For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexed the real-time mRNA quantification for three T7 promoters on a RT-PCR thermocycler by using BFQ probes with different colored fluorophores that were specific for each target. Ultimately, we created an inexpensive multiplexed method to quantify mRNA production in real-time, and future research could use these methods to measure the affinity of transcriptional repressors to their target DNA sequence.

Gardnerella vaginalis is the main pathogen that causes bacterial vaginosis. In the healthy vaginal microecological environment of a woman, the lactobacilli produce lactate and hydrogen peroxide to inhibit the growth of pathogens such as G. vaginalis. The lack of lactobacilli results in a high pH and low hydrogen peroxide in the vagina which facilitate G. vaginalis growth, leading to the imbalance of the vaginal microecology. In this study, lactate and hydrogen peroxide were added to a G. vaginalis culture medium to simulate the co-culture of the lactobacilli and G. vaginalis, and then the genes related to the stress response of G. vaginalis were identified using transcriptomics and proteomics. It was indicated that, among all the upregulated genes, most of them encoded transporters associated with the efflux of harmful substances, and the majority of the downregulated genes were related to the biofilm formation and epithelial cell adhesion. This study may help find new drug targets for G. vaginalis for the development of novel therapies for bacterial vaginosis.

Toxoplasma gondii (T. gondii) is a globally distributed protozoan parasite, and its chronic infection has been closely associated with various neuropsychiatric disorders. However, the key susceptibility factors and mechanisms by which T. gondii manipulates host behaviors remain insufficiently understood. In this study, based on the comprehensive behavioral analyses including the AI HomeCage platform, and third-generation Nanopore-seq technology, we demonstrated that the T. gondii strain TgCtwh6 induces depression-like symptoms and cognitive impairments in hosts. These effects are linearly correlated with the expression levels of the host dopamine receptor D2 (DRD2). Furthermore, the behavioral abnormalities can be alleviated by treatment with the DRD2 agonist bromocriptine. Both in vivo and in vitro infection models revealed that the downregulation of DRD2 is significantly correlated with neuroinflammation and neuronal injury in the host central nervous system (CNS). Additionally, our findings suggest that the suppression of DRD2 may be regulated by enhanced RNA m⁶A modification following infection and may function through the DRD2/CRYAB/NF-κB signaling axis. These results emphasized the critical role of DRD2 in mediating T. gondii-induced neuroinflammation and neuropsychiatric abnormalities, providing novel insights into the development of therapeutic strategies for neuropsychiatric disorders associated with parasitic infections.

Background: Oocysts serve as the primary source of Toxoplasma infection. Therefore, understanding oocyst development and exploring effective strategies to prevent oocyst excretion are crucial for controlling toxoplasmosis. Methods: In this study, shotgun metagenomics was employed to characterize the functional and compositional changes in the gut microbiota of cats during oocyst development. The Spearman correlation test was utilized to analyze the correlation between differential Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and carbohydrate-active enzymes (CAZymes) in key bacteria regulating oocyst excretion. Results: The results revealed that group A (sexual initiation stage) displayed a lower number of functional genes, which were restored to normal levels in group B (oocyst excretion stage), compared with group C (Toxoplasma-uninfected samples). The abundance of 39 KEGG pathways, 106 CAZymes, and 98 virulence factors (VFs) varied significantly among the three groups. The atrazine degradation pathway, associated with sexual development, was upregulated in group B. CAZymes involved in restoring the intestinal mucosal barrier and VFs related to iron metabolism, antibiotic resistance, and suppression of host immunity were enriched in group B. Sexual initiation and oocyst excretion resulted in reduced gut bacterial diversity and microbiota dysbiosis. Probiotics and bacteria related to linoleic acid (LA) uptake were dominant in both group A and group B. Bacteroides stercoris was the most significantly upregulated bacterium and could influence the expression of carbohydrate-binding modules (CBMs) and glycoside hydrolases (GHs) in group B. Conclusions: During the oocyst development/excretion stage, the function and composition of the cat gut microbiota changed significantly. In addition, Bacteroides stercoris may play a crucial role in oocyst excretion by regulating key candidates of CBMs and GHs. Our findings lay the foundation for investigating the regulatory mechanisms of oocyst excretion. Graphical