Treponema Endosymbionts are the Dominant Bacterial Members with Ureolytic Potential in the Gut of the Wood-Feeding Termite, Reticulitermes Hesperus

Termites are remarkable for their ability to digest cellulose from wood as their main energy source, but the extremely low nitrogen (N) content of their diet presents a major challenge for N acquisition. Besides the activity of N 2 -fixing bacteria in the gut, the recycling of N from waste products by symbiotic microbes as a complementary N-provisioning mechanism in termites remains poorly understood. In this study, we used a combination of high-throughput amplicon sequencing, quantitative PCR, and cultivation to characterize the microbial community capable of degrading urea, a common waste product, into ammonia in the guts of termites ( Reticulitermes hesperus ) from a wild and laboratory-reared colony. Taxonomic analysis indicated that a majority of the urease ( ureC ) genes in the termite gut (53.0%) matched with a Treponema endosymbiont of gut protists previously found in several other termites, suggesting an important contribution to the nutrition of essential cellulolytic protists. Furthermore, analysis of both the 16S rRNA and ureC amplicons revealed that the laboratory colony had decreased diversity and altered community composition for both prokaryotic and ureolytic microbial communities in the termite gut. Estimation by quantitative PCR showed that microbial ureC genes decreased in abundance in the laboratory-reared colony compared to the wild colony. In addition, most of our cultivated isolates appeared to originate from non-gut environments. Together, our results underscore a more important role for ureolysis by endosymbionts within protists than by free-swimming bacteria in the gut lumen of R. hesperus .

Identification of SRXN1 and KRT6A as Key Genes in Smoking-Related Non-Small-Cell Lung Cancer Through Bioinformatics and Functional Analyses

BackgroundLung cancer is the leading cause of cancer-related mortality worldwide. Although cigarette smoking is an established risk factor for lung cancer, few reliable smoking-related biomarkers for non-small-cell lung cancer (NSCLC) are available. An improved understanding of these biomarkers would further the development of new biomarker-targeted therapies and lead to improvements in overall patient survival.MethodsWe performed bioinformatic analysis to screened potential target genes, then quantitative PCR, western, siRNA, CCK-8, flow cytometry, tumorigenicity assays in nude mice were performed to validated the function.ResultsIn this study, we identified 83 smoking-related genes (SRGs) based on an integration analysis of two Gene Expression Omnibus (GEO) datasets, and 27 hub SRGs with potential carcinogenic effects by analyzing a dataset of smokers with NSCLC in The Cancer Genome Atlas (TCGA) database. A survival analysis revealed three genes with potential prognostic value, namely SRXN1, KRT6A and JAKMIP3. A univariate Cox analysis revealed significant associations of elevated SRXN1 and KRT6A expression with prognosis. A receiver operating characteristic (ROC) curve analysis indicated the high diagnostic value of SRXN1 and KRT6A for smoking and cancer. Quantitative PCR and western blotting validated the increased expression of SRXN1 and KRT6A mRNA and protein, respectively, in lung cancer cell lines and NSCLC tissues. In patients with NSCLC, SRXN1 and KRT6A expression was associated with the tumor–node–metastasis (TNM) stage, presence of metastasis, history of smoking and daily smoking consumption. Furthermore, inhibition of SRXN1 or KRT6A suppressed viability and enhanced apoptosis in the A549 human lung carcinoma cell line. Tumorigenicity assays in nude mice confirmed that the siRNA-mediated downregulation of SRXN1 and KRT6A expression inhibited tumor growth in vivo.ConclusionsIn summary, SRXN1 and KRT6A act as oncogenes in NSCLC and might be potential biomarkers of smoking exposure and the early diagnosis and prognosis of NSCLC in smokers, which is vital for lung cancer therapy.

Experimental reproduction of White Feces Syndrome in whiteleg shrimp, Penaeus vannamei

White Feces Syndrome (WFS) is an emergent disease of penaeid shrimp (Penaeus monodon and P. vannamei) that is identified by the presence of floating white fecal strings on pond water in grow-out ponds. Although the clinical manifestations of WFS are well defined, the underling etiology remains obscure. WFS has been associated with several enteric pathogens, including Enterocytozoon hepatopenaei (EHP). The association is based on studies that found areas where WFS has been reported, the prevalence and severity of EHP infection are high. In this study, we describe an experimental reproduction of WFS in P. vannamei pre-infected with EHP and challenged with a unique isolate of Vibrio parahaemolyticus isolated from the gastrointestinal tract of a shrimp displaying WFS. Upon laboratory challenge, shrimp displaying white fecal strings and white discoloration of the gastrointestinal tract were analyzed by histopathology, in-situ hybridization and quantitative PCR. Histological analysis confirmed the lesions of EHP and septic hepatopancreatic necrosis in the hepatopancreas of shrimp exposed to both pathogens. Quantitative PCR showed shrimp infected with both EHP and V. parahaemolyticus had a significantly higher load of EHP compared to shrimp infected with EHP alone. This is the first demonstration of experimental reproduction of WFS under laboratory conditions when animals are infected with EHP and V. parahaemolyticus concurrently. The data revealed a synergistic relation between EHP and V. parahaemolyticus isolate that led to the manifestation of WFS. We propose the gross signs of WFS can be used as an indicator of the presence of EHP infection in association with a particular strain of an enteric Vibrio spp. in countries where EHP is endemic.

Adapted Protocol for Saccharibacteria Cocultivation: Two New Members Join the Club of Candidate Phyla Radiation

In this study, the first TaqMan real-time quantitative PCR (qPCR) system has been developed. This technique can specifically quantify Saccharibacteria members in any sample of interest in order to investigate their prevalence.

Effect of Ag Nanoparticles on Denitrification and Microbial Community in a Paddy Soil

The extensive application of Ag nanoparticles (AgNPs) in industry, agriculture, and food processing areas increases the possibility of its release and accumulation to agroecosystem, but the effects of AgNPs to denitrification and the microbial community in paddy ecosystems are still poorly studied. In this study, microcosmic simulation experiments were established to investigate the response of soil denitrification to different levels of AgNPs (i.e., 0.1, 1, 10, and 50 mg/kg) in a paddy soil. Real-time quantitative PCR and high-throughput sequencing were conducted to reveal the microbial mechanism of the nanometer effect. The results showed that, though 0.1–10 mg/kg AgNPs had no significant effects on denitrification rate and N2O emission rate compared to CK and bulk Ag treatments, 50 mg/kg AgNPs significantly stimulated more than 60% increase of denitrification rate and N2O emission rate on the 3rd day (P < 0.05). Real-time quantitative PCR revealed that 50 mg/kg AgNPs significantly decreased the abundance of 16S bacterial rRNA gene, nirS/nirK, cnorB, and nosZ genes, but it did not change the narG gene abundance. The correlation analysis further revealed that the cumulative N2O emission was positively correlated with the ratio of all the five tested denitrifying genes to bacterial 16S rRNA gene (P < 0.05), indicating that the tolerance of narG gene to AgNPs was the key factor of the increase in denitrification in the studied soil. High-throughput sequencing showed that only the 50-mg/kg-AgNP treatment significantly changed the microbial community composition compared to bulk Ag and CK treatments. The response of microbial phylotypes to AgNPs suggested that the most critical bacteria which drove the stimulation of 50 mg/kg AgNPs on N2O emission were Firmicutes and β-proteobacteria, such as Clotridiales, Burkholderiales, and Anaerolineales. This study revealed the effects of AgNPs to denitrification in a paddy ecosystem and could provide a scientific basis for understanding of the environmental and toxicological effects of Ag nanomaterials.

High-quality RNA improves sensitivity of SARS-CoV-2 detection by colorimetric RT-LAMP

The global SARS-CoV-2 pandemic requires a rapid, reliable, and user-friendly diagnostic test to help control the spread of the virus. Reverse transcription and quantitative PCR (RT-qPCR) is currently the gold standard method for SARS-CoV-2 detection. Here, we develop a protocol based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and demonstrate increased sensitivity of this technique using fresh RNA extracts compared to RNA samples subjected to freezing/thawing cycles. We further compare RT-LAMP to RT-qPCR and demonstrate that the RT-LAMP approach has high sensitivity in fresh RNA extracts and can detect positive samples with Ct values between 8 and 35.