Srinivasan, M. | bioRxiv Science

bioRxiv2018

Differential Profiles of Soluble and Cellular Toll-like Receptors (TLRs) 2 and 4 in Chronic Periodontitis.

Chronic periodontitis is a common inflammatory disease initiated by a complex microbial biofilm and mediated by the host response causing destruction of the supporting tissues of the teeth. Host recognition of pathogens is mediated by toll-like receptors (TLRs) that bind conserved molecular patterns shared by groups of microorganisms. The oral epithelial cells respond to most periodontopathic bacteria via TLR-2 and TLR-4. Many studies have previously reported the presence of elevated numbers of viable exfoliated epithelial cells (SEC) in the saliva of patients with chronic periodontitis. In addition to the membrane-associated receptors, soluble forms of TLR-2 (sTLR-2) and TLR-4 (sTLR-4) have been identified and are thought to play a regulatory role by binding microbial ligands. sTLR-2 has been shown to arise from ectodomain shedding of the extracellular domain of the membrane receptor and sTLR-4 is thought to be an alternate spliced form. The objective of this study was to investigate the potential value of salivary sTLR-2/4 and the paired epithelial cell-associated TLR-2/4 mRNA as diagnostic markers for chronic periodontitis. Unstimulated whole saliva was collected after obtaining informed consent from 40 individuals in either periodontitis or gingivitis cohorts. The levels of sTLR-2/4 were measured by enzyme-linked immunosorbent assay (ELISA). SEC TLR-2/4 transcripts were quantitated by real time polymerase chain reaction. While levels of sTLR-2 exhibited an inverse correlation, sTLR-4 positively correlated with clinical parameters in the gingivitis cohort. Interestingly, both correlations were lost in the periodontitis cohort indicating a dysregulated host response. On the other hand, while sTLR-2 and the paired SEC associated TLR-2 mRNA exhibited a direct correlation (r2=0.62), that of sTLR4 and SEC TLR-4 mRNA exhibited an inverse correlation (r2=0.53) in the periodontitis cohort. Collectively, assessments of salivary sTLR2 and sTLR4 together with the respective transcripts in SECs could provide clinically relevant markers of disease progression from gingivitis to periodontitis.

immunology↗

bioRxiv2017

The cohesin ring uses its hinge to organize DNA using non-topological as well as topological mechanisms

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is a perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion in a large variety of mutants. In most cells where cohesin loads onto chromosomes but fails to form cohesion, loading is accompanied by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen has been neutralized not only loads onto and translocates along chromatin but also organizes it into chromatid-like threads, despite largely failing to entrap DNAs inside its ring. Thus, cohesin engages chromatin in a non-topological as well as a topological manner. Our finding that hinge mutations, but not fusions between Smc and kleisin subunits, abolish entrapment suggests that DNAs may enter cohesin rings through hinge opening. Lastly, mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesins initial recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesins hinge driven by cycles of ATP hydrolysis.

molecular biology↗

bioRxiv2017

STRT-seq-2i: dual-index 5′ single cell and nucleus RNA-seq on an addressable microwell array

Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but currently no high-throughput platform has been used successfully on archived human brain samples. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform.

molecular biology↗