Combining segmental bulk- and single-cell RNA-sequencing to define the chondrocyte gene expression signature in the murine knee joint.

OBJECTIVE: Due to the small size of the murine knee joint, extracting the chondrocyte transcriptome from articular cartilage (AC) is a major technical challenge. In this study, we demonstrate a new pragmatic approach of combining bulk RNA-sequencing (RNA-seq) and single cell (sc)RNA-seq to address this problem. DESIGN: We propose a new cutting strategy for the murine femur which produces three segments with a predictable mixed cell population, where one segment contains AC and growth plate (GP) chondrocytes, another GP chondrocytes, and the last segment only bone and bone marrow. We analysed the bulk RNA-seq of the different segments to find distinct genes between the segments. The segment containing AC chondrocytes was digested and analysed via scRNA-seq. RESULTS: Differential expression analysis using bulk RNA-seq identified 350 candidate chondrocyte gene in the AC segment. Gene set enrichment analysis of these genes revealed biological processes related- and non-related to chondrocytes, including, cartilage development (adj. P-value: 3.45E-17) and endochondral bone growth (adj. P-value 1.22E-4), respectively. ScRNA-seq of the AC segment found a cluster of 131 cells containing mainly chondrocytes. This cluster had 759 differentially expressed genes which enriched for extracellular matrix organisation (adj. P-value 7.76E-40) and other joint development processes. The intersection of the gene sets of bulk- and scRNA-seq contained 75 genes. CONCLUSIONS: Based on our results, we conclude that the combination of the two RNA-seq methods is necessary to precisely delineate the chondrocyte transcriptome and to study the disease phenotypes of chondrocytes in murine OA models in the future.

Gut memories do not fade: epigenetic regulation of lasting gut homing receptor expression in CD4+ memory T cells.

The concept of a "topographical memory" in lymphocytes implies a stable expression of homing receptors mediating trafficking of lymphocytes back to the tissue of initial activation. However, a significant plasticity of the gut-homing receptor α4ß7 was found in CD8+ T cells, questioning the concept. We now demonstrate that α4ß7 expression in murine CD4+ memory T cells is, in contrast, imprinted and remains stable in the absence of the inducing factor retinoic acid (RA) or other stimuli from mucosal environments. Repetitive rounds of RA treatment enhanced the stability of de novo induced α4ß7. A novel enhancer element in the murine Itga4 locus was identified that showed, correlating to stability, selective DNA demethylation in mucosa-seeking memory cells and methylation-dependent transcriptional activity in a reporter gene assay. This implies that epigenetic mechanisms contribute to the stabilization of α4ß7 expression. Analogous DNA methylation patterns could be observed in the human ITGA4 locus, suggesting that its epigenetic regulation is conserved between mice and men. These data prove that mucosa-specific homing mediated by α4ß7 is imprinted in CD4+ memory T cells, reinstating the validity of the concept of "topographical memory" for mucosal tissues, and imply a critical role of epigenetic mechanisms.

Non-destructive mobile monitoring of microbial contaminations on meat surfaces using porphyrin fluorescence intensities.

A non-destructive mobile system for meat quality monitoring was developed and investigated for the possible application along the whole production chain of fresh meat. Pork and lamb meat was stored at 5 °C for up to 20 days post mortem and measured with a fluorescence spectrometer. Additionally, the bacterial influence on the fluorescence signals was evaluated by different experimental procedures. Fluorescence of NADH and different porphyrins could be correlated to the growth of diverse bacteria and hence used for contamination monitoring. The increase of porphyrin fluorescence started after 9 days p.m. for pork and after 2 days p.m. for lamb meat. Based on the results, a mobile fluorescence system was built and compared with the laboratory system. The corrected function of the meat slices showed a root mean square error of 1156.97 r.u. and a mean absolute percentage error of 12.59%; for lamb the values were 470.81 r.u. and 15.55%, respectively. A mobile and non-invasive measurement system would improve the microbial security of fresh meat.