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We developed and validated a reliable, robust, and easy-to-implement quantitative method for multielemental analysis of low-volume samples. Our ICP-MS-based method comprises the analysis of 20 elements (Mg, P, S, K, Ca, V, Cr, Mn, Fe, Co, Cu, Zn, Se, Br, Rb, Sr, Mo, I, Cs, and Ba) in 10 μL of serum and 12 elements (Mg, S, Mn, Fe, Co, Cu, Zn Se, Br, Rb, Mo, and Cs) in less than 250 000 cells. As a proof-of-concept, we analyzed the elemental profiles of serum and sorted immune T cells derived from naı̈ve and tumor-bearing mice. The results indicate a tumor systemic effect on the elemental profiles of both serum and T cells. Our approach highlights promising applications of multielemental analysis in precious samples such as rare cell populations or limited volumes of biofluids that could provide a deeper understanding of the essential role of elements as cofactors in biological and pathological processes.

Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.

Background and aims: Brain white matter hyperintensities (WMHs) have been associated with an increased risk of ischemic stroke and considered as markers of brain ischemia. Progression of WMHs in asymptomatic patients with non-hemodynamically significant carotid plaque could represent a putative marker of plaque vulnerability. We prospectively evaluate progression and determinants of WMHs in this population. Methods: This prospective study included 51 asymptomatic patients with carotid stenosis

Summary In type 1 diabetes, a renewable source of human pancreatic β cells, in particular from human induced pluripotent stem cell (hiPSC) origin, would greatly benefit cell therapy. Earlier work showed that pancreatic progenitors differentiated from human embryonic stem cells in vitro can further mature to become glucose responsive following macroencapsulation and transplantation in mice. Here we took a similar approach optimizing the generation of pancreatic progenitors from hiPSCs. This work demonstrates that hiPSCs differentiated to pancreatic endoderm in vitro can be efficiently and robustly generated under large-scale conditions. The hiPSC-derived pancreatic endoderm cells (HiPECs) can further differentiate into glucose-responsive islet-like cells following macroencapsulation and in vivo implantation. The HiPECs can protect mice from streptozotocin-induced hyperglycemia and maintain normal glucose homeostasis and equilibrated plasma glucose concentrations at levels similar to the human set point. These results further validate the potential use of hiPSC-derived islet cells for application in clinical settings., Highlights • hiPSCs can be efficiently differentiated to pancreatic endoderm in a large-scale setup • HiPECs can further differentiate into glucose-responsive β cells in vivo • HiPEC-derived β cells can protect mice from STZ-induced hyperglycemia, In this article, Kraus and colleagues show that human induced pluripotent stem cells can robustly and efficiently be differentiated to pancreatic endoderm in a large-scale setup. Moreover, after macroencapsulation and further in vivo maturation, hiPSC-derived pancreatic endoderm cells can give rise to glucose-responsive β cells, which protect mice from STZ-induced hyperglycemia.

Summary Diurnal oscillations of gene expression controlled by the circadian clock and its connected feeding rhythm enable organisms to coordinate their physiologies with daily environmental cycles. While available techniques yielded crucial insights into regulation at the transcriptional level, much less is known about temporally controlled functions within the nucleus and their regulation at the protein level. Here, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC proteomics. We identified around 5,000 nuclear proteins, over 500 of which showed a diurnal accumulation. Parallel analysis of the nuclear phosphoproteome enabled the inference of the temporal activity of kinases accounting for rhythmic phosphorylation. Many identified rhythmic proteins were parts of nuclear complexes involved in transcriptional regulation, ribosome biogenesis, DNA repair, and the cell cycle and its potentially associated diurnal rhythm of hepatocyte polyploidy. Taken together, these findings provide unprecedented insights into the diurnal regulatory landscape of the mouse liver nucleus., Graphical Abstract, Highlights • SILAC nuclear proteomics uncovered new diurnal regulatory landscape of mouse liver • Regulation of the diurnal nuclear proteome is mostly post-translational • Diurnal proteins regulate transcription, ribosome biogenesis, DNA repair, and cell cycle • Hepatocyte polyploidy and size oscillate diurnally, Wang et al. quantify the temporal nuclear accumulation of proteins and phosphoproteins in the mouse liver and reveal that 13% of nuclear proteins exhibit a diurnal rhythm regulated at the post-translational level through nuclear transport of protein complexes involved in transcription, DNA repair, ribosome biogenesis, cell cycle, and polyploidy.

The development of cell-based biosensors that give insight into cell and tissue function in vivo is an attractive technology for biomedical research. Here, the development of a cell line expressing a fluorescent calcium sensor for the study of beta-cell function in vivo is reported. The bioresponsive cell model is based on INS-1E pancreatic beta-cells, stably expressing the genetically encoded cameleon-based fluorescent sensor YC3.6(cyto). Following single-cell selection and expansion, functional testing and in vitro encapsulation experiments are used to identify a suitable clone of INS-1E cells expressing the calcium sensor. This clone is transplanted subcutaneous in mouse using a cell macroencapsulation system based on flat sheet porous membranes. Cells in the implanted capsules are able to respond to glucose in vivo by secreting insulin and thereby contributing to the regulation of glycaemia in the mice. Furthermore, fluorescence imaging of explanted devices shows that encapsulated cells maintain high level expression of YC3.6(cyto) in vivo. In conclusion, these data show that encapsulated INS-1E cells stably expressing a genetically encoded calcium sensor can be successfully implanted in vivo, and therefore serve as biosensing element or in vivo model to longitudinally monitor the function of pancreatic beta-cells.

Skeletal muscle is a regenerative tissue which can repair damaged myofibers through the activation of tissue-resident muscle stem cells (MuSCs). Many muscle diseases with impaired regeneration cause excessive adipose tissue accumulation in muscle, alter the myogenic fate of MuSCs, and deregulate the cross-talk between MuSCs and fibro/adipogenic progenitors (FAPs), a bi-potent cell population which supports myogenesis and controls intra-muscular fibrosis and adipocyte formation. In order to better characterize the interaction between adipogenesis and myogenesis, we studied muscle regeneration and MuSC function in whole body Pparg null mice generated by epiblast-specific Cre/lox deletion (Pparg&Delta, /&Delta, ). We demonstrate that deletion of PPAR&gamma, completely abolishes ectopic muscle adipogenesis during regeneration and impairs MuSC expansion and myogenesis after injury. Ex vivo assays revealed that perturbed myogenesis in Pparg&Delta, mice does not primarily result from intrinsic defects of MuSCs or from perturbed myogenic support from FAPs. The immune transition from a pro- to anti-inflammatory MuSC niche during regeneration is perturbed in Pparg&Delta, mice and suggests that PPAR&gamma, signaling in macrophages can interact with ectopic adipogenesis and influence muscle regeneration. Altogether, our study demonstrates that a PPAR&gamma, dependent adipogenic response regulates muscle fat infiltration during regeneration and that PPAR&gamma, is required for MuSC function and efficient muscle repair.

Monocytes are known to play a key role in the initiation and progression of atherosclerosis and contribute to plaque destabilization through the generation of signals that promote inflammation and neoangiogenesis. In humans, studies investigating the features of circulating monocytes in advanced atherosclerotic lesions are lacking.Patients (mean age 69 years, 56% males) with intermediate asymptomatic carotid stenosis (40-70% in diameter) were evaluated for maximal stenosis in common carotid artery, carotid bulb and internal carotid artery, overall disease burden as estimated with total plaque area (TPA), greyscale and neovascularization in 244 advanced carotid plaques. Absolute counts of circulating CD14+ monocytes, of classical (CD14No correlation was found between monocytes and overall atherosclerotic burden, nor with high sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6). In contrast, plaque signs of neovascularization were associated with significantly lower counts of circulating CD14+ monocytes (297 versus 350 cells/mmNeovascularized atherosclerotic lesions selectively associate with lower blood levels of CD14+ and CD14

Background and aims Monocytes are known to play a key role in the initiation and progression of atherosclerosis and contribute to plaque destabilization through the generation of signals that promote inflammation and neoangiogenesis. In humans, studies investigating the features of circulating monocytes in advanced atherosclerotic lesions are lacking. Methods Patients (mean age 69 years, 56% males) with intermediate asymptomatic carotid stenosis (40–70% in diameter) were evaluated for maximal stenosis in common carotid artery, carotid bulb and internal carotid artery, overall disease burden as estimated with total plaque area (TPA), greyscale and neovascularization in 244 advanced carotid plaques. Absolute counts of circulating CD14+ monocytes, of classical (CD14 high CD16−), intermediate (CD14 high CD16+) and non-classical (CD14 low CD16+) monocytes and HLA-DR+ median fluorescence intensity for each subset were evaluated with flow cytometry. Results No correlation was found between monocytes and overall atherosclerotic burden, nor with high sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6). In contrast, plaque signs of neovascularization were associated with significantly lower counts of circulating CD14+ monocytes (297 versus 350 cells/mm 3 , p = 0.039) and of classical monocytes (255 versus 310 cells/mm 3 , p = 0.029). Conclusions Neovascularized atherosclerotic lesions selectively associate with lower blood levels of CD14+ and CD14 high CD16− monocytes independently of systemic inflammatory activity, as indicated by normal hsCRP levels. Whether the reduction of circulating CD14+ and CD14 high CD16− monocytes is due to a potential redistribution of these cell types into active lesions remains to be explored.