GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

Recruitment of calcineurin to the TCR positively regulates T cell activation.

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

Serine restriction alters sphingolipid diversity to constrain tumour growth.

Serine, glycine and other nonessential amino acids are critical for tumour progression, and strategies to limit their availability are emerging as potential therapies for cancer1-3. However, the molecular mechanisms driving this response remain unclear and the effects on lipid metabolism are relatively unexplored. Serine palmitoyltransferase (SPT) catalyses the de novo biosynthesis of sphingolipids but also produces noncanonical 1-deoxysphingolipids when using alanine as a substrate4,5. Deoxysphingolipids accumulate in the context of mutations in SPTLC1 or SPTLC26,7-or in conditions of low serine availability8,9-to drive neuropathy, and deoxysphinganine has previously been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and the promiscuity of SPT to modulate the endogenous synthesis of toxic deoxysphingolipids and slow tumour progression. Anchorage-independent growth reprogrammes a metabolic network involving serine, alanine and pyruvate that drives the endogenous synthesis and accumulation of deoxysphingolipids. Targeting the mitochondrial pyruvate carrier promotes alanine oxidation to mitigate deoxysphingolipid synthesis and improve spheroid growth, similar to phenotypes observed with the direct inhibition of SPT or ceramide synthesis. Restriction of dietary serine and glycine potently induces the accumulation of deoxysphingolipids while decreasing tumour growth in xenograft models in mice. Pharmacological inhibition of SPT rescues xenograft growth in mice fed diets restricted in serine and glycine, and the reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to the accumulation of deoxysphingolipids and mitigates tumour growth. The promiscuity of SPT therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which further sensitizes tumours to metabolic stress.

Neutrophil infiltration during inflammation is regulated by PILRα via modulation of integrin activation.

Acute inflammatory responses are important in host defense, whereas dysregulated inflammation results in life-threatening complications. Here we found that paired immunoglobulin-like type 2 receptor alpha (PILRα), an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), negatively regulated neutrophil infiltration during inflammation. Pilra(-/-) mice had increased neutrophil recruitment to inflammatory sites and were highly susceptible to endotoxin shock. Pilra(-/-) neutrophils showed enhanced transmigration ability and increased adhesion to the ß(2) integrin ligand ICAM-1. PILRα expressed on neutrophils constitutively associated in cis with its ligands, resulting in clustering of PILRα during stimulation with a chemoattractant. Clustering of PILRα enhanced ITIM-mediated signaling, thus modulating ß(2) integrin inside-out activation. These data demonstrate that neutrophil recruitment in inflammatory responses is regulated by PILRα via modulation of integrin activation.

Peptide-based self-assembled monolayers (SAMs): what peptides can do for SAMs and vice versa.

Self-assembled monolayers (SAMs) represent highly ordered molecular materials with versatile biochemical features and multidisciplinary applications. Research on SAMs has made much progress since the early begginings of Au substrates and alkanethiols, and numerous examples of peptide-displaying SAMs can be found in the literature. Peptides, presenting increasing structural complexity, stimuli-responsiveness, and biological relevance, represent versatile functional components in SAMs-based platforms. This review examines the major findings and progress made on the use of peptide building blocks displayed as part of SAMs with specific functions, such as selective cell adhesion, migration and differentiation, biomolecular binding, advanced biosensing, molecular electronics, antimicrobial, osteointegrative and antifouling surfaces, among others. Peptide selection and design, functionalisation strategies, as well as structural and functional characteristics from selected examples are discussed. Additionally, advanced fabrication methods for dynamic peptide spatiotemporal presentation are presented, as well as a number of characterisation techniques. All together, these features and approaches enable the preparation and use of increasingly complex peptide-based SAMs to mimic and study biological processes, and provide convergent platforms for high throughput screening discovery and validation of promising therapeutics and technologies.

Nanoporous Titanium Implant Surface Accelerates Osteogenesis via the Piezo1/Acetyl-CoA/ß-Catenin Pathway.

Osseointegration is the most important factor determining implant success. The surface modification of TiO2 nanotubes prepared by anodic oxidation has remarkable advantages in promoting bone formation. However, the mechanism behind this phenomenon is still unintelligible. Here we show that the nanomorphology exhibited open and clean nanotube structure and strong hydrophilicity, and the nanomorphology significantly facilitated the adhesion, proliferation, and osteogenesis differentiation of stem cells. Exploring the mechanism, we found that the nanomorphology can enhance mitochondrial oxidative phosphorylation (OxPhos) by activating Piezo1 and increasing intracellular Ca2+. The increase in OxPhos can significantly uplift the level of acetyl-CoA in the cytoplasm but not significantly raise the level of acetyl-CoA in the nucleus, which was beneficial for the acetylation and stability of ß-catenin and ultimately promoted osteogenesis. This study provides a new interpretation for the regulatory mechanism of stem cell osteogenesis by nanomorphology.

Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing.

Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin concentration and exposure time.

Development and characterization of the mode-of-action of inhibitory and agonist peptides targeting the voltage-gated sodium channel SCN1B beta-subunit.

Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and ß1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (ß1/ß1B) mimetic peptide, ßadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore ßadp1's mechanism and develop novel SCN1B mimetic peptides affecting ß1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in ß1-expressing cells, we observed ßadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of ßadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and ß1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. ßadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, ßadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (ß1/ß1B) mimetic peptides are reported with the potential to modulate intercellular VGSC ß1-mediated adhesion, potentially through ß1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.

Blockage of aquaporin-3 peroxiporin activity by organogold compounds affects melanoma cell adhesion, proliferation and migration.

Aquaporin-3 (AQP3) is a membrane channel with dual aquaglyceroporin/peroxiporin activity, facilitating the diffusion of water, glycerol and H2O2 across cell membranes. AQP3 shows aberrant expression in melanoma and its role in cell adhesion, migration and proliferation is well described. Gold compounds were shown to modulate AQP3 activity with reduced associated toxicity, making them promising molecules for cancer therapy. In this study, we validated the phenotype resulting from AQP3-silencing of two melanoma cell lines, MNT-1 and A375, which resulted in decreased H2O2 permeability. Subsequently, the AQP3 inhibitory effect of a new series of organogold compounds derived from Auphen, a potent AQP3 inhibitor, was first evaluated in red blood cells (RBCs) that highly express AQP3, and then in HEK-293T cells with AQP3 overexpression to ascertain the compounds' specificity. The first screening in RBCs unveiled two organogold compounds as promising blockers of AQP3 permeability. Moderate reduction of glycerol permeability but drastic inhibition of H2O2 permeability was detected for some of the gold derivatives in both AQP3-overexpressing cells and human melanoma cell lines. Additionally, all compounds were effective in impairing cell adhesion, proliferation and migration, although in a cell type-dependent manner. In conclusion, our data show that AQP3 peroxiporin activity is crucial for melanoma progression and highlight organogold compounds as promising AQP3 inhibitors with implications in melanoma cell adhesion, proliferation and migration, unveiling their potential as anticancer drugs against AQP3-overexpressing tumours. KEY POINTS: AQP3 affects cellular redox balance. Gold compounds inhibit AQP3 permeability in melanoma cells. AQP3 is involved in cell adhesion, proliferation and migration of melanoma. Blockage of AQP3 peroxiporin activity impairs melanoma cell migration. Gold compounds are potential anticancer drug leads for AQP3-overexpressing cancers.

Molecular engineering of a minimal E-cadherin inhibitor protein derived from Clostridium botulinum hemagglutinin.

Hemagglutinin (HA), a nontoxic component of the botulinum neurotoxin (BoNT) complex, binds to E-cadherin and inhibits E-cadherin-mediated cell-cell adhesion. HA is a 470 kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents. Thus, to prepare recombinant full-length HA in vitro, it is necessary to reconstitute the macromolecular complex from purified HA subcomponents, which involves multiple purification steps. In this study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from Clostridium botulinum HA with a simple purification strategy needed for production. NanoHA, containing HA2 and a truncated mutant of HA3 (amino acids 380-626; termed as HA3mini), is a 47 kDa single polypeptide (one-tenth the molecular weight of full-length HA, 470 kDa) engineered with three types of modifications: (i) a short linker sequence between the C terminus of HA2 and N terminus of HA3; (ii) a chimeric complex composed of HA2 derived from the serotype C BoNT complex and HA3mini from the serotype B BoNT complex; and (iii) three amino acid substitutions from hydrophobic to hydrophilic residues on the protein surface. We demonstrated that NanoHA inhibits E-cadherin-mediated cell-cell adhesion of epithelial cells (e.g., Caco-2 and Madin-Darby canine kidney cells) and disrupts their epithelial barrier. Finally, unlike full-length HA, NanoHA can be transported from the basolateral side to adherens junctions via passive diffusion. Overall, these results indicate that the rational design of NanoHA provides a minimal E-cadherin inhibitor with a wide variety of applications as a lead molecule and for further molecular engineering.

Carboxymethylated Desmodium styracifolium polysaccharide reduces the risk of calcium oxalate kidney stone formation by inhibiting crystal adhesion and promoting crystal endocytosis.

The inhibition of cell surface crystal adhesion and an appropriate increase in crystal endocytosis contribute to the inhibition of kidney stone formation. In this study, we investigated the effects of different degrees of carboxymethylation on these processes. An injury model was established by treating human renal proximal tubular epithelial (HK-2) cells with 98.3 ± 8.1 nm calcium oxalate dihydrate (nanoCOD) crystals. The HK-2 cells were protected with carboxy (-COOH) Desmodium styracifolium polysaccharides at 1.17% (DSP0), 7.45% (CDSP1), 12.2% (CDSP2), and 17.7% (CDSP3). Changes in biochemical indexes and effects on nanoCOD adhesion and endocytosis were detected. The protection of HK-2 cells from nanoCOD-induced oxidative damage by carboxymethylated Desmodium styracifolium polysaccharides (CDSPs) is closely related to the protection of subcellular organelles, such as mitochondria. CDSPs can reduce crystal adhesion on the cell surface and maintain appropriate crystal endocytosis, thereby reducing the risk of kidney stone formation. CDSP2 with moderate -COOH content showed the strongest protective activity among the CDSPs.

VCAM-1 mediates proximal tubule-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

Studies in animal models have suggested a linkage between the inflammatory response to injury and subsequent nephron loss during the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Failure of normal repair during the CKD transition correlates with de novo expression of vascular cell adhesion protein-1 (VCAM-1) by a subset of injured proximal tubule cells. This study identified the role of VCAM-1 expression in promoting the failed repair state. Single-cell transcriptome analysis of patients with AKI and CKD and whole kidney RNA and protein analyses of mouse models of CKD confirmed a marked increase of VCAM-1 expression in the proximal tubules of injured kidneys. In immortalized mouse proximal tubular cells and primary cultured renal cells (PCRCs), VCAM-1 expression was induced by proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. Analyses of bulk RNA sequencing of TNF-α-treated primary cultured renal cells or pseudo-bulk RNA sequencing of biopsies from Kidney Precision Medicine Project datasets indicated activation of NF-κB and an enrichment of inflammatory response and cell adhesion pathways in VCAM-1-positive cells. Pharmacological inhibition of NF-κB signaling or genetic deletion of myeloid differentiation factor 88 and TIR domain-containing adapter-inducing interferon-ß suppressed TNF-α- and IL-1ß-induced VCAM-1 expression in vitro. TNF-α stimulation or overexpression of VCAM-1 significantly increased splenocyte adhesion to the mouse proximal tubular monolayer in culture. These results demonstrate that persistence of proinflammatory cytokines after AKI can induce NF-κB-dependent VCAM-1 expression by proximal tubule cells, mediating increased immune cell adhesion to the tubule and thus promoting further tubule injury and greater risk of progression from AKI to CKD.NEW & NOTEWORTHY We demonstrated the induction of VCAM-1 and its biological function in proximal tubules. We found that proinflammatory cytokines (TNF-α and IL-1ß) significantly induced VCAM-1 expression via NF-κB signaling pathway. TNF-α treatment or overexpression of VCAM-1 in immortalized MPT cells increased CD45+ splenocyte adhesion. Pharmacological inhibition of NF-κB or genetic deletion of Vcam1 suppressed TNF-α-induced splenocyte adhesion in vitro, suggesting that VCAM-1 mediates proximal tubular-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

The influence of a modified p53 C-terminal peptide by using a tumor-targeting sequence on cellular apoptosis and tumor treatment.

The restoration of the function of p53 in tumors is a therapeutic strategy for the highly frequent mutation of the TP53 tumor suppressor gene. P460 is a wild-type peptide derived from the p53 C-terminus and has been proven to be capable of restoring the tumor suppressor function of p53. The poor accumulation of drugs in tumors is a serious hindrance to tumor treatment. For enhancing the activity of P460, the tumor-targeting sequence Arg-Gly-Asp-Arg (RGDR, C-end rule peptide) was introduced into the C-terminus of P460 to generate the new peptide P462. P462 presented better activity than P460 in inhibiting the proliferation of cancer cells and increasing the number of tumor cells undergoing apoptosis. Cell adhesion analysis and tumor imaging results revealed that P462 showed more specific and extensive binding with tumor cells and greater accumulation in tumors than the wild-type peptide. Importantly, treatment with P462 was more efficacious than that with P460 in vivo and was associated with considerably improved tumor-homing activity. This study highlights the importance of the roles of the tumor-homing sequence RGDR in the enhancement in cell attachment and tumor accumulation. The results of this work indicate that P462 could be a novel drug candidate for tumor treatment.

A facile Immunoregulatory Constructional Design by Proanthocyanidin Optimizing Directional Chitosan Microchannel.

Improving the interconnected structure and bioregulatory function of natural chitosan is beneficial for optimizing its performance in bone regeneration. Here, a facile immunoregulatory constructional design is proposed for developing instructive chitosan by directional freezing and alkaline salting out. The molecular dynamics simulation confirmed the assembly kinetics and structural features of various polyphenols and chitosan molecules. Along with the in vitro anti-inflammatory, antioxidative, promoting bone mesenchymal stem cell (BMSC) adhesion and proliferation performance, proanthocyanidin optimizing chitosan (ChiO) scaffold presented an optimal immunoregulatory structure with the directional microchannel. Transcriptome analysis in vitro further revealed the cytoskeleton- and immune-regulation effect of ChiO are the key mechanism of action on BMSC. The rabbit cranial defect model (Φ = 10 mm) after 12 weeks of implantation confirmed the significantly enhanced bone reconstitution. This facile immunoregulatory directional microchannel design provides effective guidance for developing inducible chitosan scaffolds.

DNA Nanoparticle Based 2D Biointerface to Study the Effect of Dynamic RGD Presentation on Stem Cell Adhesion and Migration.

The native extracellular matrix (ECM) undergoes constant remodeling, where adhesive ligand presentation changes over time and in space to control stem cell function. As such, it is of interest to develop 2D biointerfaces able to study these complex ligand stem-cell interactions. In this study, a novel dynamic bio interface based on DNA hybridization is developed, which can be employed to control ligand display kinetics and used to study dynamic cell-ligand interaction. In this approach, mesoporous silica nanoparticles (MSN) are functionalized with single-strand DNA (MSN-ssDNA) and spin-coated on a glass substrate to create the 2D bio interface. Cell adhesive tripeptide RGD is conjugated to complementary DNA strands (csDNA) of 9, 11, or 20 nucleotides in length, to form csDNA-RGD. The resulting 3 csDNA-RGD conjugates can hybridize with the ssDNA on the MSN surface, presenting RGD with increased ligand dissociation rates as DNA length is shortened. Slow RGD dissociation rates led to enhanced stem cell adhesion and spreading, resulting in elongated cell morphology. Cells on surfaces with slow RGD dissociation rates also exhibited higher motility, migrating in multiple directions compared to cells on surfaces with fast RGD dissociation rates. This study contributes to the existing body of knowledge on dynamic ligand-stem cell interactions.

R406 reduces lipopolysaccharide-induced neutrophil activation.

Modulating SYK has been demonstrated to have impacts on pathogenic neutrophil responses in COVID-19. During sepsis, neutrophils are vital in early bacterial clearance but also contribute to the dysregulated immune response and organ injury when hyperactivated. Here, we evaluated the impact of R406, the active metabolite of fostamatinib, on neutrophils stimulated by LPS. We demonstrate that R406 was able to effectively inhibit NETosis, degranulation, ROS generation, neutrophil adhesion, and the formation of CD16low neutrophils that have been linked to detrimental outcomes in severe sepsis. Further, the neutrophils remain metabolically active, capable of releasing cytokines, perform phagocytosis, and migrate in response to IL-8. Taken together, this data provides evidence of the potential efficacy of utilizing fostamatinib in bacterial sepsis.

The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss.

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

Enhancing Cell Aggregation and Migration via Double-Click Cross-Linking with Azide-Modified Hyaluronic Acid.

We present a novel approach to the formation of cell aggregates by employing click chemistry with water-soluble zwitterionic dibenzo cyclooctadiyne (WS-CODY) and azide-modified hyaluronic acid (HA-N3) as a linker to facilitate rapid and stable cell aggregation. By optimizing the concentrations of HA-N3 and WS-CODY, we achieved efficient cross-linking between azide-modified cell surfaces and HA-N3, generating cell aggregates within 10 min, and the resulting aggregates remained stable for up to 5 days, with cell viability maintained at approximately 80%. Systematic experiments revealed that a stoichiometric balance between HA-N3 and WS-CODY is important for effective cross-linking, highlighting the roles of both cell-surface azide modification and HA in the aggregate formation. We also investigated the genetic basis of altered cell behavior within these aggregates. Transcriptome analysis (RNA-seq) of aggregates postcultivation revealed a marked fluctuation of genes associated with 'cell migration' and 'cell adhesion', including notable changes in the expression of HYAL1, ICAM-1, CEACAM5 and RHOB. These findings suggest that HA-N3-mediated cell aggregation can induce intrinsic cellular responses that not only facilitate cell aggregate formation but also modulate cell-matrix interactions. We term this phenomenon 'chemo-resilience', The simplicity and efficacy of this click chemistry-based approach suggest it may have broad applicability for forming cell aggregates and modulating cell-matrix interactions in tissue engineering and regenerative medicine.

Ethanol exposure exacerbates 4-nitroquinoline-1-oxide induced esophageal carcinogenesis and induces invasive carcinoma with muscularis propria infiltration in a mouse model.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal cancers worldwide. Most ESCC patients are diagnosed at an advanced stage; however, current research on in vivo animal models accurately reflecting their clinical presentation is lacking. Alcohol consumption is a major risk factor for ESCC and has been used in several disease models for disease induction. In this study, we used 4-nitroquinoline-1-oxide in combination with ethanol to induce an in vivo ESCC mouse model. Esophageal tissues were stained with hematoxylin and eosin for histopathological examination and lesion scoring. In cellular experiments, cell adhesion and migration invasion ability were observed using phalloidin staining, cell scratch and transwell assays, respectively, and the expression of epithelial-mesenchymal transition-related markers was detected using quantitative reverse transcription polymerase chain reaction and western blotting. The results showed that ethanol-exposed mice lost more weight and had an increased number of esophageal nodules. Histological examination revealed that the lesion scores of the ethanol-exposed esophageal samples were significantly higher than those of the unexposed esophageal samples. Furthermore, ethanol-exposed esophageal cancer samples had more severe lesions with infiltration of tumor cells into the muscularis propria. In vitro cellular experiments showed that ethanol exposure induced cytoskeletal microfilament formation, promoted cell migration invasion elevated the expression of N-cadherin and Snail, and decreased the expression of E-cadherin. In conclusion, ethanol exposure exacerbates ESCC, promotes tumor cell infiltration into the muscularis propria, and could be an effective agent for establishing innovative models of invasive carcinoma.

Functionalization of Alginate Hydrogels with a Multifunctional Peptide Supports Mesenchymal Stem Cell Adhesion and Reduces Bacterial Colonization.

Hydrogels with cell adhesive moieties stand out as promising materials to enhance tissue healing and regeneration. Nonetheless, bacterial infections of the implants represent an unmet major concern. In the present work, we developed an alginate hydrogel modified with a multifunctional peptide containing the RGD cell adhesive motif in combination with an antibacterial peptide derived from the 1-11 region of lactoferrin (LF). The RGD-LF branched peptide was successfully anchored to the alginate backbone by carbodiimide chemistry, as demonstrated by 1H NMR and fluorescence measurements. The functionalized hydrogel presented desirable physicochemical properties (porosity, swelling and rheological behavior) to develop biomaterials for tissue engineering. The viability of mesenchymal stem cells (MSCs) on the peptide-functionalized hydrogels was excellent, with values higher than 85 % at day 1, and higher than 95 % after 14 days in culture. Moreover, the biological characterization demonstrated the ability of the hydrogels to significantly enhance ALP activity of MSCs as well as to decrease bacterial colonization of both Gram-positive and Gram-negative models. Such results prove the potential of the functionalized hydrogels as novel biomaterials for tissue engineering, simultaneously displaying cell adhesive activity and the capacity to prevent bacterial contamination, a dual bioactivity commonly not found for these types of hydrogels.