Chemokine receptor 7 contributes to T- and B-cell filtering in ageing bladder, cystitis and bladder cancer.

BACKGROUND: Research has suggested significant correlations among ageing, immune microenvironment, inflammation and tumours. However, the relationships among ageing, immune microenvironment, cystitis and bladder urothelial carcinoma (BLCA) in the bladder have rarely been reported. METHODS: Bladder single-cell and transcriptomic data from young and old mice were used for immune landscape analysis. Transcriptome, single-cell and The Cancer Genome Atlas Program datasets of BLCA and interstitial cystitis/bladder pain syndrome (IC/BPS) were used to analyse immune cell infiltration and molecular expression. Bladder tissues from mice, IC/BPS and BLCA were collected to validate the results. RESULTS: Eight types of immune cells (macrophages, B-cells, dendritic cells, T-cells, monocytes, natural killer cells, γδ T-cells and ILC2) were identified in the bladder of mice. Aged mice bladder tissues had a significantly higher number of T-cells, γδ T-cells, ILC2 and B-cells than those in the young group (P < 0.05). Three types of T-cells (NK T-cells, γδ T-cells and naïve T-cells) and three types of B-cells (follicular B-cells, plasma and memory B-cells) were identified in aged mice bladder. Chemokine receptor 7 (CCR7) is highly expressed in aged bladder, IC/BPS and BLCA (P < 0.05). CCR7 is likely to be involved in T- and B-cell infiltration in aged bladder, IC/BPS and BLCA. Interestingly, the high CCR7 expression on BLCA cell membranes was a prognostic protective factor. CONCLUSIONS: In this study, we characterised the expression profiles of immune cells in bladder tissues of aged and young mice and demonstrated that CCR7-mediated T- and B-cell filtration contributes to the development of bladder ageing, IC/BPS and BLCA.

Alteration of the gut microbiota profile in children with autism spectrum disorder in China.

Background: Autism spectrum disorder (ASD) is associated with alterations in the gut microbiome. However, there are few studies on gut microbiota of children with ASD in China, and there is a lack of consensus on the changes of bacterial species. Purpose: Autism spectrum disorder (ASD) is associated with alterations in the gut microbiome. However, there are few studies on gut microbiota of children with ASD in China, and there is a lack of consensus on the changes of bacterial species. Methods: We used 16S rRNA sequencing to analyze ASD children (2 to 12 years), HC (2 to 12 years). Results: Our findings showed that the α-diversity, composition, and relative abundance of gut microbiota in the ASD group were significantly different from those in the HC groups. Compared with the HC group, the α-diversity in the ASD group was significantly decreased. At the genus level, the relative abundance of g_Faecalibacterium, g_Blautia, g_Eubacterium_eligens_group, g_Parasutterella, g_Lachnospiraceae_NK4A136_group and g_Veillonella in ASD group was significantly increased than that in HC groups, while the relative abundance of g_Prevotella 9 and g_Agathobacter was significantly decreased than that in HC groups. In addition, KEGG pathway analysis showed that the microbial functional abnormalities in ASD patients were mainly concentrated in metabolic pathways related to fatty acid, amino acid metabolism and aromatic compound metabolism, and were partially involved in neurotransmitter metabolism. Conclusion: This study revealed the characteristics of gut microbiota of Chinese children with ASD and provided further evidence of gut microbial dysbiosis in ASD.

A novel siRNA validation system for functional screening of effective RNAi targets in mammalian cells and development of a derivative lentivirus delivery system.

RNA interference technology is a widely used tool for the regulation of gene expression at the post-transcriptional level. One major challenge is to find the effective short interfering (si)RNA for target gene rapidly and easily, and then to deliver the siRNA into cells or tissues with high efficiency. Here, we designed a novel siRNA validation vector using a dual luciferase reporter system for the functional screening of effective RNAi targets in mammalian cells. Then, based on a siRNA expression cassette, we developed a derivative lentivirus delivery system to infect the appropriate cells or tissues for the efficient knockdown of target gene expression. Based on this system, we used human IRF7 gene, a key regulatory factor for the differentiation of monocytes to macrophages, as an example. We screened for the optimal siRNA, then packaged it into a lentiviral siRNA expression system. Then, monocytes were infected and we confirmed the knockdown of IRF7 expression could inhibit the differentiation of monocytes to macrophages. To validate our method further, we also screened and identified optimal siRNA for human TLR4 gene. In summary, we developed a novel siRNA validation system to identify optimal siRNA to target genes rapidly. In addition, the lentivirus system is an efficient tool for siRNA delivery for the further study of target gene function. Taken together, this represents an efficient and user-friendly strategy to validate and deliver siRNAs.

A simple negative selection method to identify adenovirus recombinants using colony PCR

Background: The AdEasy system is a fast-track system for generating recombinant adenoviruses using the efficient homologous recombination machinery between shuttle and adenovirus backbone plasmids in Escherichia coli BJ5183 cells. The key step is homologous recombination in BJ5183 cells, which is driven by RecA activity. However, culture time is stringently limited to reduce the damage to recombinant plasmids by RecA activity. Therefore, rapid identification of recombinant adenoviruses within the limited time-period is critical. Results: We developed a simple negative selection method to identify recombinant adenoviruses using colony PCR, which improves the efficiency of adenovirus recombination screening and packaging. Conclusions: The negative selection method to identify AdEasy adenovirus recombinants by colony PCR can identify the recombined colony within a short time-period, and maximally avoid damage to the recombinant plasmid by limiting recombination time, resulting in improved adenovirus packaging.