Cell Adhesion Glycoprotein Research Articles

Exploration into Galectin-3 Driven Endocytosis and Lattices.

Essentially all plasma membrane proteins are glycosylated, and their activity is regulated by tuning their cell surface dynamics. This is achieved by glycan-binding proteins of the galectin family that either retain glycoproteins within lattices or drive their endocytic uptake via the clathrin-independent glycolipid-lectin (GL-Lect) mechanism. Here, we have used immunofluorescence-based assays to analyze how lattice and GL-Lect mechanisms affect the internalization of the cell adhesion and migration glycoprotein α5β1 integrin. In retinal pigment epithelial (RPE-1) cells, internalized α5β1 integrin is found in small peripheral endosomes under unperturbed conditions. Pharmacological compounds were used to competitively inhibit one of the galectin family members, galectin-3 (Gal3), or to inhibit the expression of glycosphingolipids, both of which are the fabric of the GL-Lect mechanism. We found that under acute inhibition conditions, endocytic uptake of α5β1 integrin was strongly reduced, in agreement with previous studies on the GL-Lect driven internalization of the protein. In contrast, upon prolonged inhibitor treatment, the uptake of α5β1 integrin was increased, and the protein was now internalized by alternative pathways into large perinuclear endosomes. Our findings suggest that under these prolonged inhibitor treatment conditions, α5β1 integrin containing galectin lattices are dissociated, leading to an altered endocytic compartmentalization.

Cadherin-5 facilitated the differentiation of human induced pluripotent stem cells into sinoatrial node-like pacemaker cells by regulating β-catenin.

Our study was conducted to investigate whether cadherin-5 (CDH5),a vascular endothelial cell adhesion glycoprotein, could facilitate the differentiation of human induced pluripotent stem cells (hiPSCs)into sinoatrial node-like pacemaker cells (SANLPCs),following previous findings of silk-fibroin hydrogel-induced direct conversion of quiescent cardiomyocytes into pacemaker cells in rats through the activation of CDH5. In this study, the differentiating hiPSCs were treated with CDH5 (40 ng/mL) between Day 5 and 7 during cardiomyocytes differentiation. The findings in the present study demonstrated that CDH5 stimulated the expression of pacemaker-specific markers while suppressing markers associated with working cardiomyocytes, resulting in an increased proportion of SANLPCs among hiPSCs-derived cardiomyocytes (hiPSC-CMs)population. Moreover, CDH5 induced typical electrophysiological characteristics resembling cardiac pacemaker cells in hiPSC-CMs.Further mechanistic investigations revealed that the enriched differentiation of hiPSCs into SANLPCs induced by CDH5 was partially reversed by iCRT14, an inhibitor of β-catenin. Therefore, based on the aforementioned findings, it could be inferred that the regulation of β-catenin by CDH5 played a crucial role in promoting the enriched differentiation of hiPSCs into SANLPCs, which presents a novel avenue for the construction of biological pacemakers in forthcoming research.

Design, Pharmacological Characterization, and Molecular Docking of Minimalist Peptidomimetic Antagonists of α4β1 Integrin.

Integrin receptors mediate cell-cell interactions via the recognition of cell-adhesion glycoproteins, as well as via the interactions of cells with proteins of the extracellular matrix, and upon activation they transduce signals bi-directionally across the cell membrane. In the case of injury, infection, or inflammation, integrins of β2 and α4 families participate in the recruitment of leukocytes, a multi-step process initiated by the capturing of rolling leukocytes and terminated by their extravasation. In particular, α4β1 integrin is deeply involved in leukocyte firm adhesion preceding extravasation. Besides its well-known role in inflammatory diseases, α4β1 integrin is also involved in cancer, being expressed in various tumors and showing an important role in cancer formation and spreading. Hence, targeting this integrin represents an opportunity for the treatment of inflammatory disorders, some autoimmune diseases, and cancer. In this context, taking inspiration from the recognition motives of α4β1 integrin with its natural ligands FN and VCAM-1, we designed minimalist α/β hybrid peptide ligands, with our approach being associated with a retro strategy. These modifications are expected to improve the compounds' stability and bioavailability. As it turned out, some of the ligands were found to be antagonists, being able to inhibit the adhesion of integrin-expressing cells to plates coated with the natural ligands without inducing any conformational switch and any activation of intracellular signaling pathways. An original model structure of the receptor was generated using protein-protein docking to evaluate the bioactive conformations of the antagonists via molecular docking. Since the experimental structure of α4β1 integrin is still unknown, the simulations might also shed light on the interactions between the receptor and its native protein ligands.

Development of 2-D and 3-D culture platforms derived from decellularized nucleus pulposus.

Bioscaffolds derived from the extracellular matrix (ECM) have shown the capacity to promote regeneration by providing tissue-specific biological instructive cues that can enhance cell survival and direct lineage-specific differentiation. This study focused on the development and characterization of two-dimensional (2-D) and three-dimensional (3-D) cell culture platforms incorporating decellularized nucleus pulposus (DNP). First, a detergent-free protocol was developed for decellularizing bovine nucleus pulposus (NP) tissues that was effective at removing cellular content while preserving key ECM constituents including collagens, glycosaminoglycans, and the cell-adhesive glycoproteins laminin and fibronectin. Next, novel 2-D coatings were generated using the DNP or commercially-sourced bovine collagen type I (COL) as a non-tissue-specific control. In addition, cryo-milled DNP or COL particles were incorporated within methacrylated chondroitin sulphate (MCS) hydrogels as a 3-D cell culture platform for exploring the effects of ECM particle composition. Culture studies showed that the 2-D coatings derived from the DNP could support cell attachment and growth, but did not maintain or rescue the phenotype of primary bovine NP cells, which de-differentiated when serially passaged in monolayer culture. Similarly, while bovine NP cells remained highly viable following encapsulation and 14 days of culture within the hydrogel composites, the incorporation of DNP particles within the MCS hydrogels was insufficient to maintain or rescue changes in NP phenotype associated with extended in vitro culture based on gene expression patterns. Overall, DNP produced with our new decellularization protocol was successfully applied to generate both 2-D and 3-D bioscaffolds; however, further studies are required to assess if these platforms can be combined with additional components of the endogenous NP microenvironment to stimulate regeneration or lineage-specific cell differentiation.

ORAL MANIFESTATIONS IN PATIENTS WITH PEMPHIGUS VULGARIS

<h3>Objectives</h3> To review in the literature the main oral manifestations in patients with pemphigus vulgaris (PV). <h3>Study Design</h3> A bibliographic survey was carried out in the Scielo, PubMed, MEDLINE, and LILACS databases between the years 2010 to 2020. <h3>Results</h3> Ninety-eight articles were collected and 10 were selected, including 4 case reports, 2 systematic reviews, 3 literature reviews, and 1 cohort study. In the etiology of PV, there are autoantibodies directed against cell adhesion glycoproteins of the epithelium, the desmogleins, causing acantholysis and the formation of intraepithelial clefts that, clinically, manifest as bubbles that break easily, leading to ulcerations and pain as the main oral findings. Nikolsky signal, a semiologic maneuver, directs the diagnosis. The proposed treatment depends on the stage of the disease, with the control of oral manifestations systematically and/or topically, aiming at the patient's well-being. <h3>Conclusions</h3> Diffuse and painful oral ulcerations prevailed as the main oral manifestations and the positive Nikolsky sign as an auxiliary diagnosis. Such a disease must be familiar to the dentist because of the possible initial manifestation, emphasizing the importance of the professional in the diagnostic process, allowing for early detection and a good prognosis.

Characterization of the Tumor Microenvironment and the Biological Processes with a Role in Prostatic Tumorigenesis.

Prostate intratumoral heterogeneity, driven by epithelial–mesenchymal plasticity, contributes to the limited treatment response, and it is therefore necessary to use the biomarkers to improve patient prognostic survival. We aimed to characterize the tumor microenvironment (T lymphocyte infiltration, intratumoral CD34, and KI-67 expressions) by immunohistochemistry methods and to study the biological mechanisms (cell cycle, cell proliferation by adhesion glycoproteins, cell apoptosis) involved in the evolution of the prostate tumor process by flow-cytometry techniques. Our results showed that proliferative activity (S-phase) revealed statistically significant lower values of prostate adenocarcinoma (PCa) and benign prostatic hyperplasia (BPH) reported at non-malignant adjacent cell samples (PCa 4.32 ± 4.91; BPH 2.35 ± 1.37 vs. C 10.23 ± 0.43, p < 0.01). Furthermore, 68% of BPH cases and 88% of patients with PCa had aneuploidy. Statistically increased values of cell proliferation (CD34+ CD61+) were observed in prostate adenocarcinoma and hyperplasia cases reported to non-malignant adjacent cell samples (PCa 28.79 ± 10.14; BPH 40.65 ± 11.88 vs. C 16.15 ± 2.58, p < 0.05). The CD42b+ cell population with a role in cell adhesion, and metastasis had a significantly increased value in PCa cases (38.39 ± 11.23) reported to controls (C 26.24 ± 0.62, p < 0.01). The intratumoral expression of CD34 showed a significantly increased pattern of PCa tissue samples reported to controls (PCa 26.12 ± 6.84 vs. C 1.50 ± 0.70, p < 0.01). Flow cytometric analysis of the cell cycle, apoptosis, and adhesion glycoproteins with a critical role in tumoral cell proliferation, T cell infiltrations, Ki-67, and CD 34 expressions by IHC methods are recommended as techniques for the efficient means of measurement for adenocarcinoma and hyperplasia prostate tissue samples and should be explored in the future.

Assessment of the Molecular Heterogeneity of E-Cadherin Expression in Invasive Lobular Breast Cancer.

Simple SummaryInvasive lobular breast cancers (ILCs) are histologically classified by their discohesive growth pattern, due to loss of the cell adhesion glycoprotein E-cadherin (CDH1), which arises via mutation in CDH1 in around half of these tumours. A subset of these tumours, however, show mixed levels of E-cadherin expression. Here, we sought to address whether the distinct parts of individual tumours showing heterogeneous E-cadherin expression harbour distinct driver alterations. Using whole genome sequencing and methylation profiling of nine such cases, we identified that these tumours are clonally related, suggesting that they are part of the spectrum of ILC tumours. CDH1 mutant tumours showed a higher mutational burden indicative of APOBEC-mediated mutagenesis. In some cases, known clinically actionable driver mutations, such as PIK3CA, were exclusive to one component. Together, these results highlight the heterogeneity underpinning this special histological breast cancer.Mutations and loss of E-cadherin protein expression define the vast majority of invasive lobular carcinomas. In a subset of these cases, the heterogeneous expression of E-cadherin is observed either as wild-type (strong membranous) expression or aberrant expression (cytoplasmic expression). However, it is unclear as to whether the two components would be driven by distinct genetic or epigenetic alterations. Here, we used whole genome DNA sequencing and methylation array profiling of two separately dissected components of nine invasive lobular carcinomas with heterogeneous E-cadherin expression. E-cadherin negative and aberrant/positive components of E-cadherin heterogeneous tumours showed a similar mutational, copy number and promoter methylation repertoire, suggesting they arise from a common ancestor, as opposed to the collision of two independent tumours. We found that the majority of E-cadherin heterogeneous tumours harboured CDH1 mutations in both the E-cadherin negative and aberrant/positive components together with somatic mutations in additional driver genes known to be enriched in both pure invasive carcinomas of no special type and invasive lobular breast cancers, whereas these were less commonly observed in CDH1 wild-type tumours. CDH1 mutant tumours also exhibited a higher mutation burden as well as increased presence of APOBEC-dependent mutational signatures 2 and 13 compared to CDH1 wild-type tumours. Together, our results suggest that regardless of E-cadherin protein expression, tumours showing heterogeneous expression of E-cadherin should be considered as part of the spectrum of invasive lobular breast cancers.

Biomaterial-induced conversion of quiescent cardiomyocytes into pacemaker cells in rats.

Pacemaker cells can be differentiated from stem cells or transdifferentiated from quiescent mature cardiac cells via genetic manipulation. Here we show that the exposure of rat quiescent ventricular cardiomyocytes to a silk-fibroin hydrogel activates the direct conversion of the quiescent cardiomyocytes to pacemaker cardiomyocytes by inducing the ectopic expression of the vascular endothelial cell-adhesion glycoprotein cadherin. The silk-fibroin-induced pacemaker cells exhibited functional and morphological features of genuine sinoatrial-node cardiomyocytes in vitro, and pacemaker cells generated via the injection of silk fibroin in the left ventricles of rats functioned as a surrogate in situ sinoatrial node. Biomaterials with suitable surface structure, mechanics and biochemistry could facilitate the scalable production of biological pacemakers for human use.

Are dietary factors involved in the association of CDH4 methylation and breast cancer risk?

DNA methylation is one of the most important epigenetic modifications in breast cancer (BC) development, and long-term dietary habits can alter DNA methylation. Cadherin-4 (CDH4, a member of the cadherin family) encodes Ca2+-dependent cell-cell adhesion glycoproteins. We conducted a case-control study (380 newly diagnosed BC and 439 cancer-free controls) to explore the relationship of CDH4 methylation in peripheral blood leukocyte DNA (PBL DNA), as well as its combined and interactive effects with dietary factors on BC risk. A case-only study (335 newly diagnosed BC) was conducted to analyse the association between CDH4 methylation in breast tissue DNA and dietary factors. CDH4 methylation was detected using quantitative methylation-specific PCR. Unconditional logistic regressions were used to analyse the association of CDH4 methylation in PBL DNA and BC risk. Cross-over analysis and unconditional logistic regression were used to calculate the combined and interactive effects between CDH4 methylation in PBL DNA and dietary factors in BC. CDH4 hypermethylation was significantly associated with increased BC risk in PBL DNA (ORadjusted (ORadj) = 2·70, (95 % CI 1·90, 3·83), P < 0·001). CDH4 hypermethylation also showed significant combined effects with the consumption of vegetables (ORadj = 4·33, (95 % CI 2·63, 7·10)), allium vegetables (ORadj = 7·00, (95 % CI 4·17, 11·77)), fish (ORadj = 7·92, (95 % CI 3·79, 16·53)), milk (ORadj = 6·30, (95 % CI 3·41, 11·66)), overnight food (ORadj = 4·63, (95 % CI 2·69, 7·99)), pork (ORadj = 5·59, (95 % CI 2·94, 10·62)) and physical activity (ORadj = 4·72, (95 % CI 2·87, 7·76)). Moreover, consuming milk was significantly related with decreased risk of CDH4 methylation (OR = 0·61, (95 % CI 0·38, 0·99)) in breast tissue. Our findings may provide direct guidance on the dietary intake for specific methylated carriers to decrease their risk for developing BC.

THE ROLE OF SERUM WISTERIA FLORIBUNDA AGGLUTINATION POSITIVE MAC-2 BINDING PROTEIN IN PREDICTION OF ESOPHAGEAL VARICES IN HEPATITIS C VIRUS-RELATED CIRRHOTIC PATIENTS

HCV infection is one of the main causes of chronic liver disease worldwide.Liver cirrhosis is the final stage of chronic liver injury with a high mortality. The transition from compensated forms to decompensated cirrhosis occurs at a rate of 5-7% per year. Once patients have developed the first episode of decompensation, complications tend to accumulate and life expectancy is dramatically decreased. Portal Hypertension is the initial and main consequence of cirrhosis and is responsible for the majority of its complications such as ascites, hepatic encephalopathy and gastroesophageal varices. Variceal haemorrhage is considered the most lethal complications of portal hypertension with a six-week mortality after an episode of bleeding ranges between 15% and 25%. So early detection and management of esophageal varices is very crucial in cirrhotic patients.Serum WFA- M2BP, is a cell adhesive glycoprotein of the extracellular matrix secreted as a ligand of galectin 3.It is secreted from many cell types, particularly hepatocytes. It has been shown to increase with the progression of liver fibrosis.

In Silico Identification of Potential Phosphorylation in the Cytoplasmic Domain of Epithelial Cell Adhesion Molecule.

The epithelial cell adhesion molecule (EpCAM) is a transmembrane cell adhesion glycoprotein, which primarily contributes to stemness, proliferation, and metastasis properties of tumor cells. Regulated intramembrane proteolysis by ADAM proteases and γ-secretase cleaves EpCAM into an ∼27 kDa soluble extracellular and an ∼4 kDa cytoplasmic domain (EpICD). After the EpICD fragment is released inside the cell, the formation of a nuclear signaling complex with the FHL2 molecule is critical for exerting its regulatory role. Trop-2, a homologous protein of EpCAM, undergoes phosphorylation in its cytoplasmic domain (Trop-IC). The phosphorylation of Trop-2 is reported to be crucial for its function. This led us to ask the fundamental question if EpCAM does undergo similar post-translational modification(PTM) like its homologous protein to carry out its diverse biological function. Here, we identify a putative phosphorylation site at Tyr297 located in the cytoplasmic domain of EpCAM. Molecular dynamic simulation (MDS) of 90 ns was carried out to understand the biological/functional relevance of the putative phosphorylation. It was observed that this phosphorylation stabilizes the α-helical structure of the EpICD. Though Tyr297 does not affect the γ-secretase mediated cleavage of EpCAM, it affects the binding of EpICD to FHL2. Docking analysis revealed that phosphorylation mediated structural stability of EpICD positively impacts its binding affinity with FHL2, which was further validated using 100 ns MDS. Phosphorylated EpICD forms higher numbers of hydrogen bonds, salt bridges, and other non-bonded interactions with FHL2, leading to enhanced interactions. This in silico study reveals a potential PTM in the EpICD, providing the basis for future research in understanding the mechanism behind the diverse biological function of EpCAM.

Prevention of Heme-Induced Human Endothelial Cell Activation By Hemopexin in Vitro

Hemoglobin (Hb) is one of the most abundant proteins in the human body. When red blood cells rupture, cell-free Hb may initiate adverse pathophysiological reactions. Pathophysiology triggered by cell-free Hb plays an important role in modifying the phenotype of sickle cell disease (SCD). SCD is caused by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA found in healthy individuals. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where it triggers lipid peroxidation and generation of cytotoxic and pro-inflammatory reaction products. These processes promote endothelial cell activation and damage. The endogenous plasma protein hemopexin exhibits the highest binding affinity for heme and binds heme in a 1:1 binding ratio. Heme bound to hemopexin is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation. To investigate the endothelial-protective function of hemopexin based on its ability to scavenge heme, we exposed human umbilical vein endothelial cells (HUVEC) in vitro to heme(NaOH) in the presence or absence of different hemopexin doses. As a read-out, different markers for endothelial cell activation were analyzed by either flow cytometry or multiplexed particle-based flow cytometry (Luminex). Briefly, confluent HUVEC were preincubated with hemopexin at different concentrations for 5 min before stimulation with heme(NaOH) for 25 min. Following stimulation cells were analyzed by flow cytometry for expression of membrane bound P-Selectin, a robust marker of endothelial cell activation. Alternatively, heme(NaOH) stimulation of hemopexin-preincubated HUVEC was conducted for 16 h and cell culture supernatants were analyzed by Luminex for three additional well-characterized plasma markers of endothelial cell activation: pro-inflammatory cytokine IL-8, cell adhesion molecule VCAM-1 and glycoprotein Von Willebrand factor (vWF). In the absence of hemopexin, heme(NaOH) consistently induced robust cell surface expression of P-Selectin and elevated levels of soluble IL-8, VCAM-1 and vWF. However, hemopexin completely blocked the stimulatory potential of heme as HUVEC exposed to pre-formed heme:hemopexin complexes showed unchanged P-Selectin expression levels compared to negative control samples. We found that hemopexin reduced heme(NaOH)-mediated P-selectin expression on HUVEC in a dose-dependent fashion. Once an equimolar ratio between heme and hemopexin was reached, P-selectin expression was abolished as shown in figure 1. In addition to P-Selectin, hemopexin also had a strong effect to reduce the heme-induced expression of IL-8, VCAM-1 and vWF to background levels. Thus, the presented data underlines on the one hand the stimulatory capacity of heme(NaOH) on endothelial cells and demonstrates on the other hand the potential of hemopexin to efficiently neutralize free heme. In a stoichiometric fashion, hemopexin potently prevents the pro-inflammatory effect of heme on endothelial cells. Hence, our study suggests a protective role of hemopexin for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis. Additional experiments are required to elucidate the effect of hemopexin on the endothelium in more detail. Combined with our other lines of data, our results further support the investigation of hemopexin as a potential therapeutic agent in the treatment of sickle cell disease. Disclosures Adam: CSL Behring AG: Current Employment. Gentinetta:CSL Behring: Current Employment. Diditchenko:CSL Behring AG: Current Employment. Schaub:CSL Behring AG: Current Employment. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment.

Abstract 5867: Transcriptome profiling of ST3GAL4 and FUT7 in multiple tumor types and prognostic value in adult acute myeloid leukemia

Abstract E-selectin is a cell adhesion glycoprotein expressed on endothelial cells and participates in the development of environmental-mediated drug resistance and poor clinical outcome in cancer. The E-selectin antagonist, uproleselan, interrupts leukemic cell homing to the vascular niche and increases susceptibility to cytotoxic therapies. Recent clinical data demonstrate a correlation between leukemic cell surface E-selectin ligands and response to uproleselan, linking E-selectin ligand expression to response. Multiple genes involved in the glycan synthesis of E-selectin ligands are highly expressed in pediatric AML. Expression levels of two of these genes, ST3GAL4 and FUT7 are associated with poor outcome and are associated with cell surface E-selectin ligand expression. In the current studies we extend transcriptome profiling of E-selectin ligand forming glycosylation genes with an emphasis on ST3GAL4 and FUT7 in different cancers. Initially, we investigated expression levels of ST3GAL4 and FUT7 in 10,258 samples covering 33 cancer types from the TCGA PanCanAtlas. ST3GAL4 and FUT7 were consistently expressed in the majority of cancers evaluated. The cancer types that expressed ST3GAL4 most highly were melanoma (uveal and skin), kidney chromophobe, adrenocortical carcinoma and bladder urothelial carcinoma, while for FUT7, it was AML, diffuse large B-cell lymphoma, thymoma, testicular germ cell tumors and head and neck squamous cell carcinoma. Of particular interest was the identification of adult AML for the highest expression of FUT7 and high expression of ST3GAL4 (mean log2 gene expression = 8.1 and 9.4, respectively). Augmented expression of FUT7 and ST3GAL4 were also observed in an analysis of 39 AML cell lines among the 1,457 cell lines comprising the Cancer Cell Line Encyclopedia RNAseq data set. The prognostic significance of FUT7 and ST3GAL4 in adult AML was further assessed using the TCGA-LAML RNAseq dataset for differential expression and associations with overall survival (OS). The data set included 151 RNAseq profiles of bone marrow samples from adult patients with AML, and within this data set the status of the FMS-like tyrosine kinase 3 (FLT3) proto-oncogene was considered. Mutational alterations of FLT3 are associated with higher risk of relapse and shorter OS compared with wild-type FLT3. ST3GAL4 and FUT7 were both identified as being upregulated (fold-change = 1.73 and 1.40, respectively) in the mutated FLT3 subset (n=46) as compared to wild type FLT-3 (p=0.000033 and 0.046, respectively). Notably in the mutated FLT-3 subset expression of FUT7 was significantly associated with a poor prognosis and decreased OS (HR = 4.56, p= 0.015). Collectively, these studies extend the prognostic importance of the E-selectin ligand glycosylation genes, ST3GAL4 and FUT7, to adult AML where these genes may be useful as predictive biomarkers. In addition, these studies suggest potential additional tumor types beyond AML where treatment protocols with uproleselan may have therapeutic benefits. Citation Format: William E. Fogler, Vince Deng, David Stewart, Michael Jarman, Simone Daminelli, Adrian Carr, John L. Magnani. Transcriptome profiling of ST3GAL4 and FUT7 in multiple tumor types and prognostic value in adult acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5867.

Self-assembled biomimetic Nano-Matrix for stem cell anchorage.

Mesenchymal stem cells (MSCs) have been widely applied in biomedicine due to their ability to differentiate into many different cell types and their ability to synthesize a broad spectrum of growth factors and cytokines that directly and indirectly influence other cells in their vicinity. To guide MSC infiltration to a bone fracture site, we developed a novel self-assembled Nano-Matrix which can be used as an injectable scaffold to repair bone fractures. The Nano-Matrix is formed by Janus base nanotubes (JBNTs) and fibronectin (FN). JBNTs are nucleobase-derived nanotubes mimicking collagen fibers, and FN is one of the cell adhesive glycoproteins which is responsible for cell-extracellular matrix interactions and guides stem cell migration and differentiation to desired cells types. Here, we demonstrated the successful fabrication and characterization of the JBNT/FN Nano-Matrix as well as its excellent bioactivity that encouraged human MSC migration and adhesion. This work lays a solid foundation for using the Nano-Matrix as an injectable approach to improve MSC retention and function during bone fracture healing.

Transcriptome Profiling of Glycosylation Genes Defines Correlation with E-Selectin Ligand Expression and Clinical Outcome in AML

E-selectin (E-sel) is a cell adhesion glycoprotein that is expressed on endothelial cells and has been implicated in therapeutic resistance. In most myeloid leukemias, leukemic blasts express E-sel ligands (EsL), which contain the glycan epitope of the carbohydrate sialyl Lex (sLex). This expression increases the likelihood of adhesion to vascular endothelial cells and facilitates sequestration in the bone marrow vascular niche, leading to cell adhesion-mediated drug resistance and poor clinical outcome. E-sel antagonists like uproleselan, interrupts leukemic cell homing to the vascular niche, increases susceptibility to cytotoxic and targeted therapies and can be potent adjuncts to therapeutics. Recent data demonstrated a correlation between leukemic cell surface levels of EsL and response to uproleselan, linking EsL expression to response. We questioned whether transcriptome profiling of EsL-forming glycosylation genes can be used to identify elevated EsL expression in patients with acute myeloid leukemia (AML), and subsequently which patients might best respond to uproleselan. RNA-seq data from patients treated in COG AAML1031 (N = 1,074) was available for evaluation. We examined transcriptome expression of 24 genes that code for enzymes involved in glycosylation of EsL. All analyses were performed in R. Cox proportional hazards models were generated using the survival package. Multidimensional flow cytometry (MDF) was used to detect cell surface EsL expression by two techniques: direct binding of an E-sel/hIg, PE labeled chimera, and the anti-sLex antibody HECA-452. Seven of the 24 genes examined had minimal expression (mean &lt;1 TPM) and were excluded from further analysis. The remaining 17 were variably expressed (Fig. 1A). To assess association of expression with outcome, univariate Cox models for overall survival (OS) were generated, using gene expression as a continuous coefficient (N = 1,061). Of the 17 genes, 7 were significantly associated with increased risk (p &lt; 0.05, Fig. 1B). ST3GAL4 and FUT7 were targeted for further evaluation, as they directly synthesize sLex (Fig. 1C), and were significantly associated with adverse outcome (HR = 1.013, p &lt; 0.0001, and HR = 1.023, p &lt; 0.0001, respectively). Patients highly expressing FUT7 (highest quartile of expression) had significantly worse outcome than low expressors (lowest 3 quartiles of expression), with a 5-year OS of 50.3% vs. 68.3% (p &lt; 0.0001, Fig. 1D). Similarly, those with high ST3GAL4 expression had a 5-year OS of 51.3%, compared to 68.1% for low expressors (p &lt; 0.0001, Fig. 1D). A subset of patients highly expressed both genes (ST3GAL4 and FUT7 high; SFhigh, N = 132). Compared to patients that did not highly express either gene (SFlow), these individuals had particularly adverse survival (45.8% OS vs 71.0% OS, p &lt; 0.0001). Patients with one of two high expressing genes (SFinter) had a 5-year OS of 55.5%, illustrating what may be a compounding unfavorable impact conferred on survival (Fig. 1E). Further investigation of clinical characteristics within these 3 groups revealed that 71.5% of infants &lt;1 year were SFlow, with only 4.66% in SFhigh. In addition, CBF-AML was greatly underrepresented in SFhigh, with 97% of both t(8;21) and inv(16) patients in SFlow, and 0% in SFhigh. To verify surface protein expression of the two genes, leukemic specimens from SFhigh patients (N = 10) and SFlow patients (N = 10) underwent cell surface expression evaluation of glycosylated EsL using two MDF assays. SFlow patients had low or undetectable levels of cell surface EsL by both assays, whereas SFhigh patients had significantly higher expression of EsL (p &lt; 0.001, Fig. 1F). This suggests a strong correlation between transcriptome measurements of EsL glycosylation genes and cell surface glycosylation levels of EsL. In summary, we have shown that multiple genes involved in the glycan synthesis of EsLs are highly expressed in pediatric AML. Two of these genes, ST3GAL4 and FUT7, are associated with poor outcome. Additionally, high expression of these genes is detectable at the transcript level and associated with cell surface EsL expression. These genes provide novel targets for overcoming drug resistance induced by the tumor microenvironment, and lend support for the use of EsL glycosylation genes as predictive biomarkers. These data also confirm the importance of E-sel in disease progression in AML and its potential as a therapeutic target. Figure 1 Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Fogler:GlycoMimetics Inc: Employment, Equity Ownership. Loken:Hematologics, Inc: Employment, Equity Ownership.

NMR-based insight into galectin-3 binding to endothelial cell adhesion molecule CD146: Evidence for noncanonical interactions with the lectin's CRD β-sandwich F-face.

Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1-D5) and a shorter, D5-deleted version of CD146 (D1-D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1-D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding β-sheet S-face (β-strands 1, 10, 3, 4, 5, 6) of the Gal-3 β-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face β-sheet (β-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with β-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin's CRD F-face in the CD146 binding process.

Structural biology of laminins.

Laminins are large cell-adhesive glycoproteins that are required for the formation and function of basement membranes in all animals. Structural studies by electron microscopy in the early 1980s revealed a cross-shaped molecule, which subsequently was shown to consist of three distinct polypeptide chains. Crystallographic studies since the mid-1990s have added atomic detail to all parts of the laminin heterotrimer. The three short arms of the cross are made up of continuous arrays of disulphide-rich domains. The globular domains at the tips of the short arms mediate laminin polymerization; the surface regions involved in this process have been identified by structure-based mutagenesis. The long arm of the cross is an α-helical coiled coil of all three chains, terminating in a cell-adhesive globular region. The molecular basis of cell adhesion to laminins has been revealed by recent structures of heterotrimeric integrin-binding fragments and of a laminin fragment bound to the carbohydrate modification of dystroglycan. The structural characterization of the laminin molecule is essentially complete, but we still have to find ways of imaging native laminin polymers at molecular resolution.

LPS decreases the expression of Monocarboxylate transporter‐1 (MCT1) in mouse monocytes

The molecule Toll‐like Receptor‐4 (TLR4) recognizes Gram negative bacteria, specifically the lipopolysaccharide component (LPS) and initiates a signal transduction cascade leading to an inflammatory response. Recent studies by this laboratory suggest the cell adhesion glycoprotein Basigin (CD147) interacts with TLR4 via their transmembrane domains. The role of Basigin in mouse retina has been well‐studied and includes an association with Monocarboxylate Transporter 1 (MCT1) for metabolic support of photoreceptor neurons. The role of Basigin in innate immune responses is less‐well studied. Therefore, the response of Basigin to LPS was assessed and compared to that of TLR4. The response of MCT1 to LPS was also assessed. It was hypothesized that Basigin and MCT1 expression would increase in response to LPS. To test this, RAW 264.7 mouse monocytes were incubated in the presence of 1 mg/mL LPS in serum‐free medium for various times ranging from 2–24 hours. Control cells received PBS. The cells were collected, and RNA was isolated for gene expression analyses via quantitative‐reverse transcription PCR (q‐RT‐PCR). The expression of TLR4 decreased at 2 hours and increased at 24 hours of LPS induction, when compared to control samples, although no changes were determined to be statistically significant. The expression of Basigin was similar between control and LPS‐treated cells at all time points, except for 24 hours of induction, when Basigin expression was significantly lower in LPS‐treated cells. MCT1 expression was also similar between control and LPS‐treated cells at all time points, except for 24 hours of induction, when MCT1 expression was significantly lower in LPS‐treated cells. These findings do not support the hypothesis. The data for TLR4 expression in response to LPS in mouse monocytes are consistent with published reports. Although it was hypothesized that metabolic responses would increase in monocytes to support immune mechanisms, the data for MCT1 suggests that metabolism is reduced in activated monocytes, perhaps as a shift of resources from general cellular maintenance to induction of an innate immune response. The observed decrease in Basigin expression also did not support the hypothesis and suggests that the molecule is either regulated directly by the innate immune response or indirectly via its role in metabolism through MCT1. Future studies will assess the expression of Basigin and MCT1 at the protein level to better analyze the role of metabolism in the innate immune response.Support or Funding InformationUNF Office of Academic AffairsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prognositic significance of P-cadherin expression in breast cancer: Protocol for a meta-analysis.

Background:P-cadherin is a calcium-dependent cell–cell adhesion glycoprotein. It has been implicated in invasiveness and metastasis. However, the clinical prognostic value of overexpression of P-cadherin in patients with breast cancer (BC) remains unsettled.Methods:A systematic literature search will be performed in all available databases to quantitatively review eligible studies and identify all relevant data, which could be used to detect the relationship between overexpression of P-cadherin and overall survival (OS), disease-free survival (DFS), and clinicopathological parameters. Hazard ratio and 95% confidence intervals (CIs) or P value will be employed as effect measures to estimate the correlation between P-cadherin and the oncologic outcomes including overall survival (OS), disease-free survival (DFS). Odds ratios (ORs) and the 95% CIs will be evaluated for the pooled analysis of the correlation between P-cadherin expression and clinicopathological features. We will use the Review Manager (Revman) 5.3.5 software (Cochrane Community, London, United Kingdom) and STATA 14 software (version 14.0; Stata Corp, College Station, TX) to perform the meta-analysis to calculate the data.Results:The review will provide a high-quality synthesis of current evidence of the prognostic role of P-cadherin in BCs. The results will be published in a peer-reviewed journal.Conclusion:We hope that the results of this study will provide significant evidence to assess whether the expression of P-cadherin is associated with poor prognosis in patients with BC.PROSPERO registration number:This meta-analysis protocol has been registered in the PROSPERO network with registration number: CRD42019119880.

SMAGP a novel biomarker of cervical cancer development and progression.

IntroductionCervical cancer, one of the most common malignant gynecological tumors, is a significant burden on the health of females worldwide. The purpose of this study was to investigate genes associated with lymph node metastasis in cervical cancer.MethodsWe report on the lymph node metastasis-associated gene, small cell adhesion glycoprotein (SMAGP), as a key regulator of cervical cancer development and progression. SMAGP expression levels were investigated in 70 cervical squamous cell carcinoma samples and 10 normal cervical squamous epithelium samples.ResultsImmunohistochemistry analysis revealed that SMAGP protein levels were significantly elevated in cervical cancer tissue compared with normal cervical squamous epithelium. Silencing of SMAGP induced cell cycle arrest, inhibited the cell proliferation and colony formation ability of cervical cancer cells in vitro and suppressed their tumorigenic potential in nude mice. In addition, SMAGP knockdown reduced expression of epithelial mesenchymal transition-related proteins, including vimentin, β-cadherin, MMP2, and Twist.ConclusionTogether, our findings demonstrate that SMAGP plays a critical role in cell proliferation and tumorigenesis and could be a new therapeutic target in cervical cancer.