New Insights on the Diurnal Mechanism of Calcification in the Stony Coral, Stylophora pistillata

Scleractinian corals are evolutionary-successful calcifying marine organisms, which utilize an endo-symbiotic relationship with photosynthetic dinoflagellate algae that supply energy products to their coral hosts. This energy further supports a higher calcification rate during the day in a process known as light enhanced calcification. Although this process has been studied for decades, the mechanisms behind it are still unknown. However, photosynthesis and respiration also cause daily fluctuations in oxygen and pH levels, resulting in the coral facing highly variable conditions. Here we correlated gene expression patterns with the physiological differences along the diel cycle to provide new insights on the daily dynamic processes, including circadian rhythm, calcification, symbiosis, cellular arrangement, metabolism, and energy budget. During daytime, when solar radiation levels are highest, we observed increased calcification rate combined with an extensive up-regulation of genes associated with reactive oxygen species, redox, metabolism, ion transporters, skeletal organic matrix, and mineral formation. During the night, we observed a vast shift toward up-regulation of genes associated with cilia movement, tissue development, cellular movement, antioxidants, protein synthesis, and skeletal organic matrix formation. Our results suggest that light enhanced calcification is related to several processes that occur across the diel cycle; during nighttime, tissue might elevate away from the skeleton, extending the calcifying space area to enable the formation of a new organic framework template. During daytime, the combination of synthesis of acid-rich proteins and a greater flux of ions to the sites of calcification facilitate the conditions for extensive mineral growth.

Galactose-Binding C-Type Lectin Promotes Cellular Aggregation of Coelomocytes in Sea Cucumber

Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus, markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.

Molecular mechanism of claudin-15 strand flexibility

Claudins are one of the major components of tight junctions that play a key role in formation and maintaining epithelial barrier function. Tight junction strands are dynamic and capable of adapting their structure in response to large-scale tissue rearrangement and cellular movement. Here, we present molecular dynamics simulations of claudin-15 strands of up to 225 nm in length in two parallel lipid membranes and characterize their mechanical properties. The persistence length of claudin-15 strands is comparable with experiments leading to a curvature of 0.12 nm−1 at room temperature. Our results indicate that lateral flexibility of claudin strands is due to an interplay of three sets of interfacial interaction networks between four linear claudin strands in the membranes. In this model, claudins are assembled into interlocking tetrameric ion channels along the strand that slide with respect to each other as the strands curve over sub-micrometer length scales. These results suggest a novel molecular mechanism underlying claudin-15 strand flexibility. It also sheds light on the inter-molecular interactions and their role in maintaining epithelial barrier function.

Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention

A study was conducted to determine the effects of a diet supplemented with fruits and vegetables (FV) on the host whole blood cell (WBC) transcriptome and the composition and function of the intestinal microbiome. Nine six-week-old pigs were fed a pig grower diet alone or supplemented with lyophilized FV equivalent to half the daily recommended amount prescribed for humans by the Dietary Guideline for Americans (DGA) for two weeks. Host transcriptome changes in the WBC were evaluated by RNA sequencing. Isolated DNA from the fecal microbiome was used for 16S rDNA taxonomic analysis and prediction of metabolomic function. Feeding an FV-supplemented diet to pigs induced differential expression of several genes associated with an increase in B-cell development and differentiation and the regulation of cellular movement, inflammatory response, and cell-to-cell signaling. Linear discriminant analysis effect size (LEfSe) in fecal microbiome samples showed differential increases in genera from Lachnospiraceae and Ruminococcaceae families within the order Clostridiales and Erysipelotrichaceae family with a predicted reduction in rgpE-glucosyltransferase protein associated with lipopolysaccharide biosynthesis in pigs fed the FV-supplemented diet. These results suggest that feeding an FV-supplemented diet for two weeks modulated markers of cellular inflammatory and immune function in the WBC transcriptome and the composition of the intestinal microbiome by increasing the abundance of bacterial taxa that have been associated with improved intestinal health.

Weakening of resistance force by cell–ECM interactions regulate cell migration directionality and pattern formation

Collective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell–cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, we analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, we showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Our results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.

Cell division in tissues enables macrophage infiltration

Migration of cells through diverse tissues is essential for development, immune response and cancer metastasis. To reach their destination, cells must overcome the resistance imposed by complex microenvironments, composed of neighboring cells and extracellular matrix (ECM). While migration through pores and tracks in ECM has been well studied, little is known about cellular traversal into confining cell-dense tissues. Here by combining quantitative live imaging with genetic and optogenetic perturbations we identify a crucial role for cell division during cell migration into tissues. We find that normal embryonic invasion by Drosophila macrophages between the ectoderm and mesoderm absolutely requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by Integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade. Decreasing or increasing the frequency of ectodermal division correspondingly either hinders or promotes macrophage invasion. Reducing the levels of focal adhesion components in the ectoderm allows macrophage entry even in the absence of division. Our study demonstrates the critical importance of division at the entry site to enable in vivo cell invasion by relieving the steric impediment caused by focal adhesions. We thus provide a new perspective on the regulation of cellular movement into tissues

Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis

Cranial neural crest (CNC) cells contribute to different cell types during embryonic development. It is unknown whether postmigratory CNC cells undergo dynamic cellular movement and how the process of cell fate decision occurs within the first pharyngeal arch (FPA). Our investigations demonstrate notable heterogeneity within the CNC cells, refine the patterning domains, and identify progenitor cells within the FPA. These progenitor cells undergo fate bifurcation that separates them into common progenitors and mesenchymal cells, which are characterized by Cdk1 and Spry2/Notch2 expression, respectively. The common progenitors undergo further bifurcations to restrict them into osteogenic/odontogenic and chondrogenic/fibroblast lineages. Disruption of a patterning domain leads to specific mandible and tooth defects, validating the binary cell fate restriction process. Different from the compartment model of mandibular morphogenesis, our data redefine heterogeneous cellular domains within the FPA, reveal dynamic cellular movement in time, and describe a sequential series of binary cell fate decision-making process.

Indoxylsulfate, a Metabolite of the Microbiome, Has Cytostatic Effects in Breast Cancer via Activation of AHR and PXR Receptors and Induction of Oxidative Stress

Changes to bacterial metabolite-elicited signaling, in oncobiosis associated with breast cancer, plays a role in facilitating the progression of the disease. We show that indoxyl-sulfate (IS), a tryptophan metabolite, has cytostatic properties in models of breast cancer. IS supplementation, in concentrations corresponding to the human serum reference range, suppressed tumor infiltration to the surrounding tissues and metastasis formation in a murine model of breast cancer. In cellular models, IS suppressed NRF2 and induced iNOS, leading to induction of oxidative and nitrosative stress, and, consequently, reduction of cell proliferation; enhanced oxidative and nitrosative stress are crucial in the subsequent cytostasis. IS also suppressed epithelial-to-mesenchymal transition vital for suppressing cellular movement and diapedesis. Furthermore, IS rendered cells hypometabolic, leading to a reduction in aldehyde-dehydrogenase positive cells. Pharmacological inhibition of the pregnane-X receptor using CH223191 and the aryl-hydrocarbon receptor using ketoconazole diminished the IS-elicited effects, suggesting that these receptors were the major receptors of IS in these models. Finally, we showed that increased expression of the human enzymes that form IS (Cyp2E1, Sult1A1, and Sult1A2) is associated with better survival in breast cancer, an effect that is lost in triple negative cases. Taken together, IS, similar to indolepropionic acid (another tryptophan metabolite), has cytostatic properties and higher expression of the metabolic machinery responsible for the formation of IS supports survival in breast cancer.

Global Transcriptional Profiling Reveals Novel Autocrine Functions of Interleukin 6 in Human Vascular Endothelial Cells

Background. Interleukin 6 (IL6) is a multifunctional cytokine produced by various cells, including vascular endothelial cells. IL6 has both pro- and non-/anti-inflammatory functions, and the response to IL6 is dependent on whether it acts via the membrane-bound IL6 receptor α (IL6Rα) (classic signaling) or the soluble form of the receptor (transsignaling). As human endothelial cells produce IL6 and at the same time express IL6Rα, we hypothesized that IL6 may have autocrine functions. Methods. Knockdown of IL6 in cultured human endothelial cells was performed using siRNA. Knockdown efficiency was evaluated using ELISA. RNA sequencing was employed to characterize the transcriptional consequence of IL6 knockdown, and Ingenuity Pathway Analysis was used to further explore the functional roles of IL6. Results. Knockdown of IL6 in cultured endothelial cells resulted in a 84-92% reduction in the release of IL6. Knockdown of IL6 resulted in dramatic changes in transcriptional pattern; knockdown of IL6 in the absence of soluble IL6Rα (sIL6Rα) led to differential regulation of 1915 genes, and knockdown of IL6 in the presence of sIL6Rα led to differential regulation of 1967 genes (fold change 1.5, false discovery rate<0.05). Pathway analysis revealed that the autocrine functions of IL6 in human endothelial cells are mainly related to basal cellular functions such as regulation of cell cycle, signaling, and cellular movement. Furthermore, we found that knockdown of IL6 activates functions related to adhesion, binding, and interaction of endothelial cells, which seem to be mediated mainly via STAT3. Conclusion. In this study, a large number of novel genes that are under autocrine regulation by IL6 in human endothelial cells were identified. Overall, our data indicate that IL6 acts in an autocrine manner to regulate basal cellular functions, such as cell cycle regulation, signaling, and cellular movement, and suggests that the autocrine functions of IL6 in human endothelial cells are mediated via IL6 classic signaling.