ART is effective against liver fibrosis using Mongolian gerbil model induced by S. japonicum infection. We identified host candidate regulators of schistosomiasis-induced liver fibrosis in response to ART through transcriptomics approach.

BNIP3 as a potential biomarker for the identification of prognosis and diagnosis in solid tumours.

BACKGROUND: Traditional radiotherapy and chemotherapy have been intensively studied for their role in the treatment of tumours. However, these therapies often cause side effects for patients, which calls for the development of novel treatment options for tumours. B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) reportedly apoptosis-inducing effects in tumour cells and is associated with the progression and treatment of multiple tumours. Nevertheless, little is known about its potential role in tumour diagnosis and targeted therapy. FINDINGS: The results of the study demonstrated that the interaction of BNIP3 with HDAC1 may affect the progression of breast invasive cancer (BRCA), sarcoma (SARC), kidney renal clear cell carcinoma (KIRC), and low-grade glioma (LGG). BNIP3 seemed to exert its effects in BRCA and SARC primarily through gene silencing and integrator complex, and in KIRC and LGG, mainly by affecting olfactory function, suggesting that targeted therapy can be developed based on the above signalling pathway and downstream molecules. INTERPRETATION: BNIP3 has emerged as a promising therapeutic and diagnostic target for BRCA, SARC, KIRC, and LGG, providing new insights into tumour molecular therapies in the clinic.

Photocatalytic degradation of industrial acrylonitrile wastewater by F-S-Bi-TiO2 catalyst of ultrafine nanoparticles dispersed with SiO2 under natural sunlight.

Highly active photocatalyst, having certain anti-ionic interfering function, of F, S and Bi doped TiO2/SiO2 was used for the first time to degrade the organic pollutants in acrylonitrile industrial wastewater under natural sunlight. The photocatalyst were prepared and characterized by UV-Vis, XRD, TEM, EDS, Nitrogen physical adsorption and XPS technique. UV-Vis analysis revealed addition of F, S and Bi into the lattice of TiO2 led to the expansion of TiO2 response in the visible region and hence the efficient separation of charge carrier. The photocatalytic potential of as prepared catalyst to degrade acrylonitrile wastewater under simulated and natural sunlight irradiation was investigated. The extent of degradation of acrylonitrile wastewater was evaluated by chemical oxygen demand (CODCr). CODCr in wastewater decreased from 88.36 to 7.20 mgL-1 via 14 h irradiation of simulated sunlight and achieved regulation discharge by 6 h under natural sunlight, illuminating our photocatalyst effectiveness for refractory industrial wastewater treatment. From TEM results, we found that SiO2 could disperse the photocatalyst with different component distributions between the surface and the bulk phase that should also be responsible for the light absorption and excellent photocatalytic performance. The XPS analysis confirmed the presence of surface hydroxyl group, oxygen vacancies.

Efficient photocatalytic degradation of acrylonitrile by Sulfur-Bismuth co-doped F-TiO2/SiO2 nanopowder.

In this study, a simple sol-gel method was applied for preparing effectual photocatalyst of S-Bi co-doped F-TiO2/SiO2 (S-Bi-F-TiO2/SiO2) nanopowder. Optimal preparation conditions were obtained by optimizing the calcination temperature and the ratio of S and Bi. The synthesized powder was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive spectrometer (EDS), X-ray photoelectron spectroscopy (XPS), brunauer-emmett-teller (BET), UV-Visible diffuse-reflectance spectroscopy (UV-Vis DRS), photoluminescence spectroscopy (PL) and ammonia adsorption and temperature-programmed desorption (NH3-TPD). The photocatalytic activity was evaluated by the degradation of acrylonitrile under simulated visible light irradiation. S-Bi-F-TiO2/SiO2 nanopowder possess excellent photocatalytic properties under visible light for the degradation of acrylonitrile, when the calcination temperature was 450 °C for 2 h and the ratio of S and Bi was 0.02: 0.007. The degradation efficiency of acrylonitrile reached to 81.9% within 6 min of visible light irradiation. Compared with F-TiO2/SiO2 sample, NH3-TPD and PL results revealed the higher photocatalytic activity for S-Bi-F-TiO2/SiO2, which is mainly due to the increase strength and number of surface acid site with S doping. The co-doping with S & Bi improved the separation of electron-hole pairs and enhanced the photocatalytic oxidizing species. The UV-Vis DRS showed stronger absorption in S-Bi co-doped F-TiO2/SiO2 catalyst as compared to F-TiO2/SiO2 catalyst. XPS results demonstrated the presence of various surface species viz. oxygen vacancies, Ti3+, Ti4+, O2- and OH group.

Adsorption and photocatalytic degradation of benzene compounds on acidic F-TiO2/SiO2 catalyst.

The adsorption and photocatalytic degradation performance of F-TiO2/SiO2 catalyst towards a series of benzene compounds were studied. The results revealed that the F-TiO2/SiO2 catalyst is superior to TiO2 P25 in adsorption capacity and photocatalytic degradation under simulant sunlight irradiation. The adsorptive capacity for chlorobenzene is the highest and the degradation rate is the greatest among these target pollutants. The increase of absorptive organic molecules on acidic F-TiO2/SiO2 catalyst benefits photocatalytic degradation. The photocatalytic reaction accords to Langmuir-Hinshelwood mechanism. The FTIR results indicated that the promoting effect of acidic centers on adsorption of benzene compounds depends on electron property of the functional groups. The electron-donating groups (-OH and -NH2) of benzene compounds are weakly adsorbed on acidic centers of the catalyst due to the competitive adsorption with H2O, while the electron-withdrawing groups (-Cl and -NO2) are adsorbed more strongly at acidic sites. The monosubstituted chlorobenzene prefers to perpendicular adsorption on acidic surface, while the disubstituted benzenes prefer to horizontal adsorption, which decreases the adsorbed amounts. A photocatalytic rate mainly depends on electron donating property of the functional group and amount of adsorptive organic molecules, but not on electron density of benzene ring.

Early Detection of Toxoplasma gondii Infection In Mongolian Gerbil By Quantitative Real-Time PCR.

Toxoplasmosis, caused by Toxoplasma gondii, is associated with several clinical syndromes, including encephalitis, chorioretinitis, and congenital infection. Toxoplasma gondii is a ubiquitous apicomplexan parasite found in both humans and animals. Mongolian gerbils, which are more susceptible to both high- and low-virulence Toxoplasma strains compared with mice, are considered useful models for assessing diagnosis and treatment methods for toxoplasmosis, as well as infection by and host defense to this organism. Here we established a quantitative real-time polymerase chain reaction (qPCR) method targeting the B1 gene for early and specific detection of T. gondii infection in Mongolian gerbil. The detection limit of the developed qPCR was approximately 1 T. gondii tachyzoite. This method was also applied to detect T. gondii genomic DNA in experimentally infected Mongolian gerbils, with positive results in blood (66.7%), liver (73.3%), lung (80.0%), spleen (80.0%), and peritoneal fluid (66.7%) samples as early as 1 day postinfection. Specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Cryptosporidium parvum, Eimeria tenella, Trypanosoma evansi, Schistosoma japonicum, Angiostrongylus cantonensis, and Strongyloides stercoralis. This study first reports the use of Mongolian gerbils as an animal model for early diagnosis of toxoplasmosis by qPCR.

SREBP­2 expression pattern contributes to susceptibility of Mongolian gerbils to hypercholesterolemia.

Gerbils are susceptible to dietary cholesterol and prone to hypercholesterolemia and non­alcoholic fatty liver disease. The present study aimed to explore the role of sterol regulatory element binding protein (SREBP)­2 and 3­hydroxy­3­methylglutaryl CoA reductase (HMGCR) in hypercholesterolemia susceptibility in gerbils. Male gerbils were fed the normal diet or a high­fat diet (HFD) for 2 weeks, or the HFD for 2 weeks followed with the normal diet for an additional 2 weeks. Serum lipid levels and hepatic fat deposition were measured, and mRNA and protein levels of SREBP­2 and HMGCR were evaluated by quantitative polymerase chain reaction and Western blotting. In addition, the role of SREBP­2 function in cholesterol synthesis from the gerbil primary hepatic cells was also investigated by modulation of SERBP­2 expression via the transfection of SREBP­2 overexpression and knockdown plasmids, respectively. The data demonstrated that the total cholesterol and low­density lipoprotein cholesterol levels in the gerbil serum samples were rapidly and significantly elevated in response to HFD. In addition, the effect of the HFD was rapidly attenuated in the gerbils following a return to the normal diet. HMGCR expression and activation were not altered by dietary cholesterol consumption in the livers from the gerbils in model or recovery groups. HMGCR expression and activation were effectively regulated in cultured hepatic cells from the gerbils. These results indicated that the activation of SREBP­2 to HMGCR was not terminated in gerbil livers during cholesterol intake. Therefore, stable SREBP­2 expression contributes to the susceptibility of gerbils to hypercholesterolemia.

Evaluation of an ischemic model in ischemia prone and general Mongolian gerbils by neurological symptom, injury, and sex difference.

BACKGROUND: In the previous study, we established an ischemia-prone gerbil population (IG), which was selectively bred to increase the incidence of unilateral carotid arterial occlusion (UCO)-induced ischemia in Mongolian gerbils. However, if the characteristics of ischemia model in IG are the same as those in general gerbils (GG), and if the neurological symptoms are associated with the neurological insults in IG is still unclear. METHODS: In the present study, we evaluated the UCO model in IG by analyzing neurological symptoms, neurological injury in the hippocampal CA1 region and compared with GG. RESULTS: The data showed that the ratios of neurological symptom scores ≥ 2 in the IG and GG groups were 65.0% vs 30.0%, respectively, and were significantly different (P < .01).The neuronal damage following a UCO ischemic insult in the IG group was more severe compared to the GG group. There was a high correlation between the neurological insults' scale and the neurological symptom score in the IG and GG groups (r = .979 and .943 in the IG and GG groups, respectively). In animals with mild neurological symptom scores (2 and 3), the neuronal insults were significantly different between female and male gerbils in both IG and GG. CONCLUSION: Our findings suggest that IG population would likely be more advantageous to establish an ischemic model.

The complete mitochondrial genome of a rare human pathogen, Aspergillus ustus.

Aspergillus ustus is among the most ubiquitous soil species and has been implicated in human infections. The complete mitochondrial genome of A. ustus has been determined by high-throughput sequencing technology in this work. Our study revealed that the mitochondrial genome of A. ustus is 33,007 bp long, with AT content of 74.84%, which consists of a usual set of mitochondrial proteins and RNA genes, including large and small ribosomal RNAs, 15 proteins and 20 tRNA genes and contains two introns. Notably, it also contains three hypothetical proteins without obvious homology to any known proteins. All structural genes are located on one strand and are apparently transcribed in one direction. The complete mitochondrial genomes of A. ustus would be useful for future investigation of the genetics, evolution and clinical identification of Aspergillus species.

A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

Protective role of supplement with foreign Bifidobacterium and Lactobacillus in experimental hepatic ischemia-reperfusion injury.

BACKGROUND AND AIM: Intestinal microflora play a crucial role in some severe liver diseases. The purpose of this study was to evaluate the effects of a Lactobacillus strain and a Bifidobacterium strain on ischemia-reperfusion (I/R) liver injury. METHODS: Rats were divided into six groups. Each group received either Bifidobacterium Catenulatum ZYB0401; Lactobacillus Fermentum ZYL0401; a mixture of these two bacterial strains; gentamicin; or saline by daily gavage for 7 days. On the sixth day, all rats, except those in the control group, were subjected to 20 min of liver ischemia. After 22 h of hepatic reperfusion, liver enzymes and histology, malondialdehyde (MDA), superoxide dismutase (SOD), endotoxemia, serum tumor necrosis factor-alpha (TNF-alpha), intestinal bacteria, intestinal mucosal ultrastructure, and bacterial translocation were studied. RESULTS: All administered bacteria increased intestinal Bifidobacterium and Lactobacillus, decreased endotoxemia (P < 0.01), alanine aminotransferase (ALT) (P < 0.01), and markedly ameliorated liver histology and intestinal mucosal ultrastructure. Only rats treated with Bifidobacterium Catenulatum ZYB0401 and Lactobacillus Fermentum ZYL0401 showed reduced incidence of bacterial translocation to the kidney (P < 0.05), associated with decreased serum TNF-alpha and liver MDA (P < 0.05) and increased liver SOD (P < 0.05) compared to the I/R group. Gentamicin decreased almost all kinds of intestinal bacteria (P < 0.01) and decreased ALT (P < 0.01) and serum TNF-alpha, but failed to reduce both endotoxemia and the incidence of bacterial translocation and had no effects on liver MDA and SOD. CONCLUSION: Bifidobacterium Catenulatum ZYB0401 in combination with Lactobacillus Fermentum ZYL0401 could be useful in restoring intestinal microflora and in preventing liver injury in hepatic I/R of rats.

Effects of Salvia miltiorrhiza on intestinal microflora in rats with ischemia/reperfusion liver injury.

BACKGROUND: Hepatic ischemia/reperfusion injury may induce intestinal microflora imbalance. Salvia miltiorrhiza is effective in promoting blood circulation and counteracting peroxidation in tissues. The aim of the present study was to determine the effects of Salvia miltiorrhiza on intestinal microflora, endotoxemia, and bacterial translocation in rats with hepatic I/R injury. METHODS: Sprague-Dawley rats in specific pathogen free grade were divided into 3 groups: group I(n=6) for sham operation; groups II(n=10) and III(n=7) for liver ischemia for 20 minutes and reperfusion for 22 hours. Group III was also pretreated with 4 ml/day of Salvia miltiorrhiza solution (250 mg/kg) by daily gavage for 7 days. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) and superoxide dismutase(SOD) in liver tissues, serum endotoxin, intestinal bacterial counts, intestinal mucosal histology and bacterial translocation were studied. RESULTS: The levels of ALT, AST, plasma endotoxin and MDA in liver tissues were decreased more markedly in group III (57.57+/-18.08 U/L, 147.57+/-40.84 U/L, 0.42+/-0.144 EU/ml and 0.52+/-0.19 nmol/mg-prot respectively) in group II(122.8+/-80.12 U/L, 295.9+/-216.92 U/L, 0.80+/-0.262 EU/ml and 0.72+/-0.12 nmol/mg-prot; P<0.05-0.01 respectively). Liver SOD activity was increased more significantly in group III (318.47+/-64.62 U/mg-prot) than in group II(240.76+/-63.67 U/mg-prot, P<0.05). The counts of Bifidobacteria and Bacteroides increased more significantly in group III than in group II, but were similar to those in group I. Bacterial translocation to the kidney in group II was 50%(5/10), whereas no bacterial translocation to the kidney occurred in the other two groups (P<0.01). Ileal mucosal structure was markedly ameliorated in group III as compared with group II. CONCLUSIONS: Salviae miltiorrhiza could partially restore intestinal microflora balance, improve intestinal mucosal integrity, and reduce bacterial translocation and plasma endotoxin in rats with hepatic ischemia/reperfusion injury.