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.

Calbindin S100A16 Promotes Renal Cell Carcinoma Progression and Angiogenesis via the VEGF/VEGFR2 Signaling Pathway.

Purpose: Recent research has indicated that the calcium-binding protein S100A16 promotes carcinogenesis and tumor growth in several forms of cancer. The objective of this study was to examine the relationship between S100A16 and renal cell cancer. Methods: By using The Cancer Genome Atlas (TCGA) database, the differentially expressed gene S100A16 was identified, and its appearance and link to the prognosis of persons with renal cancer were confirmed. Cox regression was used in multivariate analysis, and a nomogram was developed for internal validation. The correlation between S100A16 and immune cells was analyzed in the TIMER database. Moreover, the potential mechanism of action was investigated utilizing GO and KEGG enrichment analyses. Proliferation, migration, and angiogenesis were investigated in vitro, and the involvement of S100A16 in the undesirable biological events of renal cell carcinoma (RCC) was further explored. Results: S100A16 was the differentially expressed molecule identified through database screening. Malignant tissues showed higher S100A16 expression than noncancerous tissues, and S100A16 expression was mostly localized in the cytoplasm. According to the TCGA and KM-plotter datasets, patients with RCC and low S100A16 expression had superior OS, PFI, and DSS. The C-index of the nomogram was 0.754 (0.726-0.782), and the accuracy of the prediction model was high. The TIMER database shows that the expression of S100A16 is associated with immune infiltration and may play an important role in promoting tumor cell immune escape in the RCC tumor microenvironment. S100A16 may influence the biological processes of RCC via the VEGF/VEGFR2 signaling route and PI3K-Akt signaling pathway and through P53 alteration and cell cycle according to the gene enrichment technique. In vitro cytological experiments demonstrated that S100A16 knockdown can inhibit the proliferation and migration of renal cancer cells and the expression levels of VEGF, VEGFR2, and phosphorylated AKT within renal cancer cells, thereby inhibiting angiogenesis in renal cancer cells and resulting in a poor prognosis of RCC. Conclusion: A decrease in S100A16 expression may dramatically increase the OS, PFI, and DSS of patients with RCC and may thus be used as a biomarker for predicting RCC. It may be associated with the immune infiltration of RCC and play a crucial role in the immune evasion of tumor cells within the RCC microenvironment. Intervention of s100a16 can promote the progression and angiogenesis of renal cell carcinoma through the VEGF/VEGFR2 signal transduction pathway and lead to poor prognosis of renal cell carcinoma. These findings suggest a potential target for the development of anticancer strategies for renal cancer.

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.

Tetrandrine attenuates ischemia/reperfusion­induced neuronal damage in the subacute phase.

Ischemic stroke, the third leading cause of disability globally, imposes a notable economic burden. Tetrandrine (Tet), which has been widely used clinically, exhibits potential protective effects against stroke. However, there has been little pre­clinical research to evaluate the therapeutic effects of Tet on stroke. The present study investigated the beneficial effect of Tet on ischemia­reperfusion (I/R) injury and its underlying mechanism in rats. Rats were subjected to occlusion of the middle cerebral artery, then treated with Tet (30 mg/kg/day, intraperitoneal) in the subacute phase for 7 days. In order to detect the effects of Tet on the behavior of rats, modified neurological severity score and longa behavior, grasping capability and inclined plane tests were conducted on days 1, 3 and 7 following cerebral ischemia. In addition, neuronal apoptosis in the cortex and hippocampus following ischemia was assessed by Nissl staining and TUNEL assay. Finally, oxidative stress was evaluated by measurement of free radicals and immunofluorescence staining of LC3 was used to assess autophagy. Tet improved neurological function and decreased infarct volume in I/R injury rats. Tet also prevented neuronal apoptosis in the cortex and hippocampus region. In addition, Tet protected against oxidative damage following ischemia, which was reflected by decreased levels of nitric oxide and malondialdehyde and increased levels of glutathione (GSH) and GSH peroxidase. In addition, the expression levels of the autophagy marker LC3 decreased in the Tet treatment group. In conclusion, Tet attenuated I/R­induced neuronal damage in the subacute phase by decreasing oxidative stress, apoptosis and autophagy.

Application of Gastroscopy Based on Nano Carbon in the Remedy of Chronic Pulmonary Heart Disease Caused by Helicobacter Pylori.

Chronic pulmonary heart disease is a common respiratory disease. Helicobacter pylori infection can lead to the occurrence of chronic pulmonary heart disease. However, most drugs for chronic pulmonary heart disease caused by helicobacter pylori are lack of tissue specificity. At the same time, due to the blocked blood circulation in the ischemic area, the distribution of drugs in the ischemic area is often not ideal. The gastroscope technology of nano carbon can make the drug release in the focus as much as possible, and can achieve the goal of targeted treatment. This paper mainly studies the application of the gastroscope technology based on nano carbon in the remedy of chronic pulmonary heart disease caused by helicobacter pylori. After 8 weeks of treatment, the indexes of right heart function in the two groups: TAPSE and RVMPI were better than before treatment, and the improvement degree in the remedy group was more obvious than that in the control group (P < 0.05). The improvement degree of E/A and PASP was not statistically significant, the difference between the two groups was not significant (P > 0.05). In addition, the gastroscope of nano carbon can also reduce UA and TG in blood. The overall response rate was 93.75% in the remedy group, which was higher than that in the control group (P < 0.05).

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.

Oral administration of ampelopsin protects against acute brain injury in rats following focal cerebral ischemia.

Ampelopsin (AMP) is isolated from the Chinese medicinal herb Ampelopsis grossedentata (Hand-Mazz) and has been associated with numerous biological and pharmacological activities. However, it is not clear whether AMP has a direct protective effect on cerebral ischemia reperfusion injury. Therefore, the present study investigated its role in acute brain injury following focal cerebral ischemia in rats. The current study induced transient focal cerebral ischemia by performing middle cerebral artery occlusion (MCAO) for 60 min, followed by 24 h of reperfusion. Rats were exposed to 40, 80 and 160 mg/kg AMP by oral administration 30 min prior to MCAO and the cysteinyl leukotriene receptor 1-antagonist, pranlukast (0.1 mg/kg, i.p.) was used as a positive control. Neurological deficit scores were observed and an inclined board test was used to assess behavioral dysfunction. The coronal slices were stained with 3,5-triphenyltetrazolium chloride to determine the infarct volume and brain edema. Neuronal morphology was assessed in brain sections stained with cresyl violet and degenerating neurons were identified using Fluoro-Jade B staining. Blood-brain barrier permeability was determined with immunoglobulin (Ig)G immunohistochemistry. Interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α) in serum and cerebrospinal fluid were measured using ELISA kits. AMP at 80 and 160 mg/kg attenuated neurological deficits, reduced infarct volume, brain edema, IgG exudation and neuron degeneration and loss. Similar to pranlukast, AMP also inhibited the MCAO-induced IL-1ß and TNF-α release. Thus, AMP has a neuroprotective effect on acute brain injury following focal cerebral ischemia in rats at an effective oral dose of 80-160 mg/kg. The results of the current study indicate a therapeutic role for AMP in the treatment of ischemic stroke.