Cell Adhesion Processes Research Articles

Continuum and stochastic approach for cell adhesion process based on Eulerian fluid-capsule coupling with Lagrangian markers

This paper presents a novel development for a full Eulerian coupling method between a fluid and capsule with hyperelastic membrane to address a cell adhesion process. The capsule motion in the fluid field and their mechanical interactions are expressed in the Eulerian framework, while solving the capsule adhesion process by stochastic events of ligand-receptor bindings in a discrete level, with using Lagrangian markers distributed on the implicit surface of the capsule. In addition to this, numerical improvements of the full Eulerian method for calculating an interface normal vector and dealing with multiple capsules by introducing the temporarily constructed level set function. A numerical validity of the present development is investigated through a comparison with available numerical data in a validation problem, and a capability for the capsule adhesion is shown in a single capsule adhesion and platelet adhesion in a suspension with blood cells.

PO-152 Differentiation of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) by gene co-expression analysis of notch signalling targets

IntroductionLung carcinomas are very aggressive malignancies, which remain leading cancer-related cause of death among men and women worldwide. Regarding histological classification LUSC and LUAD account for the majority of lung tumours among non-small cell carcinomas (NSCLC). Notch signalling is evolutionarily-conserved pathway that regulates many cellular processes such as proliferation, differentiation and epithelial-to-mesenchymal transition (EMT). To date, aberrant Notch signalling has been linked with wide variety of malignancies, including lung tumours. However the association between co-expression profile of Notch downstream effector and lung cancer subtypes remains unclear. Therefore the aim of our study was to investigate functional gene co-expression networks to search for candidate biomarkers or therapeutic targets.Material and methodsIn our analysis we used RNASeq expression and clinical data downloaded from The Cancer Genome Atlas (TGCA). Gene Set Enrichment Analysis (GSEA) performed for canonical pathways pointed to Notch pathway as one of the most significant. Subsequently we analysed expression of downstream Notch effectors, 2949 HES/HEY transcription factors targets. To this extent used Weighted Gene Co-expression Network Analysis (WGCNA) to find differences in gene expression profile between LUSC and LUAD.Results and discussionsThe analysis of Notch pathway downstream targets which expression is regulated by HES/HEY transcription factors, identified 9 gene modules highly correlated with cancer type, with two of them as the most promising. Functional analysis revealed that among the differentially expressed genes were those involved in proliferation, cell cycle regulation, DNA repair, EMT, adhesion and metabolic processes, for example: TP63, PIK3CA, ADAM23, DLG1, FXR1, SENP5, TTK, BIRC5, KIF18A, KIF14, KIF4A, MCM4, MCM10. For one module the highly connected hub gene is TP63, acts as oncogene in many tumour types including squamous cell carcinomas, and for the second module is KIF4A. Interestingly, all listed genes were found to be overexpressed in LUSC and downexpressed in LUAD.ConclusionOur analysis could be valuable for better understanding of the molecular mechanism of lung carcinoma as well as Notch signalling in lung cancer with emphasis of pathway gene expression as useful biomarker for differentiating cancer progression in lung cancer subtypes.AcknowledgmentThis study was founded by the National Science Centre, Poland nr 2016/23/N/NZ2/02575 and Medical University of Lodz 503/0-078-02/503-01-004

Binding of intercellular adhesion molecule 1 to β2-integrin regulates distinct cell adhesion processes on hepatic and cerebral endothelium.

Flowing polymorphonuclear neutrophils (PMNs) are forced to recruit toward inflamed tissue and adhere to vascular endothelial cells, which is primarily mediated by the binding of β2-integrins to ICAM-1. This process is distinct among different organs such as liver and brain; however, the underlying kinetic and mechanical mechanisms regulating tissue-specific recruitment of PMNs remain unclear. Here, binding kinetics measurement showed that ICAM-1 on murine hepatic sinusoidal endothelial cells (LSECs) bound to lymphocyte function-associated antigen-1 (LFA-1) with higher on- and off-rates but lower effective affinity compared with macrophage-1 antigen (Mac-1), whereas ICAM-1 on cerebral endothelial cells (BMECs or bEnd.3 cells) bound to LFA-1 with higher on-rates, similar off-rates, and higher effective affinity compared with Mac-1. Physiologically, free crawling tests of PMN onto LSEC, BMEC, or bEnd.3 monolayers were consistent with those kinetics differences between two β2-integrins interacting with hepatic sinusoid or cerebral endothelium. Numerical calculations and Monte Carlo simulations validated tissue-specific contributions of β2-integrin-ICAM-1 kinetics to PMN crawling on hepatic sinusoid or cerebral endothelium. Thus, this work first quantified the biophysical regulation of PMN adhesion in hepatic sinusoids compared with cerebral endothelium.

Yessotoxin: risk assessment for public health. Justification of regulations of content in seafood

Yessotoxin and its derivatives (about 90) are isolated from algae belonging to the species Protoceratium reticulatum, Gonyaulax cf. Spinifera, Lingulodinium polyedrum and from invertebrate organisms that feed on these algae. Previously yessotoxin have been associated with the group of diarrheal toxins. Later studies of the possible impact of yessotoxin on the activity of alkaline phosphatase allowed to exclude them from this group. Yessotoxin causes a violation of calcium entry in the cells, which, in turn, effects the calcium-calmodulin system and thus influences into homeostasis of the organism as a whole. It was shown that yessotoxin induces a biphasic change in the concentration of adenosine monophosphate, an initial increase with a subsequent relative decrease, within some minutes after adding the toxin to the lymphocytes cell culture. Yessotoxin has effects on immune system; which is manifested in an increase of cytokines level, by inducing the expression of the genes encoding them. Yessotoxin have impact into processes of cell adhesion via E-cadherin and, thus, could be an important factor in the development of Alzheimer's disease. It has been established that yessotoxin caused the development of apoptosis. In those cases all three mechanisms of cell death took place - apoptosis, paraptosis and autophagy. Yessotoxin's acute toxicity doses according to different data are from 100 to 500-750 μg per 1kg of body weight. Yessotoxin's acute reference dose (ARfD) - 25 μg/kg of body weight per day. The results of the analysis of yessotoxin level in shellfish meat showed that none of the studied samples contained more than 3.75 mg yessotoxin equivalents/kg shellfish meat. This level has been adopted by the European Union as the maximum acceptable level of yessotoxin in shellfish meat (EU Regulation N 786/2013). Presented data on the mechanism of action, toxicity and prevalence of yessotoxins make it necessary to establish regulations of their content in seafood, placed on the markets of the Eurasian Economic Union.

A hypervariable immunoglobulin superfamily member from Crassostrea gigas functions as pattern recognition receptor with opsonic activity

Immunoglobulin superfamily (IgSF), an extensive collection of proteins possessing at least one immunoglobulin-like (Ig-like) domain, performs a wide range of functions in recognition, binding or adhesion process of cells. In the present study, a cysteine-rich motif associated immunoglobulin domain containing protein (designated CgCAICP-1) was identified in Pacific oyster Crassostrea gigas. The deduced protein sequence of CgCAICP-1 contained 534 amino acidresidues, with three Ig domains which were designated as IG1, IG2 and IG3, and a cysteine-rich motif between the first and second Ig domain. The mRNA transcripts of CgCAICP-1 were highly expressed in hemocytes and up-regulated significantly (p < 0.05) after the stimulation of lipopolysaccharides (LPS), but not peptidoglycan (PGN). The recombinant CgCAICP-1 protein (rCgCAICP-1) exhibited binding activity to various pathogen-associated molecular patterns (PAMPs) including LPS, PGN, mannose (Man) and D-galactose (D-Gal), and microorganisms including Vibrio splendidus, Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Pichia pastoris. The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. splendidus and Gram-positive bacteria M. luteus were significantly enhanced (p < 0.05) after pre-incubation of microbes with rCgCAICP-1. Furthermore, the transcripts of CgCAICP-1 exhibited high level of polymorphism among individuals. The ratio of nonsynonymous and synonymous distances (dN/dS) for AA’BCC’D strands of IG1 (the possible binding sites 1, pbs1) across all allelic variants was 2.09 (p < 0.05), while the ratio for the non-pbs regions was less than 1.0. The 1248 bp fragment amplified from the 5′ end of CgCAICP-1 open reading frame (ORF) from 24 transcript variants could be divided artificially into seven regions of 50 elements, and all of the allelic variants might be derived from these elements by point mutation and recombination processes. These results collectively suggested that CgCAICP-1 might function as an important pattern recognition receptor (PRR) to recognize various PAMPs and facilitated the phagocytosis of oyster hemocytes towards both Gram-positive and Gram-negative bacteria. Diverse isoforms of CgCAICP-1 were generated through point mutation and recombination processes and maintained by balancing selection, which would provide a broader spectrum of interaction surface and be associated with immune resistance of oysters to infectious pathogens.

High-Resolution Adhesion Kinetics of EGCG-Exposed Tumor Cells on Biomimetic Interfaces: Comparative Monitoring of Cell Viability Using Label-Free Biosensor and Classic End-Point Assays.

A high-throughput label-free resonant waveguide grating biosensor, the Epic BenchTop, was utilized to in situ monitor the adhesion process of cancer cells on Arg-Gly-Asp tripeptide displaying biomimetic polymer surfaces. Using highly adherent human cervical adenocarcinoma (HeLa) cells as a model system, cell adhesion kinetic data with outstanding temporal resolution were obtained. We found that pre-exposing the cells to various concentrations of the main extract of green tea, the (−)-epigallocatechin gallate (EGCG), largely affected the temporal evolution of the adhesion process. For unexposed and low dosed cells, sigmoid shaped spreading kinetics was recorded. Higher dose of EGCG resulted in a complete absence of the sigmoidal character, and displayed adsorption-like kinetics. By using the first derivatives of the kinetic curves, a simple model was developed to quantify the sigmoidal character and the transition from sigmoidal to adsorption-like kinetics. The calculations showed that the transition happened at EGCG concentration of around 60 μg/mL. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide end-point assay, we concluded that EGCG is cytostatic but not cytotoxic. The effect of EGCG was also characterized by flow cytometry. We concluded that, using the introduced label-free methodology, the shape of the cell adhesion kinetic curves can be used to quantify in vitro cell viability in a fast, cost-effective, and highly sensitive manner.

MKLN1 splicing defect in dogs with lethal acrodermatitis

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of ~1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN1:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo.

Metabolic Reprogramming in Amyotrophic Lateral Sclerosis

Mitochondrial dysfunction in the spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the neurometabolic alterations during early stages of the disease remain unknown. Here, we investigated the bioenergetic and proteomic changes in ALS mouse motor neurons and patients’ skin fibroblasts. We first observed that SODG93A mice presymptomatic motor neurons display alterations in the coupling efficiency of oxidative phosphorylation, along with fragmentation of the mitochondrial network. The proteome of presymptomatic ALS mice motor neurons also revealed a peculiar metabolic signature with upregulation of most energy-transducing enzymes, including the fatty acid oxidation (FAO) and the ketogenic components HADHA and ACAT2, respectively. Accordingly, FAO inhibition altered cell viability specifically in ALS mice motor neurons, while uncoupling protein 2 (UCP2) inhibition recovered cellular ATP levels and mitochondrial network morphology. These findings suggest a novel hypothesis of ALS bioenergetics linking FAO and UCP2. Lastly, we provide a unique set of data comparing the molecular alterations found in human ALS patients’ skin fibroblasts and SODG93A mouse motor neurons, revealing conserved changes in protein translation, folding and assembly, tRNA aminoacylation and cell adhesion processes.

Down-regulation of FN1 inhibits colorectal carcinogenesis by suppressing proliferation, migration, and invasion.

Fibronectin 1 (FN1) is involved in cell adhesion and migration processes including embryogenesis, wound healing, blood coagulation, host defense, metastasis, and implicated in various biochemical processes. However, its effects on the development and progression of human cancer, especially colorectal cancer (CRC), are unclear. To evaluate the relationship between the expression of FN1 and the histopathologic parameters of patients with CRC or the proliferation, migration, and invasion of colorectal cancer cell lines, we screened FN1 as a new candidate gene which promotes development of CRC, in an independent dataset (The Human Protein Atlas website). Here, we reported that FN1 was elevated in CRC tissues compared with normal colon tissues. Further, FN1 expression level was correlated with age, lymph vascular invasion, and survival rate. Knockdown of FN1 in two CRC cell lines, LOVO, and SW1116, significantly inhibited cell proliferation, migration and invasion, and induced cell apoptosis. Western blot analysis showed that down-regulation of FN1 significantly decreased the expression of Bcl-2, MMP-9, Twist, and increased the expression of Bax, Caspase-3, and E-cadherin in LOVO and SW1116 cells. Then, we found that the protein ITGA5 was identified as a binding partner of FN1 and ITGA5 overexpression reversed FN1-induced tumorigenesis of CRC in vitro. Taken together, FN1 suppressed apoptosis and promoted viability, invasion, and migration in CRC through interacting with ITGA5. FN1 may be a prognostic factor and potential target for CRC treatment.

DNA methylation signatures of pulmonary arterial smooth muscle cells in chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening disease, which is often underpinned by vascular remodeling. Pulmonary arterial smooth muscle cells (PASMCs) are the main participants in vascular remodeling. However, their biological role in CTEPH is not entirely clear. In the present study, we analyzed the whole epigenome-wide DNA methylation profile of cultured PASMCs from CTEPH and control cell lines with the Illumina Human Methylation 450K BeadChip. A total of 6,829 significantly differentially methylated probes (DMPs) were detected between these two groups. Among these, 4,246 DMPs were hypermethylated, while 2,583 DMPs were hypomethylated. The functional enrichment analysis of 1,743 DMPs in the promoter regions and corresponding genes indicated that DNA hypermethylation and hypomethylation might be involved in the regulation of genes that have multifarious biological roles, including roles in cancer-related diseases, the regulation of the actin cytoskeleton, cell adhesion, and pattern specification processes. The observed methylations were categorized into the most important functions, including those involved in cell proliferation, immunity, and migration. We speculate that these methylations were most likely involved in the possible pathophysiology of CTEPH. Gene interaction analysis pertaining to signal networks confirmed that PIK3CA and PIK3R1 were important mediators in these whole networks. The mRNA levels of PIK3CA, HIC1, and SSH1 were verified by qPCR and corresponded with DNA methylation differences. Understanding epigenetic features associated with CTEPH may provide new insights into the mechanism that underlie this condition.

Lis1 dysfunction leads to traction force reduction and cytoskeletal disorganization during cell migration

Cell migration is a critical process during development, tissue repair, and cancer metastasis. It requires complex processes of cell adhesion, cytoskeletal dynamics, and force generation. Lis1 plays an important role in the migration of neurons, fibroblasts and other cell types, and is essential for normal development of the cerebral cortex. Mutations in human LIS1 gene cause classical lissencephaly (smooth brain), resulting from defects in neuronal migration. However, how Lis1 may affect force generation in migrating cells is still not fully understood. Using traction force microscopy (TFM) with live cell imaging to measure cellular traction force in migrating NIH3T3 cells, we showed that Lis1 knockdown (KD) by RNA interference (RNAi) caused reductions in cell migration and traction force against the extracellular matrix (ECM). Immunostaining of cytoskeletal components in Lis1 KD cells showed disorganization of microtubules and actin filaments. Interestingly, focal adhesions at the cell periphery were significantly reduced. These results suggest that Lis1 is important for cellular traction force generation through the regulation of cytoskeleton organization and focal adhesion formation in migrating cells.

Temozolomide affects Extracellular Vesicles Released by Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most aggressive primary tumour within the brain as well as the most common and lethal cerebral cancer, mainly because of treatment failure. Indeed, tumour recurrence is inevitable and fatal in a short period of time. Glioblastoma stem-like cells (GSCs) are thought to participate in tumour initiation, expansion, resistance to treatments, including to the alkylating chemotherapeutic agent temozolomide, and relapse. Here, we assessed whether extracellular vesicles (EVs) released by GSCs could disseminate factors involved in resistance mechanisms. We first characterized EVs either circulating in peripheral blood from newly diagnosed patients or released by patient-derived temozolomide-resistant GSCs. We found that EVs from both sources were mainly composed of particles homogeneous in size (50–100 nm), while they were more abundant in liquid biopsies from GBM patients, as compared to healthy donors. Further, mass spectrometry analysis from GSC-derived EVs unveiled that particles from control and temozolomide-treated cells share core components of EVs, as well as ribosome- and proteasome-associated networks. More strikingly, temozolomide treatment led to the enrichment of EVs with cargoes dedicated to cell adhesion processes. Thus, while relatively inefficient in killing GSCs in vitro, temozolomide could instead increase the release of pro-tumoral information.

Unfound Associated Resonant Model and Its Impact on Response of a Quartz Crystal Microbalance in the Liquid Phase.

Quartz crystal microbalance (QCM) is an important tool to detect in real time the mass change at the nanogram level. However, for a QCM operated in the liquid phase, the Sauerbrey equation is usually disturbed by the changes in liquid properties and the longitudinal wave effect. Herein, we report another unfound associated high-frequency resonance (HFR) model for the QCM, with the intensity 2 orders of magnitude higher than that of the fundamental peak in the liquid phase. The HFR model exhibits obvious impact on the response of QCM in the thickness-shear model (TSM), especially for overtones. The frequency of HFR peak is decreased dramatically with increasing conductivity or permittivity of the liquid phase, resulting in considerable additional frequency shifts in the TSM as baseline drift. Compared to that with a faraway HFR peak, the overlapping of HFR peak to a TSM overtone results in the frequency shifts of ±50-70 kHz with its intensity enhancement by 3 orders of magnitude in the later. The HFR behavior is explained by an equivalent circuit model including leading wire inductance, liquid inductance, and static capacitance of QCM. Taking into account the HFR model, the positive frequency shifts of the QCM at high overtones during the cell adhesion process is understandable. Combining the TSM and HFR is an effective way to improve the stability of QCM and provides more reliable information from the responses of QCM. The HFR may have potential application in chemical and biological sensors.

Tuning surface properties of bone biomaterials to manipulate osteoblastic cell adhesion and the signaling pathways for the enhancement of early osseointegration.

Osteoblast cell adhesion is the initial step of early osseointegration responding to bone material implants. Enhancing the osteoblastic cell adhesion has become one of the prime aims when optimizing the surface properties of bone biomaterials. The traditional strategy focuses in improving the physical attachment of osteoblastic cells onto the surfaces of biomaterials. However, instead of a simple cell physical attachment, the osteoblastic cell adhesion has been revealed to be a sophisticated system. Despite the well-documented effect of bone biomaterial surface modifications on adhesion, few studies have focused on the underlying molecular mechanisms. Physicochemical signals from biomaterials can be transduced into intracellular signaling network and further initiate the early response cascade towards the implants, which includes cell survival, migration, proliferation, and differentiation. Adhesion is vital in determining the early osseointegration between host bone tissue and implanted bone biomaterials via regulating involving signaling pathways. Therefore, the modulation of early adhesion behavior should not simply target in physical attachment, but emphasize in the manipulation of downstream signaling pathways, to regulate early osseointegration. This review firstly summarized the basic biological principles of osteoblastic cell adhesion process and the activated downstream cell signaling pathways. The effects of different biomaterial physicochemical properties on osteoblastic cell adhesion were then reviewed. This review provided up-to-date research outcomes in the adhesion behavior of osteoblastic cells on bone biomaterials with different physicochemical properties. The strategy is optimised from traditionally focusing in physical cell adhesion to the proposed strategy that manipulating cell adhesion and the downstream signaling network for the enhancement of early osseointegration.

Equilibrium Structure and Mechanics of the Cellular Glycocalyx

Cells can employ distinct modes of cell migration, ranging from mesenchymal-like or amoeboid-like single cell motility to collective migration. Each mode is characterized by a unique balance of cell-matrix and cell-cell adhesion. Carbohydrates called glycans are concentrated on the cell surface in a complex structure called the glycocalyx. While the glycocalyx is known to impede cell adhesion by acting as a steric barrier, the mechanical response of the glycocalyx and its molecular constituents to force is largely unknown. Therefore, we have developed a physical model of the glycocalyx to understand and predict its deformation and reorganization under compressive loads, such as those that would be experienced by cells in confined three-dimensional environments. Simulations of our model predict that the glycocalyx can sustain substantial pressures without undergoing significant deformations, but beyond a critical pressure it experiences a pressure-sensitive response, followed by increased resistance at high strains. This suggests that cells could switch between weakly adherent and highly adherent states depending on the degree of confinement and the compression of the glycocalyx. Additionally, the simulations predict the formation of load-bearing clusters of glycoproteins in favorable regions away from the cytoskeletal membrane attachments. We elucidate the effects of the biophysical properties of the glycocalyx and the cell membrane on the mechanical behavior. Increasing the stiffness and the density of glycoproteins and the membrane bending modulus and the cytoskeletal density or decreasing the glycoprotein length increases the collective stiffness of the glycocalyx and the pressure required to generate significant deformations. Together, our results demonstrate how bulk material properties of the glycocalyx emerge from the physical properties of its molecular constituents and how these constituents rearrange under load. The understanding developed here has broad relevance in cell migration, adhesion, communication, and other receptor-mediated processes in the glycocalyx.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues.

MiR-449a Suppresses Tamoxifen Resistance in Human Breast Cancer Cells by Targeting ADAM22

Background/Aims: Most of estrogen receptor positive breast cancer patients respond well initially to endocrine therapies, but often develop resistance during treatment with selective estrogen receptor modulators (SERMs) such as tamoxifen. Altered expression and functions of microRNAs (miRNAs) have been reportedly associated with tamoxifen resistance. Thus, it is necessary to further elucidate the function and mechanism of miRNAs in tamoxifen resistance. Methods: Tamoxifen sensitivity was validated by using Cell Counting Kit-8 in tamoxifen-sensitive breast cancer cells (MCF-7, T47D) and tamoxifen-resistant cells (MCF-7/TAM, T47D/ TAM). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression level of miR-449a in tamoxifen-sensitive/-resistant cells and patient serums. Dual-luciferase assay was used to identify the binding of miR-449a and predicted gene ADAM22. The expression level of ADAM22 was determined by qRT-PCR and western blotting in miR-449a +/- breast cancer cells. Subsequently, rescue experiments were carried out to identify the function of ADAM22 in miR-449a-reduced tamoxifen resistance. Finally, Gene ontology (GO) and Protein-protein interaction analyses were performed to evaluate the potential mechanisms of ADAM22 in regulating tamoxifen resistance. Results: MiR-449a levels were downregulated significantly in tamoxifen-resistant breast cancer cells when compared with their parental cells, as well as in clinical breast cancer serum samples. Overexpression of miR-449a re-sensitized the tamoxifen-resistant breast cancer cells, while inhibition of miR-449a conferred tamoxifen resistance in parental cells. Luciferase assay identified ADAM22 as a direct target gene of miR-449a. Additionally, silencing of ADAM22 could reverse tamoxifen resistance induced by miR-449a inhibition in ER-positive breast cancer cells. GO analysis results showed ADAM22 was mainly enriched in the biological processes of cell adhesion, cell differentiation, gliogenesis and so on. Protein-protein interaction analyses appeared that ADAM22 might regulate tamoxifen resistance through PPARG, LGI1, KRAS and LYN. Conclusion: Decreased miR-449a causes the upregulation of ADAM22, which induces tamoxifen resistance of breast cancer cells. These results suggest that miR-449a, functioning by targeting ADAM22, contributes to the mechanisms underlying breast cancer endocrine resistance, which may provide a potential therapeutic strategy in ER-positive breast cancers.

Microfluidic auto-alignment of protein patterns for dissecting multi-receptor crosstalk in platelets.

Cell adhesion plays a critical role in many cellular functions, such as the hemostatic/thrombotic process, inflammatory reactions, and adaptive immune response. Many cell adhesion processes involve crosstalk between multiple ligand-receptor systems through intracellular signaling. To elucidate such crosstalk requires analysis of the synergistic or antagonistic effects of binding and signalling of multi-receptor species. Current techniques for these analyses, e.g., atomic force microscopy (AFM) and biomembrane force probe (BFP) assays, are either labor-intensive, low-throughput, or limited in the types of ligands they can interrogate. Circumventing these limitations requires a technique for manipulating ligand interactions with and measuring the functional response of a population of cells. In this work, we have developed a microfluidic platform for studying the binding and signaling of multi-receptor species by separating their actions in space and time. The platform directs cells through a single channel and uses sequentially presented ligands for pre-processing and stimulating cells, followed by reporting of cell activation states and functional consequences. Our method precisely patterns multiple proteins in different spatial regions without gaps. We demonstrate the utility of our method by using this platform to analyze the crosstalk between platelet receptors, glycoprotein Ib and IIb-IIIa, in the context of platelet adhesion and signaling under flow. We show the clinical utility of this platform by applying it to analyze whole blood samples and to assess differences in the activation of platelets between healthy and diabetic patients.

Role of Galectins in Multiple Myeloma.

Galectins are a family of lectins that bind β-galactose-containing glycoconjugates and are characterized by carbohydrate-recognition domains (CRDs). Galectins exploit several biological functions, including angiogenesis, regulation of immune cell activities and cell adhesion, in both physiological and pathological processes, as tumor progression. Multiple myeloma (MM) is a plasma cell (PC) malignancy characterized by the tight adhesion between tumoral PCs and bone marrow (BM) microenvironment, leading to the increase of PC survival and drug resistance, MM-induced neo-angiogenesis, immunosuppression and osteolytic bone lesions. In this review, we explore the expression profiles and the roles of galectin-1, galectin-3, galectin-8 and galectin-9 in the pathophysiology of MM. We focus on the role of these lectins in the interplay between MM and BM microenvironment cells showing their involvement in MM progression mainly through the regulation of PC survival and MM-induced angiogenesis and osteoclastogenesis. The translational impact of these pre-clinical pieces of evidence is supported by recent data that indicate galectins could be new attractive targets to block MM cell growth in vivo and by the evidence that the expression levels of LGALS1 and LGALS8, genes encoding for galectin-1 and galectin-8 respectively, correlate to MM patients’ survival.

Identification of Key Genes and Pathways in Tongue Squamous Cell Carcinoma Using Bioinformatics Analysis.

BackgroundTongue squamous cell carcinoma (TSCC) is a major type of oral cancers and has remained an intractable cancer over the past decades. The aim of this study was to identify differentially expressed genes (DEGs) during TSCC and reveal their potential mechanisms.Material/MethodsThe gene expression profiles of GSE13601 were downloaded from the GEO database. The GSE13601 dataset contains 57 samples, including 31 tongue SCC samples and 26 matched normal mucosa samples. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were performed; Cytoscape software was used for the protein-protein interaction (PPI) network and module analysis of the DEGs.ResultsWe identified a total of 1,050 upregulated DEGs (uDEGs) and 702 downregulated DEGs (dDEGs) of TSCC. The GO analysis results showed that uDEGs were significantly enriched in the following biological processes (BP): signal transduction, positive or negative regulation of cell proliferation, and negative regulation of cell proliferation. The dDEGs were significantly enriched in the following biological processes: signal transduction, cell adhesion, and apoptotic process. The KEGG pathway analysis showed that uDEGs were enriched in metabolic pathways, pathways in cancer, and PI3K-Akt signaling pathway, while the dDEGs were enriched in focal adhesion and ECM-receptor interaction. The top centrality hub genes RAC1, APP, EGFR, KNG1, AGT, and HRAS were identified from the PPI network. Module analysis revealed that TSCC was associated with significant pathways, including neuroactive ligand-receptor interaction, calcium signaling pathway, and chemokine signaling pathway.ConclusionsThe present study identified key genes and signal pathways, which deepen our understanding of the molecular mechanisms of carcinogenesis and development of the disease, and might be used as diagnostic and therapeutic molecular biomarkers for TSCC.