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.

CysLT2R Antagonist HAMI 3379 Ameliorates Post-Stroke Depression through NLRP3 Inflammasome/Pyroptosis Pathway in Gerbils.

Post-stroke depression (PSD) is a kind of prevalent emotional disorder following stroke that usually results in slow functional recovery and even increased mortality. We had reported that the cysteinyl leukotriene receptor 2 (CysLT2R) antagonist HAMI3379 (HM3379) contributes to the improvement of neurological injury. The present study was designed to investigate the role of HM3379 in PSD-induced chronic neuroinflammation and related mechanisms in gerbils. The gerbils were subjected to transient global cerebral ischemia (tGCI) and spatial restraint stress to induce the PSD model. They were randomized to receive the vehicle or HM3379 (0.1 mg/kg, i.p.) for a consecutive 14 days. In the PSD-treated gerbils, HM3379 had noteworthy efficacy in improving the modified neurological severity score (mNSS) and depression-like behaviors, including the sucrose preference test and the forced swim test. HM3379 administration significantly mitigated neuron loss, lessened TUNEL-positive neurons, and reduced the activation of microglia in the cerebral cortex. Importantly, HM3379 downregulated protein expressions of the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome and pyroptosis including NLRP3, cleaved caspase-1, interleukin-1ß (IL-1ß), IL-18, cleaved gasdermin-N domain (GSDMD-N), and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC). Mechanistically, HM3379 could repress pyroptosis via inhibiting NLRP3 inflammasome activation under oxygen-glucose deprivation (OGD) stimulation. Knockdown of CysLT2R by short hairpin RNA (shRNA) or overexpression of CysLT2R by lentivirus (LV)-CysLT2R could abolish or restore the anti-depression effect of HM3379. Our results demonstrated that the selective CysLT2R antagonist HM3379 has beneficial effects on PSD, partially by suppressing the NLRP3 inflammasome/pyroptosis pathway.

Nicotinamide riboside exerts protective effect against aging-induced NAFLD-like hepatic dysfunction in mice.

BACKGROUND & AIMS: Aging is one of the risk factors of non-alcoholic fatty liver disease (NAFLD). Yet, the mechanism underlying the aging-associated NAFLD-like syndrome is not fully understood. Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, has protective effects against aging. Here, we investigated the actions of NAD precursors nicotinamide riboside (NR) on the development of aging-induced NAFLD. METHODS: NR supplemented food (2.5 g/kg food) was applied to aged mice for three months while normal chow to the other groups. Body weight, food intake, liver weight and fat pat mass were measured. The serum concentrations of lipid content, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and NAD were determined by biochemical assays. Pathological assessment and immunohistochemistry analysis of hepatic tissues were used to evaluate the effect of NR on NAFLD development and inflammatory infiltration. RESULTS: NR repletion significantly reduced fat pat mass in aged mice, while not altered the body weight, food intake, and liver weight. NR repletion significantly rescued the NAD reduction in aged mice. The total cholesterol and triglyceride levels could be lowered by NR repletion in aged mice. The AST level was also significantly reduced by NR repletion in aged group, while the ALT level lowered but without significance. Notably, moderate NAFLD phenotypes, including steatosis and hepatic fibrosis could be markedly corrected by NR repletion. In addition, Kupffer cells accumulated and inflammatory infiltration could also be remarkably reversed by NR repletion in aged mice. CONCLUSION: Aging was associated with NAFLD-like phenotypes in mice, which could be reversed by oral NR repletion. Therefore, oral NR uptake might be a promising strategy to halt the progression of NAFLD.

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.