Single cell multi-omics characterise discrete human tendon cells populations that persist in vitro and on fibrous scaffolds.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3_PAPPA, POSTN_SCX, DCN_LUM and ITGA7_NES. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+ and POSTN_SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN_LUM, POSTN_SCX and PTX3_ PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.

Differential regulation of CaMKII inhibitor beta protein expression after exposure to a novel context and during contextual fear memory formation.

Understanding of the molecular basis of long-term fear memory (fear LTM) formation provides targets in the treatment of emotional disorders. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is one of the key synaptic molecules involved in fear LTM formation. There are two endogenous inhibitor proteins of CaMKII, CaMKII N alpha and N beta, which can regulate CaMKII activity in vitro. However, the physiological role of these endogenous inhibitors is not known. Here, we have investigated whether CaMKII N beta protein expression is regulated after contextual fear conditioning or exposure to a novel context. Using a novel CaMKII N beta-specific antibody, CaMKII N beta expression was analysed in the naïve mouse brain as well as in the amygdala and hippocampus after conditioning and context exposure. We show that in naïve mouse forebrain CaMKII N beta protein is expressed at its highest levels in olfactory bulb, prefrontal and piriform cortices, amygdala and thalamus. The protein is expressed both in dendrites and cell bodies. CaMKII N beta expression is rapidly and transiently up-regulated in the hippocampus after context exposure. In the amygdala, its expression is regulated only by contextual fear conditioning and not by exposure to a novel context. In conclusion, we show that CaMKII N beta expression is differentially regulated by novelty and contextual fear conditioning, providing further insight into molecular basis of fear LTM.

Expression studies of CLN3 protein.

Expression of the gene for Batten disease (CLN3) was studied in Escherichia coli and in a cell-free rabbit reticulocyte expression systems. A full-length recombinant fusion CLN3 protein was not produced in the bacterial systems used. However, both N-terminal fragment encompassing 246 amino acids and short C-terminal fragment containing 428-438 amino acids of the CLN3 protein were successfully overexpressed in bacteria. Further studies showed that the C-terminal sequence of the CLN3 protein corresponding to the 356-438 amino acid residues was responsible for inhibition of protein synthesis in bacteria. The full-length CLN3 gene product was readily synthesized in vitro in the cell-free rabbit reticulocyte expression system. The product obtained, corresponding to core CLN3 protein, showed an approximate molecular weight of 43 kDa. Immunoprecipitation of this product with pAb to 4-19 amino acids of the CLN3 protein allows us to suggest that CLN3 protein translation starts at Met-1.