Cellular Adhesion Assay Research Articles

The effect of augmenting suture material with magnesium and platelet-rich plasma on the in vitro adhesion and proliferation potential of subacromial bursa-derived progenitor cells

Connective tissue subacromial bursa-derived progenitor cells (SBDCs) have been suggested as a potent biologic augment to promote healing of the repaired rotator cuff tendon. Maximizing the amount of retained progenitor cells at the tendon repair site is essential for ensuring an optimal healing environment, warranting a search for proadhesive and proliferative adjuvants. The purpose was to evaluate the effect of magnesium (Mg), platelet-rich plasma (PRP), and a combination of both adjuvants on the invitro cellular adhesion and proliferation potential of SBDCs on suture material commonly used in rotator cuff surgery. SBDCs were isolated from subacromial bursa samples harvested during rotator cuff repair and cultured in growth media. Commercially available collagen-coated nonabsorbable flat-braided suture was cut into 1-inch pieces, placed into 48-well culture dishes, and sterilized under ultraviolet light. Either a one-time dose of 5 mM sterile Mg, 0.2 mL of PRP, or a combination of both adjuvants was added, while a group without treatment served as a negative control. Cellular proliferation and adhesion assays on suture material were performed for each treatment condition. Augmenting the suture with Mg resulted in a significantly increased cellular adhesion (total number of attached cells) of SBDCs compared to PRP alone (31,527±19,884 vs. 13,619±8808; P<.001), no treatment (31,527±19,884 vs. 21,643±8194; P=.016), and combination of both adjuvants (31,527±19,884 vs. 17,121±11,935; P<.001). Further, augmentation with Mg achieved a significant increase in cellular proliferation (absorbance) of SBDCs on suture material when compared to the PRP (0.516±0.207 vs. 0.424±0.131; P=.001) and no treatment (0.516±0.207 vs. 0.383±0.094; P<.001) group. The combination of Mg and PRP showed a significantly higher proliferation potential compared to PRP alone (0.512±0.194 vs. 0.424±0.131; P=.001) and no treatment (0.512±0.194 vs. 0.383±0.094; P<.001). There were no significant differences in the remaining intergroup comparisons (P>.05, respectively). Augmenting suture material with Mg resulted in a significantly increased cellular adhesion of SBDCs compared to untreated suture material, as well as augmentation with PRP alone or a combination of both adjuvants. Further, Mg with or without PRP augmentation achieved a significant increase in the cellular proliferation of SBDCs on suture material compared to untreated sutures and augmentation with PRP alone. Application of Mg may be a clinically feasible approach to optimizing the use of SBDCs as a biological augment in rotator cuff repair, while combined augmentation with PRP may harness the full potential for optimized tissue recovery due to the high concentration of PRP-derived growth factors.

Destroying glutathione peroxidase improves the oxidative stress resistance and pathogenicity of Listeria monocytogenes.

Glutathione peroxidase is abundant in eukaryotes as an important antioxidant enzyme. However, prokaryotic glutathione peroxidase has not been thoroughly studied. Listeria monocytogenes is a facultative intracellular pathogen that is capable of causing listeriosis in animals as well as humans. Despite the fact that L. monocytogenes encodes a putative glutathione peroxidase, GSH-Px (encoded by lmo0983)), the functions of the enzyme are still unknown. Here we revealed the unusual roles of L. monocytogenes GSH-Px in bacterial antioxidants and pathogenicity. L. monocytogenes Lm850658 was taken as the parental strain to construct the gsh-px deletion strain and related complement strain. The effect of the gsh-px gene on the resistance of L. monocytogenes to oxidative stress was determined by measuring the concentrations of glutathione and assaying the stress survival rates under different oxidative conditions. In addition, the pathogenicity of L. monocytogenes was determined by cellular adhesion and invasion assays and mice virulence tests, and the expression of virulence factors was determined by Western blot. Deficiency of GSH-Px not only increased glutathione concentrations in L. monocytogenes but also enhanced its resistance to oxidative stress when exposed to copper and iron ions. In addition, the absence of gsh-px significantly improved the adhesion and invasion efficiency of L. monocytogenes to Caco-2 cells. More importantly, L. monocytogenes lacking GSH-Px could colonize and proliferate more efficiently in mice livers and spleens, enhancing the pathogenicity of L. monocytogenes by increasing the expression of virulence factors like InlA, InlB, and LLO. Taken together, we confirmed that GSH-Px of L. monocytogenes has a counter-intuitive effect on the antioxidant capacity and pathogenicity.

Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder

Adhesion G protein-coupled receptors (aGPCRs) are cell-surface proteins with large extracellular regions that bind to multiple ligands to regulate key biological functions including neurodevelopment and organogenesis. Modulating a single function of a specific aGPCR isoform while affecting no other function and no other receptor is not trivial. Here, we engineered an antibody, termed LK30, that binds to the extracellular region of the aGPCR ADGRL3, and specifically acts as an agonist for ADGRL3 but not for its isoform, ADGRL1. The LK30/ADGRL3 complex structure revealed that the LK30 binding site on ADGRL3 overlaps with the binding site for an ADGRL3 ligand – teneurin. In cellular-adhesion assays, LK30 specifically broke the trans-cellular interaction of ADGRL3 with teneurin, but not with another ADGRL3 ligand – FLRT3. Our work provides proof of concept for the modulation of isoform- and ligand-specific aGPCR functions using unique tools, and thus establishes a foundation for the development of fine-tuned aGPCR-targeted therapeutics.

Biosynthesis of ZnO NPs from pumpkin seeds’ extract and elucidation of its anticancer potential against breast cancer

Abstract The cost-effective and environmental friendly biosynthesis of metal nanoparticles is becoming increasingly important in biomedical science. This study biosynthesized zinc oxide nanoparticles (ZnO NPs) from pumpkin seed extract and assessed its anticancer activity by using different molecular biology experiments in human breast cancer (MDA-MB-231) cell lines. The well-known analytical techniques, such as UV-visible spectrophotometry, energy-dispersive X-ray spectroscopy, field emission scanning electron microscopy, and transmission electron microscopy (TEM), were used to confirm the synthesis and characterize the morphological and structural features of biogenic ZnO NPs. The formation of spherical nanoparticles with an average size of 50–60 nm was recorded using scanning electron microscopy and TEM. We observed the dose-dependent increase in cytotoxicity of ZnO NPs in the MDA-MB-231 cell line with a 50% inhibitory concentration of 10 µg/mL. Moreover, the ZnO NPs also showed significant cell morphology changes, apoptosis induction, and reactive oxygen species production at the highest tested concentration. The cellular adhesion and migration assay indicated cell death and inhibition in the migration of breast cancer cells in response to ZnO NPs’ treatment. These results clearly demonstrated the significant anticancer potential of ZnO NPs against the studied breast cancer cell line. However, an extension of this study is recommended in different cancer models and, based on the results, in vivo validation should be done.

Molecular and cellular outcomes of quercetin actions on healthy and tumor osteoblasts

There is a global trend in the use of natural bioactive compounds to complement conventional therapies in bone diseases. In this work, we studied the effects of the phytoestrogen quercetin (QUE) in healthy and tumor osteoblasts. We found that QUE (1 μM, 48 h) significantly increased the cell number and the viability of healthy human osteoblasts (hFOB cells) determined by a trypan blue and a MTS assay, respectively, among other concentrations tested. In addition, wound healing and cellular adhesion assays also demonstrated that 1 μM of QUE significantly stimulated both parameters in osteoblasts. Moreover, osteoblast differentiation was also triggered by QUE in an osteogenic medium by measuring alkaline phosphatase activity, calcium deposition, and collagen levels. Herein, a concentration of 0.01 μM of QUE showed an increment in these differentiation markers and an activation of AKT/GSK3β/β-catenin pathway, determined by a Western blot analysis. In addition, immunocytochemistry and subcellular fraction studies indicated an increase of β-catenin localization in the plasma membrane after QUE treatment. Otherwise, QUE (20–100 μM) decreased the cell number and the viability in tumor osteoblasts (ROS 17/2.8 cells) after 48 h. Furthermore, QUE (100 μM) decreased AKT(Ser473) and the pro-apoptotic protein BAD(Ser136) phosphorylation. In addition, the ERK1/2 phosphorylation increased leading to osteosarcoma cell death since pre-treatment with the MEK inhibitor PD98059 had reverted QUE effect. Altogether, these results indicate that low concentrations of QUE stimulate osteoblastogenesis but have no effect on the growth of tumor osteoblast cells, for which only high concentrations are efficient.

Titanium Nanobowl-Based Nest-Like Nanofiber Structure Prepared at Room Temperature and Pressure Promotes Osseointegration of Beagle Implants.

Nest-like nanofiber structures have potential applications in surface modifications of titanium implants. In this study, nest-like nanofiber structures were prepared on a titanium surface at room temperature and pressure by using the nanobowl template-assisted method combined with alkali etching. The characterization and biocompatibility of this material were analyzed by cellular adhesion, death, CCK-8, ALP, and RT-PCR assays in vitro, and osseointegration was evaluated by micro-CT and fluorescent labeling in vivo. The results showed that this nest-like nanofiber structure has a firmer and asperate surface than nanotubes, which leads to better cellular adhesion, proliferation, and differentiation capacity. In a beagle alveolar bone implant model, the nest-like nanofiber structure showed a better osseointegration capacity. In conclusion, this nest-like nanofiber structure has potential applications in dental implantology.

Augmenting Suture Tape Used in Rotator Cuff Surgery With Magnesium Increases in Vitro Cellular Adhesion of Human Subacromial Bursal Tissue

PurposeTo evaluate the effect of magnesium on cellular adhesion and proliferation of human subacromial bursal tissue (SBT), osteoblasts, and tenocytes on nonabsorbable suture tape commonly used in rotator cuff surgery.MethodsHuman SBT cells, primary human osteoblasts (HOBs), and primary human tenocytes were isolated from tissue samples and cultured in growth media. Commercially available collagen-coated nonabsorbable suture tape was cut into one-inch pieces, placed into 48-well culture dishes, sterilized under ultraviolet light, and treated with (+) or without (–) magnesium. For the (+) magnesium group, a one-time dose of 5 mM sterile magnesium chloride was added. Subsequently, cells were plated at a density of 20,000 cells/cm2. For each cell source (SBT, HOBs, tenocytes) cellular proliferation and adhesion assays on suture tape treated (+) or (–) magnesium were performed.ResultsSBT, HOBs, and tenocytes each demonstrated the ability to adhere and proliferate on suture tape. Augmenting suture tape with magnesium resulted in a significantly increased cellular adhesion of SBT compared with nonaugmented sutures (P = .001), whereas no significant differences were observed for HOBs (P = .081) and tenocytes (P = .907). Augmentation with magnesium demonstrated no significant difference in cellular proliferation of SBT (P = .856), HOBs (P = .672), and tenocytes (P = .251) compared with nonaugmented sutures.ConclusionsSBT, osteoblasts, and tenocytes each demonstrated the ability to adhere and proliferate on suture tape. In addition, augmenting the suture with magnesium resulted in a significantly increased cellular adhesion of SBT compared with nonaugmented sutures, whereas no significant differences were observed for osteoblasts and tenocytes. Further, magnesium did not impair the proliferative activity of SBT, osteoblasts, and tenocytes on suture tape used in rotator cuff surgery.Clinical RelevanceModifying the surface of the suture used for repair with application of magnesium may be an inexpensive and technically feasible option to improve the use of SBT for biologic augmentation of rotator cuff repair.

Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, and they continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of expression of mitochondrial DNA and generation of mitochondrial reactive oxygen species indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, inhibition of OxPhos induced transfer of mitochondria derived from mesenchymal stem cells (MSCs) to AML cells via tunneling nanotubes under direct-contact coculture conditions. Inhibition of OxPhos also induced mitochondrial fission and increased functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, so we used electron microscopy to observe mitochondrial transport to the leading edge of protrusions of AML cells migrating toward MSCs. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased mitochondrial transfer of MSCs to AML cells triggered by OxPhos inhibition. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

AML-108: Exogenous Mitochondrial Transfer and Endogenous Mitochondrial Fission Facilitate Resistance to OxPhos Inhibition in AML

<h3>Context:</h3> Oxidative phosphorylation (OxPhos) inhibition induces mitochondrial trafficking from BM stromal cells to AML and is accompanied by mitochondrial fission and mitophagy. The mitochondrial coupling and crosstalk with BM stromal cells facilitate the development of resistance to OxPhos inhibition in AML cells. <h3>Objective:</h3> Acute myeloid leukemia (AML) cells are highly dependent on OxPhos for survival and continually adapt to fluctuations in nutrient and oxygen availability in the environment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. <h3>Design:</h3> Primary AML cells from 26 patients, primary normal MSCs, and AML cell lines were utilized and treated with the OxPhos inhibitor IACS-0107598 and/or cytarabine. Cap analysis of gene expression transcriptome analyses for AML PDXs, primary samples, and AML cell lines has been performed. We performed an extracellular flux assay to measure the oxygen consumption rate. To visualize mitochondria, AML cell lines and MSCs were stably transfected with mitochondria-targeted PDHA1-GFP and -dsRed, respectively. Electron microscopy and immunoelectron microscopy analysis have been performed. <h3>Results:</h3> Cellular adhesion, growth, and apoptosis assays, along with measurements of mtDNA expression and mitochondrial reactive oxygen species generation, indicated that direct interactions with BM stromal cells triggered the compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, OxPhos inhibition induced (1) transfer of mesenchymal stem cell (MSC)-derived mitochondria to AML cells via tunneling nanotubes under direct-contact co-culture conditions and (2) mitochondrial fission with an increase in functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, and we observed mitochondrial transport to the leading edge of protrusions of migrating AML cells toward MSCs by electron microscopy analysis. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased IACS-010759-triggered mitochondrial transfer from MSCs to AML cells. <h3>Conclusions:</h3> Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells and of endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption.

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

MicroRNA‑744‑5p inhibits glioblastoma malignancy by suppressing replication factor C subunit 2

Glioblastoma (GBM) is the most common malignant primary brain tumor, accounting for ~57% of all gliomas and 48% of all malignant primary central nervous system tumors in the United States. Abnormal expression of the replication factor C subunit 2 (RFC2) gene and microRNA (miR)-744-5p is associated with tumorigenic characteristics, including cellular proliferation, migration and invasiveness. However, the mechanism underlying the interaction between miR-744-5p and RFC2 in GBM remains unknown. Reverse transcription-quantitative (RT-q) PCR analysis of RFC2 and miR-744-5p was performed using GBM tumor tissues and cells, and the association between miR-744-5p and RFC2 was determined by dual-luciferase reporter assay. Cell Counting Kit 8, 5-bromo-2-deoxyuridine (BrdU), wound-healing and cellular adhesion assays, as well as the detection of caspase-3 activity and western blotting were used to detect cellular proliferation, migration and adhesion, caspase-3 activity, and Bax and Bcl-2 protein expression, respectively, in GBM cells. The results of the present study demonstrated that RFC2 expression was increased in GBM tissues and cell lines. Overexpression of RFC2 promoted cellular proliferation, migration, adhesion and an increase in Bcl-2 protein levels, and suppressed cellular caspase-3 activity and Bax protein expression, while silencing RFC2 resulted in the opposite effect. The effects of miR-744-5p inhibition were similar to those of RFC2 overexpression. Moreover, miR-744-5p was found to target RFC2 in GBM cells, and inhibiting the expression of RFC2 suppressed GBM tumorigenesis. In conclusion, the present study demonstrated that miR-744-5p targets RFC2 and suppresses the progression of GBM by repressing cellular proliferation, migration and Bcl-2 protein expression, and effectively promoting caspase-3 activity and Bax protein expression. These findings suggest a new target for the clinical treatment and improved prognosis of patients with GBM in the future.

Silk fibroin and ceramic scaffolds: Comparative in vitro studies for bone regeneration.

Synthetic bone void fillers based on calcium ceramics are used to fill cavities in the bone and promote bone regeneration. More recently, silk fibroin (SF), a protein polymer obtained from Bombyx mori silkworm, has emerged as a promising material in bone void filling. In this work, we have compared the safety and efficacy of two types of silk fibroin‐based bone void fillers with currently used and commercially available ceramic bone void fillers (based on calcium sulphate, beta tricalcium phosphate, and beta tricalcium phosphate with hydroxyapatite). Further, we have also evaluated these two types of SF scaffolds, which have strikingly different structural attributes. The biocompatibility of these scaffolds was comparable as assessed by cytotoxicity assay, cellular adhesion assay, and immunogenic assay. Ability of the scaffolds to support differentiation of human mesenchymal stem cells (hMSCs) into an osteoblastic lineage was also evaluated in an in vitro differentiation experiment using reverse transcriptase polymerase chain reaction analysis. These results revealed that cells cultured on SF scaffolds exhibit higher expression of early to late markers such as Runx2, BMPs, collagen, osterix, osteopontin, and osteocalcin as compared with ceramic‐based scaffolds. This observation was further validated by studying the expression of alkaline phosphatase and calcium deposition. We also show that scaffolds made from same material of SF, but characterized by very different pore architectures, have diverse outcome in stem cell differentiation.

CC16 Binding to α4β1 Integrin Protects against Mycoplasma pneumoniae Infection.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4β1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

The Emerging Role of C‐peptide on Immune Cells

Diabetes affects 30 million individuals in the U.S. today. Approximately 10% of these cases are type 1 diabetes (T1D). T1D is an auto‐immune disease that destroys the insulin‐producing cells of the pancreas (β‐cells), resulting in increased levels of blood glucose (hyperglycemia). Consequently, people with T1D suffer from many vascular health complications, such as nephropathy, neuropathy, retinopathy, and cardiovascular disease. In addition, individuals with T1D are immune deficient. It is hypothesized that hyperglycemia effects the expression of glucose transporter 1 (GLUT1), a known glucose transporter of circulating blood cells including immune cells. This could lead to important immune cells residing in an energy deficient state.C‐peptide is a 31‐chain amino acid peptide co‐produced with insulin in the pancreatic β‐cells. Previous work from our group has shown that C‐peptide increases glucose absorption into, and ATP release from, red blood cells (RBCs). However, we have also shown that for C‐peptide to interact specifically with RBCs to elicit a biological response, it must be co‐administered with Zn2+ and albumin. Here, we measured the interaction between C‐peptide and immune cells (Tcells and neutrophils) in the presence or absence of Zn2+ and albumin. We also investigated Zn2+ binding to T‐cells and neutrophils in the presence or absence of C‐peptide and albumin. Furthermore, we studied the effect of C‐peptide on immune‐cell activation by performing functional studies to measure cell adhesion and migration. These functional studies were enabled by a novel 3D‐printed in vitro model system.T‐cells and neutrophils were collected from whole blood samples from consenting adults and separated from other cell types by immunomagnetic separation. Immune cells were incubated with varying combinations of C‐peptide, Zn2+, and albumin. C‐peptide binding was determined using enzyme‐linked immunosorbent assay (ELISA). Zn2+ binding was determined radiometrically using 65Zn2+, and radiation was counted using a gamma counter. Cellular adhesion and migration assays will be quantified using fluorescence spectroscopy. Our results preliminarily suggest that albumin is required for delivery of C‐peptide, but not Zn2+. With albumin present we show 2.0 × 104 C‐peptide molecules bound per neutrophil. Interestingly, when albumin is absent, we observed no significant binding of C‐peptide to neutrophils. However, there appears to be additional Zn2+ added to the neutrophils when in the presence of both the albumin and C‐peptide system, suggesting a specific binding site for both on the neutrophil. Nearly identical numbers were observed for C‐peptide binding to T‐cells.Support or Funding InformationNIH

Potential for combined delivery of riboflavin and all-trans retinoic acid, from silk fibroin for corneal bioengineering.

Millions of people worldwide suffer from vision impairing conditions resulting from corneal injury or disease. Silk fibroin (SF) is an emerging biopolymer that has been used for several applications including the fabrication of bioengineered corneas and ocular prostheses. To improve the cell response to SF, riboflavin (RF) and all-trans retinoic acid (RA) were coupled onto SF matrices. RF is a photo-initiator that has previously been combined with ultraviolet light to crosslink corneal collagen while RA has been used to regulate the phenotype of corneal stromal cells and their extracellular matrix deposition. Different concentrations of RF and RA were respectively photo-crosslinked and covalently bound through carbodiimide coupling onto 2% SF matrices. The effect of incorporating these molecules on the physical, chemical and mechanical properties of the matrices was evaluated. The biological response of human corneal stromal cells to the matrices was examined using cellular adhesion assays, proliferation assays, cytoskeleton staining, gene expression analysis and immunocytochemical staining. RF and RA both led to changes in the surface nanostructure and hydrophilicity while just RF increased the material stiffness. Cells cultured on the matrices containing both biomolecules displayed improved cellular proliferation, increased GAG deposition and increased expression of keratocyte genes that are normally associated with healthy corneal stromal tissue. These in vitro studies serve as a starting point for the optimization of loading bioactive molecules on SF based matrices for formulating clinically relevant ocular implants.

Humans from Wuchereria bancrofti endemic area elicit substantial immune response to proteins of the filarial parasite Brugia malayi and its endosymbiont Wolbachia

BackgroundIn the past, immune responses to several Brugia malayi immunodominant antigens have been characterized in filaria-infected populations; however, little is known regarding Wolbachia proteins. We earlier cloned and characterized few B. malayi (trehalose-6-phosphate phosphatase, Bm-TPP and heavy chain myosin, BmAF-Myo) and Wolbachia (translation initiation factor-1, Wol Tl IF-1 and NAD+-dependent DNA ligase, wBm-LigA) proteins and investigated the immune responses, which they triggered in animal models. The current study emphasizes on immunological characteristics of these proteins in three major categories of filarial endemic zones: endemic normal (EN, asymptomatic, amicrofilaraemic; putatively immune), microfilariae carriers (MF, asymptomatic but microfilaraemic), and chronic filarial patients (CP, symptomatic and mostly amicrofilaraemic).MethodsImmunoblotting and ELISA were carried out to measure IgG and isotype antibodies against these recombinant proteins in various clinical categories. Involvement of serum antibodies in infective larvae killing was assessed by antibody-dependent cellular adhesion and cytotoxicity assay. Cellular immune response was investigated by in vitro proliferation of peripheral blood mononuclear cells (PBMCs) and reactive oxygen species (ROS) generation in these cells after stimulation.ResultsImmune responses of EN and CP displayed almost similar level of IgG to Wol Tl IF-1 while other three proteins had higher serum IgG in EN individuals only. Specific IgA, IgG1, IgG3 and IgM to Bm-TPP were high in EN subjects, while BmAF-Myo additionally showed elevated IgG2. Enhanced IgA and IgG3 were detected in both EN and CP individuals in response to Wol Tl IF-1 antigen, but IgG1 and IgM were high only in EN individuals. wBm-LigA and BmAF-Myo exhibited almost similar pattern of antibody responses. PBMC isolated from EN subjects exhibited higher proliferation and ROS generation when stimulated with all three proteins except for Wol Tl IF-1.ConclusionsOverall, these findings display high immunogenicity of all four proteins in human subjects and revealed that the EN population was exposed to both B. malayi and Wolbachia proteins simultaneously. In addition, immune responses to Wol Tl IF-1 suggest possible role of this factor in Wolbachia-induced pathological responses while immune responses to other three proteins suggest that these can be explored further as vaccine candidates.

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development.

Modulating wall shear stress gradient via equilateral triangular channel for in situ cellular adhesion assay.

This study introduces an equilateral triangular channel (ETRIC), a novel microfluidic channel with an equilateral triangular cross-section, for cell adhesion assay by modulating the wall shear stress (WSS) gradient. The channel can generate a parabolic WSS gradient perpendicular to the flow direction at a single flow rate, and cell detachment can be in situ screened in response to spatially different levels of WSS. The existence of a simple form of exact solution for the velocity field inside the entire ETRIC region enables the easy design and modulation of the WSS levels at the bottom surface; therefore, the detachment of the cells can be investigated at the pre-defined observation window in real time. The exact solution for the velocity field was validated by comparing the analytical velocity profile with those obtained from both numerical simulation and experimental particle image velocimetry. The parabolic WSS gradient can be generated stably and consistently over time at a steady-state condition and easily modulated by changing the flow rate for the given ETRIC geometry. The WSS gradient in the ETRIC is in a symmetric parabolic form, and this symmetry feature doubles the experimental data, thereby efficiently minimizing the number of experiments. Finally, a WSS gradient ranging from 0 to 160 dyn/cm2 was generated through the present ETRIC, which enables not only to measure the adhesion strength but also to investigate the time-dependent detachment of NIH-3T3 cells attached on the glass.

Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Signaling.

Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes.

Anticancer activity of cryptotanshinone on acute lymphoblastic leukemia cells.

Cryptotanshinone, a well-known diterpene quinone from a widely used traditional Chinese herb named Salvia miltiorrhiza, has been reported for its therapeutical potentials on diverse activities. In this study, pharmacological effects of cryptotanshinone on acute lymphoblastic leukemia cells were investigated. IC50 values of 5.0 and 4.8 were obtained in CEM/ADR5000 and CCRF-CEM. Microarray-based mRNA expression revealed that cryptotanshinone regulated genes associated with cell cycle, DNA damage, reactive oxygen species (ROS), NFκB signaling and cellular movement. The involvement of these pathways in the mode of action of cryptotanshinone was subsequently validated by additional independent in vitro studies. Cryptotanshinone stimulated ROS generation and induced DNA damage. It arrested cells in G2/M phase of the cell cycle and induced apoptosis as measured by annexin V-FITC-conjugating fluorescence. The induction of the intrinsic apoptotic pathway by cryptotanshinone was proved by loss of mitochondrial membrane potential and increased cleavage of caspase 3/7, caspase 9 and poly ADP ribose polymerase(PARP). DNA-binding motif analysis of the microarray-retrieved deregulated genes in the promoter region revealed NFκB as potential transcription factor involved in cryptotanshinone's mode of action. Molecular docking and Western blotting provided supportive evidence, suggesting that cryptotanshinone binds to IKK-β and inhibits the translocation of p65 from the cytosol to the nucleus. In addition, cryptotanshinone inhibited cellular movement as shown by a fibronectin-based cellular adhesion assay, indicating that this compound exerts anti-invasive features. In conclusion, cryptotanshinone exerts profound cytotoxicity, which is caused by multispecific modes of actions, including G2/M arrest, apoptosis and inhibition of cellular movement. The inhibitory activities of this compound may be explained by inhibition of NFκB, which orchestrates all these mechanisms.