Mediate Cell Adhesion Research Articles

Lactic Acid Bacteria Surface Proteins in the Mechanisms of Cell Adhesion and Immunoregulation

ABSTRACTThis study delves into the role of lactic acid bacteria (LAB) surface proteins in cell adhesion and immunoregulation. Using fluorescence microscopy, we observed distinct adhesion patterns on various cell types. LAB surface proteins demonstrated concentration‐dependent inhibition of Salmonella adhesion, with LAB69 exhibiting potent antagonistic effects. Genetic expression analysis revealed nuanced responses in key genes (MD2, TLR4, IL‐10, MUC3, MIF) across different cell types, highlighting the diverse immunomodulatory effects of LAB surface proteins. Modulation of pro‐inflammatory (TNF‐α) and anti‐inflammatory (IL‐10) cytokines further emphasized the complex interplay. In conclusion, this study underscores the pivotal role of LAB surface proteins in mediating cell adhesion and immunoregulation, providing a foundation for isolating specific immunomodulatory molecules within LAB surface proteins for potential applications in microbial ecological agents.

High-resolution snapshots of the talin auto-inhibitory states suggest roles in cell adhesion and signaling

Talin regulates crucial cellular functions, including cell adhesion and motility, and affects human diseases. Triggered by mechanical forces, talin plays crucial roles in facilitating the formation of focal adhesions and recruiting essential focal adhesion regulatory elements such as vinculin. The structural flexibility allows talin to fine-tune its signaling responses. This study presents our 2.7 Å cryoEM structures of talin, which surprisingly uncovers several auto-inhibitory states. Contrary to previous suggestions, our structures reveal that (1) the first and last three domains are not involved in maintaining talin in its closed state and are mobile, (2) the talin F-actin and membrane binding domain are loosely attached and thus available for binding, and (3) the main force-sensing domain is oriented with its vinculin binding sites ready for release. These structural snapshots offer insights and advancements in understanding the dynamic talin activation mechanism, which is crucial for mediating cell adhesion.

Bidirectional Allostery Mechanism in Catch-Bond Formation of CD44 Mediated Cell Adhesion.

Catch-bonds, whereby noncovalent ligand-receptor interactions are counterintuitively reinforced by tensile forces, play a major role in cell adhesion under mechanical stress. A basic prerequisite for catch-bond formation, as implicated in classic catch-bond models, is that force-induced remodeling of the ligand binding interface occurs prior to bond rupture. However, what strategy receptor proteins utilize to meet such specific kinetic control remains elusive. Here we report a bidirectional allostery mechanism of catch-bond formation based on theoretical and molecular dynamics simulation studies. Binding of ligand allosterically reduces the threshold force for unlocking of otherwise stably folded force-sensing element (i.e., forward allostery), so that a much smaller tensile force can trigger the conformational switching of receptor protein to high binding-strength state via backward allosteric coupling before bond rupture. Such bidirectional allostery fulfills the specific kinetic control required by catch-bond formation and is likely to be commonly utilized in cell adhesion. The essential thermodynamic and kinetic features of receptor proteins essential for catch-bond formation were identified.

The novel ECM protein SNED1 mediates cell adhesion via α5β1 integrin.

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays critical roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by α5β1integrin. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis.

Recruitment of Vitronectin by Bacterial Pathogens: A Comprehensive Overview.

The key factor that enables pathogenic bacteria to establish successful infections lies largely in their ability to escape the host's immune response and adhere to host surfaces. Vitronectin (Vn) is a multidomain glycoprotein ubiquitously present in blood and the extracellular matrix of several tissues, where it plays important roles as a regulator of membrane attack complex (MAC) formation and as a mediator of cell adhesion. Vn has emerged as an intriguing target for several microorganisms. Vn binding by bacterial receptors confers protection from lysis resulting from MAC deposition. Furthermore, through its Arg-Gly-Asp (RGD) motif, Vn can bind several host cell integrins. Therefore, Vn recruited to the bacterial cell functions as a molecular bridge between bacteria and host surfaces, where it triggers several host signaling events that could promote bacterial internalization. Each bacterium uses different receptors that recognize specific Vn domains. In this review, we update the current knowledge of Vn receptors of major bacterial pathogens, emphasizing the role they may play in the host upon Vn binding. Focusing on the structural properties of bacterial proteins, we provide details on the residues involved in their interaction with Vn. Furthermore, we discuss the possible involvement of Vn adsorption on biomaterials in promoting bacterial adhesion on abiotic surfaces and infection.

Identification and functional characterization of laminin receptor in the mud crab, Scylla paramamosain, in response to MCDV-1 challenge

Laminin receptor (LR), which mediating cell adhesion to the extracellular matrix, plays a crucial role in cell signaling and regulatory functions. In the present study, a laminin receptor gene (SpLR) was cloned and characterized from the mud crab (Scylla paramamosain). The full length of SpLR contained an open reading frame (ORF) of 960 bp encoding 319 amino acids, a 5′ untranslated region (UTR) of 66 bp and a 3′ UTR of 49 bp. The predicted protein comprised two Ribosomal-S2 domains and a 40S-SA-C domain. The mRNA of SpLR was highly expressed in the gill, followed by the hepatopancreas. The expression of SpLR was up-regulated after mud crab dicistrovirus-1(MCDV-1) infection. Knocking down SpLR in vivo by RNA interference significantly down-regulated the expression of the immune genes SpJAK, SpSTAT, SpToll1, SpALF1 and SpALF5. This study shown that the expression level of SpToll1 and SpCAM in SpLR-interfered group significantly increased after MCDV-1 infection. Moreover, silencing of SpLR in vivo decreased the MCDV-1 replication and increased the survival rate of mud crabs after MCDV-1 infection. These findings collectively suggest a pivotal role for SpLR in the mud crab's response to MCDV-1 infection. By influencing the expression of critical innate immune factors and impacting viral replication dynamics, SpLR emerges as a key player in the intricate host-pathogen interaction, providing valuable insights into the molecular mechanisms underlying MCDV-1 pathogenesis in mud crabs.

Lysine Methyltransferase 9 (KMT9) Is an Actionable Target in Muscle-Invasive Bladder Cancer.

Novel treatment modalities are imperative for the challenging management of muscle-invasive and metastatic BC to improve patient survival rates. The recently identified KMT9, an obligate heterodimer composed of KMT9α and KMT9β, regulates the growth of various types of tumors such as prostate, lung, and colon cancer. While the overexpression of KMT9α was previously observed to be associated with aggressive basal-like MIBC in an analysis of patients' tissue samples, a potential functional role of KMT9 in this type of cancer has not been investigated to date. In this study, we show that KMT9 regulates proliferation, migration, and invasion of various MIBC cell lines with different genetic mutations. KMT9α depletion results in the differential expression of genes regulating the cell cycle, cell adhesion, and migration. Differentially expressed genes include oncogenes such as EGFR and AKT1 as well as mediators of cell adhesion or migration such as DAG1 and ITGA6. Reduced cell proliferation upon KMT9α depletion is also observed in Pten/Trp53 knockout bladder tumor organoids, which cannot be rescued with an enzymatically inactive KMT9α mutant. In accordance with the idea that the catalytic activity of KMT9 is required for the control of cellular processes in MIBC, a recently developed small-molecule inhibitor of KMT9 (KMI169) also impairs cancer cell proliferation. Since KMT9α depletion also restricts the growth of xenografts in mice, our data suggest that KMT9 is an actionable novel therapeutic target for the treatment of MIBC.

Significance of a minor pilin PilV in biofilm cohesion of Geobacter sulfurreducens

Bacterial adhesion plays a vital role in forming and shaping the structure of electroactive biofilms that are essential for the performance of bioelectrochemical systems (BESs). Type IV pili are known to mediate cell adhesion in many Gram-negative bacteria, but the mechanism of pili-mediated cell adhesion of Geobacter species on anode surface remains unclear. Herein, a minor pilin PilV2 was found to be essential for cell adhesion ability of Geobacter sulfurreducens since the lack of pilV2 gene depressed the cell adhesion capability by 81.2% in microplate and the anodic biofilm density by 23.1 % at −0.1 V and 37.7 % at −0.3 V in BESs. The less cohesiveness of mutant biofilms increased the charge transfer resistance and biofilm resistance, which correspondingly lowered current generation of the pilV2-deficient strain by up to 63.2 % compared with that of the wild-type strain in BESs. The deletion of pilV2 posed an insignificant effect on the production of extracellular polysaccharides, pili, extracellular cytochromes and electron shuttles that are involved in biofilm formation or extracellular electron transfer (EET) process. This study demonstrated the significance of pilV2 gene in cell adhesion and biofilm formation of G. sulfurreducens, as well as the importance of pili-mediated adhesion for EET of electroactive biofilm.

Observing Dynamic Conformational Changes within the Coiled-Coil Domain of Different Laminin Isoforms Using High-Speed Atomic Force Microscopy.

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques.

Dual P-Selectin and Complement Inhibition with Subcutaneous IHP-102 Treatment Potently Reduces Lung Vaso-Occlusion in Sickle Cell Disease Mice

One of the most debilitating manifestations of sickle cell disease (SCD) are the excruciatingly painful vaso-occlusive episodes (VOEs), commonly referred to as “pain crises.” Vaso-occlusion, which underlies the pathomechanism of VOE, also causes downstream tissue and organ damage resulting in complications that plummet life-expectancy by more than 20 years. Current treatment options for VOE are limited and all require chronic administration, often with intolerable side-effects, and none fully prevent VOE. An acute VOE rescue therapy that could be administered in the home-setting would transform care for the more than 100,000 people in the U.S. and over 7 million worldwide who suffer from this inherited condition. IHP-102 is a novel glycan-based drug with dual anti-P-selectin and anti-complement activity. It is bioavailable by subcutaneous route of administration and could therefore be self-administered at the earliest sign of VOE. Early intervention in VOE is critical, before ischemia and tissue damage becomes irreversible. This treatment paradigm would also circumvent the wholly inadequate treatment experience that the SCD community has widely described, being treated as an opioid “drug-seeker” when engaging the healthcare system during a debilitating VOE. There are multiple pathomechanisms of VOE. P-selectin is a clinically validated therapeutic target for partially attenuating VOE, while complement has more recently been identified as an important driver of VOE, in part due to hemolysis and ischemia. While several therapeutics targeting P-selectin or complement are currently in clinical development, no therapy targets both pathomechanisms. We have previously shown that IHP-102 inhibits P-selectin mediated cell adhesion in vitro (IC50 = 0.7 µg/mL) and that intravenous administration of IHP-102 reduces lung vaso-occlusion by 75% in Townes SCD mice challenged with oxy-hemoglobin (oxy-Hb). Here, we extend these studies with subcutaneous delivery of IHP-102, achieving greater than 75% reduction in lung vaso-occlusion. This magnitude of effect is greater than that achieved in P-selectin deficient Townes SCD mice (~50% reduction), thus indicating additional activity of IHP-102 beyond P-selectin inhibition. In studies using human serum, IHP-102 inhibits complement by upstream mechanisms, including through inhibition of Factor B. Factor B is responsible for alternative pathway amplification of complement. Its fragment Bb has been found in elevated concentrations during VOE in patient serum, suggesting it plays an important role in the pathomechanism of VOE. By inhibiting Factor B, IHP-102 reduces downstream complement fragments of C3 and C5, which are widely recognized complement targets given their critical role in the complement system and promoting inflammation. The dual anti-complement and anti-P-selectin activity of IHP-102, together with its potential for home-based treatment, offers great potential to fill a major treatment gap for acute VOE. Its potent in vivo activity suggests that this dual target approach could provide robust therapeutic benefit and warrants further investigation toward clinical trials.

Targeting the JAK2V617F Hematopoietic Stem Cell through Beta1 Integrin

The major barrier to progress in treatment of primary myelofibrosis (PMF) is the lack of effective therapies targeting the malignant JAK2 V617F hematopoietic stem cell (HSC), the source of disease progression and relapse. Expression of JAK2 V617F induces abnormal proliferation and differentiation of HSC, affecting all blood lineages but most prominently the erythro-megakaryocytic lineage, leading to clonal megakaryocyte proliferation, erythrocytosis and thrombocytosis. In our previously published work ( Matsuura et al, Blood 2020) we identified elevated expression of the α5 subunit of the α5β1 integrin receptor as a driver of JAK2 V617F megakaryocyte proliferation. Expression of β1 integrin subunit was similar in wild-type (WT) and JAK2 V617F megakaryocytes, but activation of β1 integrin, detected by the conformation-specific 9EG7 antibody, was elevated in JAK2 V617F megakaryocytes. Following up on those findings, we detected elevated β1 integrin activation in long-term hematopoietic stem cells (HSC LT, LSKCD48-CD150+) from JAK2 V617F transgenic mice ( Xing et al, Blood 2008) when compared to WT controls. To determine whether inhibition of β1 integrin in vivo reduces the number of HSC LT in JAK2 V617F transgenic mice, we examined HSC LT 24 hours after intravenous administration of 2mg/Kg of anti-β1 integrin inhibitory antibody HMβ1-1. This regimen led to a sharp reduction in number and percentage of HSC LT in JAK2 V617F transgenic mice, to 45% of control antibody-treated animals. The effect on WT HSC LT was negligible. HSC ST (LSKCD48-CD150-) and MPP2 (LSKCD48+CD150+) were also reduced, but the reduction was not statistically significant. MPP3/4 (LSKCD48+CD150-) and LSK were not affected. Megakaryocyte progenitors (LKCD41+CD150+) were significantly reduced. Inhibition of β1 integrin also sharply reduced peripheral blood platelet numbers in WT animals. The reduction in JAK2 V617F transgenic mice was not statistically significant. A longer observation time of 7 days demonstrated that while WT animals were able to mount a robust regenerative response with slight but significant increases in HSC LT and normalization of platelet numbers, in JAK2 V617F transgenic mice the number of HSC LT remained depressed, as did downstream stem/progenitor populations at various significance levels. To gain insight into the mechanisms that lead to reduction in HSC LT, we examined apoptosis, cell cycle and HSC mobilization, all processes known to be affected by β1 integrin. Although basal expression of the apoptotic marker annexin V was decreased in JAK2 V617F HSC LT compared to WT, no differences were detected upon administration of HMβ1-1 in both genotypes at 4 hours post-administration. Similarly, peripheral blood colony assays revealed a basally higher level of stem/progenitor mobilization in JAK2 V617F, which did not significantly increase upon HMβ1-1 administration. In contrast, the cell cycle profile, examined at 4 hours post-administration to account for the earliest effects, demonstrated a decrease in G0 phase cells and increase in S/G2/M cells indicating cell cycle induction and exit from quiescence in both WT and JAK2 V617F HSC LT. The results indicate that inhibition of β1 integrin disrupts the adhesion-mediated cell cycle suppression in both WT and JAK2 V617F HSC LT leading to exit from quiescence. However, the downstream consequences of cell division in HSC LT differ significantly, with an apparent loss of regenerative potential in JAK2 V617F HSC LT, at least at the observed timepoints. Longer observation times are required to determine whether continuous β1 integrin inhibition can lead to JAK2 V617F HSC LT exhaustion. More sensitive assays to monitor mobilization are warranted to quantify possible minute amounts of mobilized HSC LT. Our preclinical studies indicate that β1 integrin is an attractive target to specifically reduce the number of JAK2 V617F HSC LTin vivo. The role of β1 integrin in HSC LT has not been thoroughly studied due to lack of phenotype in β1 integrin-deficient HSC ( Brakebush et al, Immunity 2002). Our studies using the anti-β1 integrin antibody in WT HSC LT confirm previous findings. However, β1 integrin seems to be critical for maintenance of JAK2 V617F HSC LT, offering a possible therapeutic window. Understanding how β1 integrin supports quiescence in HSC LT will bring us a step closer to understanding the mechanisms that drive malignancy and fitness in JAK2 V617F HSC LT.

Behavior and surface properties of microalgae indicate environmental changes

We conducted a microcosm experiment under near-natural conditions to investigate the effects of environmental stressors temperature and salinity on the response of three microalgal species. The response of microalgae to increased temperature and altered salinity is species-specific, with cell barrier complexity playing an important protective role. At decreasing salinity, the green flagellate cells of Dunaliella tertiolecta vibrate around the point and become stiff while their physiological activity is at its highest, which may indicate that organic matter is being released into the medium. At the highest salinity, Tetraselmis suecica showed the highest growth rate along with the hydrophobic behavior of the cells, which is a favorable condition for the formation of aggregates. Under these conditions, the cells could enter the cyst stage, while the content of polar lipids could mediate cell adhesion to the natural interfaces. Cylindrotheca closterium, a diatom with an organosilicate cell wall, also showed the highest physiological activity at decreasing salinity. However, the cells were softest and exhibited hydrophilic behavior, implying that released organic material could alter the surface properties of the diatom. This study provides insight into the behavioral, mechanical, and molecular changes of microalgae under near-natural conditions, which may contribute to a fundamental understanding of how biochemistry and mechanics interact to control the functional behavior of microalgal cells in climatically altered aquatic systems.

Surface roughness and its role in mediating cell adhesion on cobalt‐chromium‐molybdenum alloys

AbstractCo‐Cr‐Mo ally (CCM) is commonly used for orthopaedic and dental implants due to its excellent mechanical properties and corrosion resistance. However, the influence of surface roughness on cell attachment and proliferation remains unclear. This study aimed to elucidate the impact of surface roughness of CCM on the attachment and proliferation of osteoblasts. CCM samples with different values of surface rouges were prepared by polishing. MC3T3‐E1 mouse osteoblasts were used for cell culture experiments. Cell attachment, morphology, and the expression of actin stress fibres, vinculin, and distribution of yes‐associated protein were analysed. Our results suggest that surface roughness does not significantly affect cell attachment and proliferation on CCM, unlike on titanium. Thus implies that other properties of CCM, such as physicochemical properties, may play a more substantial role in modulating cell behaviour. This study provides important insights into the design of CCM implants, suggesting that approaches beyond tuning surface roughness may be necessary to improve biocompatibility and osseointegration.

LINC00922 decoys SIRT3 to facilitate the metastasis of colorectal cancer through up-regulation the H3K27 crotonylation of ETS1 promoter

BackgroundLysine crotonylation (Kcr) is up-regulation in colorectal cancer (CRC) tissues, while its specific contribution remains uncertain. This study aimed to elucidate the role and mechanism of crotonylation on Lys27 of histone H3 (H3K27cr) in facilitating CRC metastasis.MethodsImmunohistochemistry was employed to investigate the correlation between H3K27cr and CRC metastasis. Both in vitro and in vivo assays employing loss function or gain function approaches were conducted to elucidate the role of LINC00922 in promoting CRC metastasis. ScRNA-seq analysis and immunoprecipitation analyses were employed to explore the underlying mechanism by which LINC00922 facilitates CRC metastasis through H3K27cr.ResultsClinically, H3K27cr was upregulated in metastatic CRC tissues and positively correlated with advanced clinical stages. Functionally, knockdown of LINC00922 inhibited migration of CRC cells both in vitro and in vivo. Furthermore, the supplementation of NaCr restored the migration and invasion levels of LINC00922 stable knockdown cells by restoring the H3K27cr level. Mechanistically, LINC00922 promoted invasion and migration through H3K27cr mediated cell adhesion molecules (CAMs) in epithelial cells. Notably, LINC00922 interacted with the protein sirtuin 3 (SIRT3) and obstructed its binding to the promoter region of ETS1, leading to an elevation in the level of H3K27cr in this promoter region and the subsequent activation of ETS1 transcription.ConclusionsOur findings uncovered a novel regulatory function of H3K27cr, regulated by LINC00922, in facilitating CRC metastasis. This discovery contributed to a deeper comprehension of the involvement of histone crotonylation in the metastatic process of CRC.

Multiscale computational framework to investigate integrin mechanosensing and cell adhesion

Integrin mechanosensing plays an instrumental role in cell behavior, phenotype, and fate by transmitting mechanical signals that trigger downstream molecular and cellular changes. For instance, force transfer along key amino acid residues can mediate cell adhesion. Disrupting key binding sites within α5β1 integrin’s binding partner, fibronectin (FN) diminishes adhesive strength. While past studies have shown the importance of these residues in cell adhesion, the relationship between the dynamics of these residues and how integrin distributes force across the cell surface remains less explored. Here, we present a multiscale mechanical model to investigate the mechanical coupling between integrin nanoscale dynamics and whole-cell adhesion mechanics. Our framework leverages molecular dynamics simulations to investigate residues within α5β1-FN during stretching and the finite element method to visualize the whole-cell adhesion mechanics. The forces per integrin across the cell surface of the whole-cell model were consistent with past atomic force microscopy and Förster resonance energy transfer measurements from the literature. The molecular dynamics simulations also confirmed past studies that implicate two key sites within FN that maintain cell adhesion: the synergy site and arginine-glycine-aspartic acid (RGD) motif. Our study contributed to our understanding of molecular mechanisms by which these sites collaborate to mediate whole-cell integrin adhesion dynamics. Specifically, we showed how FN unfolding, residue binding/unbinding, and molecular structure contribute to α5β1-FN’s nonlinear force–extension behavior during stretching. Our computational framework could be used to explain how the dynamics of key residues influence cell differentiation or how uniquely designed protein structures could dynamically limit the spread of metastatic cells.

MicroRNAs Can Influence Ovarian Cancer Progression by Dysregulating Integrin Activity.

Ovarian cancer is a deadly disease that affects thousands of women worldwide. Integrins, transmembrane receptors that mediate cell adhesion and signaling, play important roles in ovarian cancer progression, metastasis, and drug resistance. Dysregulated expression of integrins is implicated in various cellular processes, such as cell migration, invasion, and proliferation. Emerging evidence suggests that microRNAs (miRNAs) can regulate integrin expression and function, thus affecting various physiological and pathological processes, including ovarian cancer. In this article, we review the current understanding of integrin-mediated cellular processes in ovarian cancer and the roles of miRNAs in regulating integrins. We also discuss the therapeutic potential of targeting miRNAs that regulate integrins for the treatment of ovarian cancer. Targeting miRNAs that regulate integrins or downstream signaling pathways of integrins may provide novel therapeutic strategies for inhibiting integrin-mediated ovarian cancer progression.

Self-Assembly of RGD-Functionalized Recombinant Spider Silk Protein into Microspheres in Physiological Buffer and in the Presence of Hyaluronic Acid.

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.

Platelet-Derived Exosomes Alleviate Knee Osteoarthritis by Attenuating Cartilage Degeneration and Subchondral Bone Loss.

Osteoarthritis (OA) is the most prevalent chronic degenerative joint disease among the aged population. However, current treatments for OA are limited to alleviating symptoms, with no therapies that prevent and regenerate cartilage deterioration. To assess the effects of platelet-derived exosomes (Plt-exos) on OA and then to explore the potential molecular mechanism. Controlled laboratory study. Exosomes derived from human apheresis platelets were isolated and identified. The effects of Plt-exos in protecting chondrocytes under interleukin 1β stimulation were evaluated by analyzing the proliferation and migration in human primary chondrocytes. RNA sequencing was later performed in vitro for primary chondrocytes to reveal the underlying mechanisms of Plt-exo treatment. Anterior cruciate ligament transection was used to construct an OA mice model, and intra-articular injection of Plt-exos was given once a week for 6 weeks. Mice were sacrificed 4 weeks after the last injection. Histologic and immunohistochemistry staining and micro-computed tomography analysis were performed to assess alterations of articular cartilage and subchondral bone. Plt-exos significantly promoted proliferation and migration of chondrocytes within a dose-dependent manner, as well as dramatically promoted cartilage regeneration and attenuated abnormal tibial subchondral bone remodeling, thus slowing the progression of OA. After being treated with Plt-exos, 1797 genes were differentially expressed in chondrocytes (923 upregulated and 874 downregulated genes). Functional enrichment results and hub genes were mainly involved in anti-inflammatory effects, mediating cell adhesion, stimulating cartilage repair, promoting anabolism, and inhibiting catabolism. Our results demonstrated that Plt-exos promoted chondrocyte proliferation and migration in vitro, as well as attenuated cartilage degeneration, improved the microarchitecture of subchondral bone, and retarded OA progression in vivo. Our study illustrated that the administered Plt-exos could alleviate knee OA by attenuating cartilage degeneration and subchondral bone loss, possibly serving as a novel promising treatment for OA in the future.

Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4

AbstractWe have previously shown that the extracellular domains of the four syndecans are intrinsically disordered, and adopt a wide range of conformations. We report here the building of coarse‐grained models of the extracellular domains of human syndecans 2 and 4 using small‐angle X‐ray scattering restraints. One, two or three heparan sulfate (HS) hexadecasaccharides, (IdoA[2S]GlcNS[6S])8, were attached to three serine residues of the core proteins, resulting in eight variants for each syndecan that were used for all‐atom molecular dynamics (MD) simulations (0.5–1 µs). Syndecan‐4 had a larger conformational diversity than syndecan‐2, and remained extended during MD simulations in absence of HS whereas syndecan‐2 adopted more compact conformations. Their core proteins thus appeared to be structurally distinct. The HS chains also behave differently, the middle chain being more flexible in syndecan‐4, and the third chain being able to interact with the core protein regions mediating cell adhesion. The cell adhesion sites on both core proteins were flexible, with or without HS chains, the NXIP motif of syndecan‐2 being located in a particularly flexible region. In conclusion, the HS chains induce moderate changes in the conformational dynamics of both syndecans, depending on the number of HS chains and their location on the core protein, and on the core protein itself.

Transposon mutagenesis of atypical enteroaggregative Escherichia coli reveals a hemagglutinin-associated protein that mediates cell adhesion and contributes to the Galleria mellonella virulence.

Twenty-two atypical enteroaggregative Escherichia coli isolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genes afpA2 and afpR were detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified in E. coli strain 7-233-03_S3_C2. Therefore, the ORF was named orfHA. The regions flanking orfHA were sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. An orfHA mutant (Q015BΔorfHA) was constructed from strain Q015B. Q015BΔorfHA strain did not adhere to HeLa cells, whereas Q015BΔ orfHA transformed with a pACYC184 plasmid carrying orfHA restored the AA/DA phenotype of strain Q015B. Furthermore, the Q015ΔorfHA mutant had a marked effect on the ability of strain Q015B to kill the larvae of Galleria mellonella. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in the G. mellonella model.