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Flow Cytometry-Assessed PD1/PDL1 Reputation throughout Tumor-Infiltrating Lymphocytes: A Link With the Analysis associated with Calm Huge B-Cell Lymphoma.
It is challenging to rapidly identify immune responses that reflect the state and capability of immune cells due to complex heterogeneity of immune cells and their plasticity to pathogens and modulating molecules. Thus, high-throughput and easy-to-use cell culture and analysis platforms are highly desired for characterizing complex immune responses and elucidating their underlying mechanisms as well. In response to this need, we have developed a micropillar chip and a 384-pillar plate, printed mouse macrophage, RAW 264.7 cell line in alginate on the pillar plate platforms, and established multiplex cell-based assays to rapidly measure cell viability, expression of cell surface markers, and secretion of cytokines upon stimulation with model compound, lipopolysaccharide (LPS), as well as synthetic N-glycan polymers that mimic native glycoconjugates and could bind to lectin receptors on RAW 264.7 cells. Interestingly, changes in RAW 264.7 cell viability, expression levels of cell surface makers, and release of cytokines measured from the pillar plate platforms in the presence and absence of LPS were well correlated with those obtained from their counterpart, the 96-well plate with 2D-cultured macrophages. With this approach, we identified that α2,3-linked N-sialyllactose polymer has significant macrophage modulation activity among the N-glycan polymers tested. Ribociclib CDK inhibitor Therefore, we successfully demonstrated that our pillar plate platforms with 3D-cultured macrophages can streamline immune cell imaging and analysis in high throughput in response to compound stimulation. We envision that the pillar plate platforms could potentially be used for rapid characterization of immune cell responses and for screening immune cell-modulating molecules.Epithelial-mesenchymal transition (EMT) is an important contributor to drug resistance in ovarian cancer. The aims of this study were to explore the potential role of the miR-302 cluster in modulating EMT and cisplatin resistance in ovarian cancer. We used qRT-PCR and western blotting to show that miR-302 expression was lower in chemoresistant than in chemosensitive cells, and miR-302 was upregulated in chemosensitive, but not chemoresistant ovarian cancer cells in response to cisplatin treatment. We identified ATAD2 as a target of miR-302 and showed that ectopic expression of miR-302 increased cisplatin sensitivity and inhibited EMT and the invasiveness of cisplatin-resistant cells in vitro by targeting ATAD2. Knockdown of ATAD2 restored cisplatin sensitivity and reversed EMT/metastasis in cisplatin-resistant cells, as shown by western blotting and invasion/migration assays. The effect of miR-302 overexpression on EMT and invasiveness was mediated by the modulation of β-catenin nuclear expression. Immunofluorescence analysis showed that ATAD2 overexpression reversed the miR-302-induced downregulation of nuclear β-catenin in cisplatin resistant cells. A xenograft tumor model was used to show that miR-302 increases the antitumor effect of cisplatin in vivo. Taken together, these results identify a potential regulatory axis involving miR-302 and ATAD2 with a role in chemoresistance, indicating that activation of miR-302 or inactivation of ATAD2 could serve as a novel approach to reverse cisplatin resistance in ovarian cancer.Atomic force microscopy is an increasingly attractive tool to study how peptides disrupt membranes. Often performed on reconstituted lipid bilayers, it provides access to time and length scales that allow dynamic investigations with nanometre resolution. Over the last decade, AFM studies have enabled visualisation of membrane disruption mechanisms by antimicrobial or host defence peptides, including peptides that target malignant cells and biofilms. Moreover, the emergence of high-speed modalities of the technique broadens the scope of investigations to antimicrobial kinetics as well as the imaging of peptide action on live cells in real time. This review describes how methodological advances in AFM facilitate new insights into membrane disruption mechanisms.AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process. Using NMR and molecular dynamics, we show that AIIt and its pseudoheterodimer interacts with two molecules of short cytoplasmic domain isoform peptides, and that interaction depends on the binding motif 454-Phe-Gly-Lys-Thr-457 where Phe-454 binds in a hydrophobic pocket of AIIt, the null mutation Phe454Ala reduces binding by 2.5 fold, and the pseudophosphorylation mutant Thr457Glu reduces binding by three fold. Since these two residues in CEACAM1-SF were also found to play a role in the binding of calmodulin and G-actin at the membrane, we hypothesize a sequential set of three interactions are responsible for regulation of cis- to trans-dimerization of CEACAM1. The hydrophobic binding pocket in AIIt corresponds to a previously identified binding pocket for a peptide found in SMARCA3 and AHNAK, suggesting a conserved functional motif in AIIt allowing multiple proteins to reversibly interact with integral membrane proteins in a calcium dependent manner.The cranial neural crest (CNC) arises within the developing central nervous system, but then migrates away from the neural tube in three consecutive streams termed mandibular, hyoid and branchial, respectively, according to the order along the anteroposterior axis. While the process of neural crest emigration generally follows a conserved anterior to posterior sequence across vertebrates, we find that ray-finned fishes (bichir, sterlet, gar, and pike) exhibit several heterochronies in the timing and order of CNC emergence that influences their subsequent migratory patterns. First, emigration of the cranial neural crest in these fishes occurs prematurely compared to other vertebrates, already initiating during early neurulation and well before neural tube closure. Second, delamination of the hyoid stream occurs prior to the more anterior mandibular stream; this is associated with early morphogenesis of key hyoid structures like external gills (bichir), a large opercular flap (gar) or first forming cartilage (pike).
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