Redox-regulation of intrinsic prion expression in multicellular prostate tumor spheroids

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.     

Spatial development of gingival fibroblasts and dental pulp cells: Effect of extracellular matrix

Cells sensing changes in their microenvironmental stiffness and composition alter their responses, accordingly. This study determines whether gingival fibroblasts (GFs) and dental pulp mesenchymal stem cells (DPMSCs) support the formation of continuous layers in vitro by mimicking the stiffness and protein composition of their native extracellular matrix (ECM). Immortalized cells were incubated with (i) 0–100% Matrigel-ECM (M-ECM) for 7-28d, and with (ii) collagen and fibrin matrices for 14d. Cultures were analyzed by phase-contrast, fluorescence and confocal microscopies. The diameters and surface areas were measured via ImageJ. Self-renewal markers were detected by RT-PCR and immunocytochemistry assays. GFs and DPMSCs developed spheroids interconnected by elongated cell bundles or layers, respectively, expressing the self-renewal markers. Increased matrix stiffness resulted in spheroids replacement by the interconnecting cells/layers. Both cells required 100% M-ECM to reduce their spheroid diameter. However, it reduced the surface area of the interconnecting layers. Those differences led to extended, spindle-shaped GFs vs. compact, ring-shaped DPMSCs constructs. Collagen and fibrin matrices developed continuous layers of tightly connected cells vs. distinctive scattered cell aggregates, respectively. The ability of GFs and DPMSCs to create tissue-like multicellular layers at various matrix conditions may be imprinted by cells’ adaptation to mechanical forces and composition in vivo.     

Using size‐controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice

Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane‐based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor‐bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 μm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 μm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin β1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 10⁴ colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor‐bearing mouse models.     

Swarming motility

Swarming involves differentiation of vegetative cells into hyperflagellated swarm cells that undergo rapid and coordinated population migration across solid surfaces. Cell density, surface contact, and physiological signals all provide critical stimuli, and close cell alignment and the production of secreted migration factors facilitate mass translocation. Flagella biogenesis is central to swarming, and the flhDC flagellar master operon is the focal point of a regulatory network governing differentiation and migration.     

多细胞生物干细胞不对称分裂的内在调节机制研究进展

多细胞生物的发育是从一个受精卵分化成多种类型细胞的过程。细胞多样性形成的基础是不等分裂,不等分裂是干细胞自我更新和自我维持的关键。干细胞不等分裂有细胞内和细胞外两种调节机制。果蝇神经干细胞增殖和分化、植物胚胎发育、表皮气孔形成及根内皮层的分化,是研究不等细胞分裂调节机制最多的发育背景。本综述介绍了果蝇神经干细胞和植物胚胎发育早期、表皮气孔发生及根皮层内皮层中细胞不等分裂内在调节机制的研究进展。     

Extracellular calcium as an integrator of tissue function

The past several decades of research into calcium signaling have focused on intracellular calcium (Ca_i²⁺), revealing both exquisite spatial and dynamic control of this potent second messenger. Our understanding of Ca_i²⁺ signaling has benefited from the evolution of cell culture methods, development of high affinity fluorescent calcium indicators (both membrane-permeant small molecules and genetically encoded proteins), and high-resolution fluorescence microscopy. As our understanding of single cell calcium dynamics has increased, translational efforts have attempted to push calcium signaling studies back into tissues, organs and whole animals. Emerging results from these more complicated, diffusion-limited systems have begun to define a role for extracellular calcium (Ca_o²⁺) as an agonist, spurred by the cloning and characterization of a G protein-coupled receptor activated by Ca_o²⁺ (the calcium sensing receptor, CaR). Here, we review the current state-of-the art for measurement of Ca_o²⁺ fluctuations, and the evidence that fluctuations in Ca_o²⁺ can act as primary signals regulating cell function. Current results suggest that Ca_o²⁺ in bone and epidermis may act as a chemotactic homing signal, targeting cells to the appropriate tissue locations prior to initiation of the differentiation program. Ca_i²⁺ signaling-mediated Ca_o²⁺ fluctuations in interstitial spaces may integrate cell signaling responses in multicellular networks through activation of CaR. Appreciation of the importance of Ca_o²⁺ fluctuations in coordinating cell function will likely spur identification of additional, niche-specific Ca²⁺ sensors, and provide unique insights into the regulation of multicellular signaling networks.     

Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus–bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.     

Engineered living materials (ELMs) design: From function allocation to dynamic behavior modulation

Natural materials possess many distinctive “living” attributes, such as self-growth, self-healing, environmental responsiveness, and evolvability, that are beyond the reach of many existing synthetic materials. The emerging field of engineered living materials (ELMs) takes inspiration from nature and harnesses engineered living systems to produce dynamic and responsive materials with genetically programmable functionalities. Here, we identify and review two main directions for the rational design of ELMs: first, engineering of living materials with enhanced performances by incorporating functional material modules, including engineered biological building blocks (proteins, polysaccharides, and nucleic acids) or well-defined artificial materials; second, engineering of smart ELMs that can sense and respond to their surroundings by programming dynamic cellular behaviors regulated via cell–cell or cell–environment interactions. We next discuss the strengths and challenges of current ELMs and conclude by providing a perspective of future directions in this promising area.