The critical regulation of the peripheral circadian gene implicated in osteoarthritis (OA) has been recently recognized; however, the causative role and clinical potential of the peripheral circadian rhythm attributable to such effects remain elusive. The purpose of this study was to elucidate the role of a circadian gene Bmal1 in human cartilage and pathophysiology of osteoarthritis. In our present study, the mRNA and protein levels of circadian rhythm genes, including nicotinamide adenine dinucleotide oxidase (NAD(+)) and sirtuin 1 (Sirt1), in human knee articular cartilage were determined. In OA cartilage, the levels of both Bmal1 and NAD(+) decreased significantly, which resulted in the inhibition of nicotinamide phosphoribosyltransferase activity and Sirt1 expression. Furthermore, the knockdown of Bmal1 was sufficient to decrease the level of NAD(+) and aggravate OA-like gene expression changes under the stimulation of IL-1β. The overexpression of Bmal1 relieved the alteration induced by IL-1β, which was consistent with the effect of the inhibition of Rev-Erbα (known as NR1D1, nuclear receptor subfamily 1, group D). On the other hand, the transfection of Sirt1 small interfering RNA not only resulted in a reduction of the protein expression of Bmal1 and a moderate increase of period 2 (per2) and Rev-Erbα but also further exacerbated the survival of cells and the expression of cartilage matrix-degrading enzymes induced by IL-1β. Overexpression of Sirt1 restored the metabolic imbalance of chondrocytes caused by IL-1β. These observations suggest that Bmal1 is a key clock gene to involve in cartilage homeostasis mediated through sirt1 and that manipulating circadian rhythm gene expression implicates an innovative strategy to develop novel therapeutic agents against cartilage diseases.

Compared with traditional monolayer cell culture, the three-dimensional tumor spheroid has emerged as an essential in vitro model for cancer research due to the recapitulation of the architecture and physiology of solid human tumors. Herein, by implementing the rapid prototyping of a benchtop 3D printer, we developed a new strategy to generate and analyze tumor spheroids on a commonly used multi-well plate. In this method, the printed artifact can be directly mounted on a 96/384-well plate, enables hanging drop-based spheroid formation, avoiding the tedious fabrication process from micromechanical systems. Besides long-term spheroid culture (20 days), this method supports subsequent analysis of tumor spheroid by seamlessly dripping from the printed array, thereby eliminating the need for spheroids retrieval for downstream characterization. We demonstrated several tumor spheroid-based assays, including tumoroid drug testing, metastasis on or inside extracellular matrix gel, and tumor transendothelial (TEM) assay. Based on quantitative phenotypical and molecular analysis without any precarious retrieval and transfer, we found that the malignant breast cancer (MDA-MB-231) cell aggregate presents a more metastatic morphological phenotype than the non-malignant breast cancer (MCF-7) and colonial cancer (HCT-116) cell spheroid, and shows an up-regulation of epithelial-mesenchymal transition (EMT) relevant genes (fold change > 2). Finally, we validated this tumor malignancy by the TEM assay, which could be easily performed using our approach. This methodology could provide a useful workflow for expediting tumoroid modeled in vitro assay, allowing the “Lab-on-a-Cloud” scenario for routine study.

BackgroundThe trehalose (Tre) pathway has strong effects on growth and development in plants through regulation of carbon metabolism. Altering either Tre or trehalose 6-phosphate (T6P) can improve growth and productivity of plants as observed under different water availability. As yet, there are no reports of the effects of modification of Tre orT6P on plant performance under limiting nutrition.ResultsHere we report that nitrogen (N) metabolism is positively affected by exogenous application of Tre in nitrogen-deficient growing conditions. Spraying foliage of tobacco (Nicotiana tabacum) with trehalose partially alleviated symptoms of nitrogen deficiency through upregulation of nitrate and ammonia assimilation and increasing activities of nitrate reductase (NR), glycolate oxidase (GO), glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) with concomitant changes in ammonium (NH4 +) and nitrate (NO3 −) concentrations, glutamine and amino acids. Chlorophyll and total nitrogen content of leaves and rates of photosynthesis were increased compared to nitrogen-deficient plants without applied Tre. Total plant biomass accumulation was also higher in Tre -fed nitrogen-deficient plants, with a smaller proportion of dry weight partitioned to roots, compared to nitrogen-deficient plants without applied Tre. Consistent with higher nitrogen assimilation and growth, Tre application reduced foliar starch. Minimal effects of Tre feeding were observed on nitrogen-sufficient plants.ConclusionsThe data show, for the first time, significant stimulatory effects of exogenous Tre on nitrogen metabolism and growth in plants growing under deficient nitrogen. Under such adverse conditions metabolism is regulated for survival rather than productivity. Application of Tre can alter this regulation towards maintenance of productive functions under low nitrogen. This has implications for considering approaches to modifying the Tre pathway for to improve crop nitrogen-use efficiency and production.

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