Extracellular matrix components direct porcine muscle stem cell behavior

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.     

Continual assembly of desmosomes within stable intercellular contacts of epithelial A-431 cells

Subclones of human carcinoma-derived A-431 cell line stably producing fusion proteins consisting of the enhanced green fluorescent protein and either human desmoglein 2 (Dsg-GFP) or human plakoglobin (GFP-Pg) were used to examine the behavior of desmosomes in living cells. Immunofluorescence microscopy of the fixed cells showed that both fusion proteins, which were expressed in significantly lower levels relative to their endogenous counterparts, were efficiently recruited into desmosomes. Time-lapse confocal imaging of these cells reveals that such GFP-labeled desmosomes (GFP desmosomes) are stable structures which exhibit various dynamic and motile activities. The most notable are independent lateral mobility and fusion. Furthermore, the continual assembly of new nascent desmosomes is observed within stable contacts located at the middle of the epithelial sheet. A new GFP desmosome appears as a closely apposed group of fine patches which after a few minutes aggregate into a single structure. These three dynamic processes resulted in constant changes of desmosome distribution, numbers, and sizes. In addition, fluorescence recovery after photobleaching experiments showed that fine patches of desmosomal proteins may participate in desmosome maintenance. Such a diverse range of dynamic activities of desmosomes apparently produces flexible but tight cell-cell adhesion required for different morphogenetic events in epithelial structures.This work was supported by grant AR44016-04 from the National Institutes of Health     

The relation of temperature and lipid composition to cell adhesion

We have examined as a function of temperature the effect of changes in the composition of the fatty acid chains of membrane phospholipids on the rate of cell to cell adhesion in the neuronal cell line B103. The rate of cell to cell adhesion in this cell line is highly temperature dependent but is not influenced by changes in the fatty acid composition of the plasma membrane generated by growing the cells either in the presence of oleic acid or elaidic acid. In contrast the temperature dependence of the rate of cell to cell adhesion, measured in a monolayer adhesion assay, is highly dependent on the shear force used during the assay. A two-step model of cell to cell adhesion involving multiple adhesion ligands is presented which can be used to explain these observations.     

不同程度慢性阻塞性肺疾病患者血清FIB、RDW、MDW及IL-6的表达水平及其与肺功能、急性发作的相关性分析

摘要 目的：分析不同程度慢性阻塞性肺疾病（COPD）患者血清纤维蛋白原（FIB）、红细胞分布宽度（RDW）、单核细胞体积分布宽度（MDW）及白介素6（IL-6）的表达水平及其与肺功能、急性发作的相关性。方法：选择我院自2021年4月至2023年4月接诊的148例COPD患者纳入观察组,根据气流受限严重程度分为轻-中度气流受限组（96例）和重-极重度气流受限组（52例）;另选同期的148例健康体检者纳入对照组。检测所有入选者血清FIB、RDW、MDW及IL-6的表达水平,比较不同组别之间血清FIB、RDW、MDW及IL-6的表达水平,使用Pearson相关性分析血清FIB、RDW、MDW及IL-6与肺功能指标[第一秒用力呼气容积与用力肺活量（FEV₁/FVC）占预计值百分比和第一秒用力呼气容积占预计值百分比（FEV₁%）]的关系,ROC曲线分析血清FIB、RDW、MDW及IL-6对急性发作的预测效能。结果：观察组血清FIB、RDW、MDW及IL-6的表达水平均高于对照组（P<0.05）;重-极重度气流受限组血清FIB、RDW、MDW及IL-6的表达水平均高于轻-中度气流受限组（P<0.05）;经Pearson相关性分析,COPD患者血清FIB、RDW、MDW及IL-6的表达水平与FEV₁/FVC占预计值百分比、FEV₁%均呈负相关（P<0.05）;经ROC曲线分析,血清FIB、RDW、MDW联合IL-6预测COPD急性发作的敏感度为66.35%,特异度为90.42%、AUC为0.912。结论：不同程度COPD患者血清FIB、RDW、MDW及IL-6的表达水平存在差异,血清FIB、RDW、MDW及IL-6与其肺功能密切相关,联合对急性发作具有一定预测价值。     

Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix

Extracellular matrix plays a critical role in cellular development by providing signaling cues that direct morphogenesis. In order to study both the cues that natural matrix provides and endothelial cell responses to that information, human fetal lung fibroblasts were used to produce a fibrous three-dimensional matrix. Following the removal of the fibroblasts by detergent extraction, protein and proteoglycan constituents of the remaining matrix were identified by immunofluorescence and immunoblotting. Matrix components included fibronectin, tenascin-C, collagen I, collagen IV, collagen VI, versican, and decorin. Colocalization analysis suggested that fibronectin was a uniquely distributed matrix protein. Morphology, three-dimensional matrix adhesions, and integrin-mediated signaling during vasculogenesis were then studied in human endothelial cells seeded onto the fibroblast-derived matrix. Elongated morphology and decreased cell area were noted, as compared with cells on fibronectin-coated coverslips. Cell-matrix adhesions contained vinculin, pY397-FAK, and pY410-p130Cas, and all of these colocalized more with fibronectin than tenascin-C, collagen I, or collagen VI. Additionally, the endothelial cells remodeled the fibroblast-derived matrix and formed networks of tubes with demonstrable lumens. Matrix adhesions in these tubes also predominantly colocalized with fibronectin. The pattern of membrane type 1 matrix metalloprotease expression in the endothelial cells suggested its involvement in the matrix remodeling that occurred during tubulogenesis. These results indicated that information in fibroblast-derived matrix promoted vasculogenic behavior.     

Proteomic Insights into Endometrial Receptivity and Embryo‐Endometrial Epithelium Interaction for Implantation Reveal Critical Determinants of Fertility

In vitro fertilization has overcome infertility issues for many couples. However, achieving implantation of a viable embryo into the maternal endometrium remains a limiting step in optimizing pregnancy success. The molecular mechanisms which characterize the transient state of endometrial receptivity, critical in enabling embryo‐endometrial interactions, and proteins which underpin adhesion at the implantation interface, are limited in humans despite these temporally regulated processes fundamental to life. Hence, failure of implantation remains the “final frontier” in infertility. A human coculture model is utilized utilizing spheroids of a trophectoderm (trophoblast stem) cell line, derived from pre‐implantation human embryos, and primary human endometrial epithelial cells, to functionally identify “fertile” versus “infertile” endometrial epithelium based on adhesion between these cell types. Quantitative proteomics identified proteins associated with human endometrial epithelial receptivity (“epithelial receptome”) and trophectoderm adhesion (“adhesome”). As validation, key “epithelial receptome” proteins (MAGT‐1/CDA/LGMN/KYNU/PC4) localized to the epithelium of receptive phase (mid‐secretory) endometrium obtained from fertile, normally cycling women but is largely absent from non‐receptive (proliferative) phase tissues. Factors involved in embryo‐epithelium interaction in successive temporal stages of endometrial receptivity and implantation are demonstrated and potential targets for improving fertility are provided, enhancing potential to become pregnant either naturally or in a clinical setting.     

Conditioning adhesive contacts between the neutrophils and the endotheliocytes by Staphylococcus aureus

We have developed a model for evaluating the integral intercellular interactions in the “endotheliocyte‐neutrophil” system and have shown the high variability of adhesion contacts in different donors associated with different expression profiles of neutrophils. Two methods (forсe spectroscopy‐spectroscopy and scanning ion‐conductance microscopy) showed a decrease in the rigidity of the membrane‐cytoskeletal complex of neutrophils under the influence of Staphylococcus aureus 2879 M. Adding this strain to the “endotheliocyte‐neutrophil” system caused a statistically significant decrease in the adhesion force and adhesion work, which indicates a change in the expression profile and physicochemical properties of membranes of both types of interacting cells (neutrophils and endotheliocytes).