Combining current knowledge on DNA methylation-based age estimation towards the development of a superior forensic DNA intelligence tool.

The estimation of chronological age from biological fluids has been an important quest for forensic scientists worldwide, with recent approaches exploiting the variability of DNA methylation patterns with age in order to develop the next generation of forensic 'DNA intelligence' tools for this application. Drawing from the conclusions of previous work utilising massively parallel sequencing (MPS) for this analysis, this work introduces a DNA methylation-based age estimation method for blood that exhibits the best combination of prediction accuracy and sensitivity reported to date. Statistical evaluation of markers from 51 studies using microarray data from over 4000 individuals, followed by validation using in-house generated MPS data, revealed a final set of 11 markers with the greatest potential for accurate age estimation from minimal DNA material. Utilising an algorithm based on support vector machines, the proposed model achieved an average error (MAE) of 3.3 years, with this level of accuracy retained down to 5 ng of starting DNA input (~ 1 ng PCR input). The accuracy of the model was retained (MAE = 3.8 years) in a separate test set of 88 samples of Spanish origin, while predictions for donors of greater forensic interest (< 55 years of age) displayed even higher accuracy (MAE = 2.6 years). Finally, no sex-related bias was observed for this model, while there were also no signs of variation observed between control and disease-associated populations for schizophrenia, rheumatoid arthritis, frontal temporal dementia and progressive supranuclear palsy in microarray data relating to the 11 markers.

Cxcl9l and Cxcr3.2 regulate recruitment of osteoclast progenitors to bone matrix in a medaka osteoporosis model.

Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka (Oryzias latipes) osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits mpeg1-positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in cxcr3.2 prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.

Lineage tracing of col10a1 cells identifies distinct progenitor populations for osteoblasts and joint cells in the regenerating fin of medaka (Oryzias latipes).

The caudal fin of teleost fish regenerates fully within two weeks of amputation. While various cell lineages have been identified and characterized in the regenerating fin, the origin of bone cells remains debated. Here, we analysed collagen10a1 (col10a1) expressing cells in the regenerating fin of the medaka (Oryzias latipes) and tested whether they represent an alternative progenitor source for regenerating osteoblasts. Under normal conditions, col10a1 cells are positioned along fin ray segments and in intersegmental regions. Lineage tracing in the amputated fin revealed that col10a1 cells from the stump contribute to the regenerating bony fin rays. However, ablation of col10a1 cells did not abolish fin regeneration suggesting that col10a1 expressing osteoblast progenitors are dispensable for regeneration. Intriguingly, however, after ablation of osterix (osx)/sp7-col10a1 double-positive osteoblasts, col10a1 cells exclusively gave rise to joint cells in the intersegmental region thus identifying a pool of lineage-restricted joint progenitor cells. To identify additional sources for regenerating osteoblasts, we performed clonal lineage analysis. Our data provide the first evidence that after ablation of mature osteoblasts in medaka, transdifferentiation does not account for de novo osteoblast generation. Instead, our findings suggest the presence of lineage restricted progenitor pools in medaka, similar to the situation in zebrafish. After osteoblast ablation, these pools become activated and give rise to fin ray osteoblasts and intersegmental joint cells during regeneration. In summary, we conclude that col10a1-positive cells do not represent an exclusive source for osteoblasts but are progenitors of joint cells in the regenerating fin.