Inhibited Cell Attachment Research Articles

Talin2 binds to non-muscle myosin IIa and regulates cell attachment and fibronectin secretion

Talin2 is localized to large focal adhesions and is indispensable for traction force generation, invadopodium formation, cell invasion as well as metastasis. Talin2 has a higher affinity toward β-integrin tails than talin1. Moreover, disruption of the talin2-β-integrin interaction inhibits traction force generation, invadopodium formation and cell invasion, indicating that a strong talin2-β-integrin interaction is required for talin2 to fulfill these functions. Nevertheless, the role of talin2 in mediation of these processes remains unknown. Here we show that talin2 binds to the N-terminus of non-muscle myosin IIA (NMIIA) through its F3 subdomain. Moreover, talin2 co-localizes with NMIIA at cell edges as well as at some cytoplasmic spots. Talin2 also co-localizes with cortactin, an invadopodium marker. Furthermore, overexpression of NMIIA promoted the talin2 head binding to the β1-integrin tail, whereas knockdown of NMIIA reduced fibronectin and matrix metalloproteinase secretion as well as inhibited cell attachment on fibronectin-coated substrates. These results suggest that talin2 binds to NMIIA to control the secretion of extracellular matrix proteins and this interaction modulates cell adhesion.

An Antibody Neutralization Determinant on Domain III and the First α-Helical Domain in the Stem-Anchor Region of Tembusu Virus Envelope Protein.

Previous studies identified three neutralizing epitopes on domains I, II, and III of the Tembusu virus (TMUV) envelope (E). More evidence is needed to understand the molecular basis of Ab-mediated neutralization and protection against TMUV. In this study, we observed a neutralizing mAb, 6C8, that neutralized TMUV infection primarily by inhibiting cell attachment. In immunofluorescence assays, 6C8 recognized the premembrane and E proteins coexpressed in HEK-293T cells, but failed to react with premembrane or E expressed individually. Epitope mapping identified nine E protein residues positioned on BC/EF loops and F/G strands in domain III and the first α-helical domain in the stem region. Further investigation with mutant viruses showed that 6C8 pressure resulted in mutations at residues 330 of BC loop and 409 of the first α-helical domain, although 6C8 only exhibited a moderate neutralizing activity in BHK-21 cells and a weak protective activity in BALB/c mice and Shaoxing duck models. Mutations A330S and T409M conferred high- and low-level 6C8 resistance, respectively, whereas the combination of A330S and T409M mutations conferred moderate-level 6C8 resistance. As a result, a quasispecies comprising three groups of antigenic variants appeared in BHK-21 cell-derived viral stocks after repeated passages of TMUV strain Y in the presence of 6C8 treatment. Taken together, these findings have raised a concern about Ab-induced antigenic variations in vivo, and they have revealed information concerning the conformational structure of the 6C8 epitope and its role in constraint on antigenic variations. The present work contributes to a better understanding of the complexity of the TMUV immunogen.

ANTIBACTERIAL AND ANTIBIOFILM ACTIVITY OF MELINJO LEAF WATER EXTRACT AGAINST PSEUDOMONAS AERUGINOSA BACTERIA

Pseudomonas aeruginosa is a multi-drug resistant (MDR) human pathogen and known for their biofilm growth making it difficult to eliminate. This research aims to determine the effectiveness of water extract of melinjo leaf (Gnetum gnemon L) on inhibition of cell attachment, growth and degradation of the biofilm using Crystal Violet (CV) biofilm Assay. Reasearch results showed that water extract of melinjo leaf (Gnetum gnemon L) contains saponin, alkaloid, tanin dan steroidal that showed antibacterial activity and antibiofilm against P. aeruginosa. Addition extract can inhibit the attachment cell of 49,80% with a concentration of 25%, temperature of 37,5 oC and the contact time of 45 minutes. With a concentration of 25% (v/v), temperature of 50 °C and the contact time of 3 days, extract of melinjo leaves can inhibit the growth of biofilms of 43,09% and able to degrade the biofilm 43.04% with a concentration of 25% (v/v), temperature 37.5°C and contact time 45 minutes

Carboxylic Acid End-Capped Brushes on Titanium via Interface-Mediated RAFT Polymerization and Cell–Surface Interactions

Polymer brushes with tailored surface functionalities are important materials to manipulate the interactions between cells and surfaces in a biomedical context. To enhance the control of the conjugation of cell-adhesive peptides to the polymer brush terminus, poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes were fabricated via interface-mediated reversible addition-fragmentation chain transfer (RAFT) polymerization on titanium substrates. Due to the immobilization of 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid as a chain transfer agent (CTA) on the titanium substrates before the subsequent RAFT polymerization, terminal carboxylic acid groups were introduced in the brushes. The brushes obtained were characterized by ellipsometry, static water contact angle measurements, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FT-IR) spectroscopy. PDEGMA brushes allow the facile conjugation of peptides containing the arginine-glycine-aspartic acid (GRGDS) sequence, which afforded the peptide-specific attachment of NIH 3T3 fibroblasts. By contrast, the inhibition of cell attachment was observed on PDEGMA brushes with 7 nm dry thickness conjugated with arginine-alanine-aspartic acid (GRADS). The strategy of surface functional group modification with controllable antifouling or cell-adhesive properties will allow a versatile bio-functionalization approach independent of the underlying surface condition as polymeric biomaterials, among others, in titanium-based medical implant devices.

Decoding antibacterial and antibiofilm properties of cinnamon and cardamom essential oils: a combined molecular docking and experimental study

This study sets out to compare the antibacterial and antibiofilm profiles of Ci/Ca EOs alone and in combination together against infectious bacterial strains. MIC assay was carried out to survey the effectiveness of prepared EOs by two-fold serial dilution method and MTT evaluation. Synergic antibacterial properties of EOs against target strains were studied by using checkerboard titration method. Biofilm growth and development were evaluated using CV and XTT reduction assays. Antibacterial activity was observed for EOs against both bacterial strains with stronger activity for CiEO against both bacteria. The synergistic antibacterial effect was observed only against B. subtilis. Based on the FIC index, combinations could not inhibit the growth of E. coli. The pure EOs and their combination inhibited cell attachment for both studied bacteria with stronger effect on E. coli. CV and XTT reduction assays results showed that Ci EO and its combination with CaEO had the highest antibiofilm activity at lowest MIC value 0.08% and 0.04/0.02% against biofilm formed by E. coli and B. subtilis respectively, indicating a high antibiofilm potential. Computational docking analyses also postulated that the active constituents of evaluated EOs have the potential to interact with different bacterial targets, suggested binding mode of action of EOs metabolites. By and large, synergistic anti-biofilm properties of EOs may provide further options for developing novel formula to inhibit a variety of infectious clinical and industrial strains without (or less) toxicity effects on human body.Graphical

Quantitative Phytochemical Analysis Reveals Significant Antibiofilm Activity in Pleione maculata, an Endangered Medicinal Orchid

Pleione maculata has no scientific reports on quantitative phytochemical and antibiofilm activity till date. The objective of the study was to quantify and determine medicinally important bioactivity in P. maculata and analyse its anti-biofilm activity against clinical isolates Staphyloccocus aureus, Klebsiella pneumoniae and Proteus mirabilis. P. maculata exhibited the highest Total Antioxidant Capacity (TAC) about 193.98±0.1 mg, highest Total Phenolic Content (TPC) at 552±0.0 mg and Total Flavonoid Content (TFC) were observed highest at 879.5±0.2 mg. The acetone and ethyl acetate extracts of P. maculata pseudobulb showed distinct and significant zone of inhibition (ZOI) against drug-resistant S.aureus about 16±0.00 mm (MIC 0.875 mg/mL), ZOI of acetonitrile pseudobulb extract against P. mirabilis was 15.33±0.4 mm (MIC 1 mg/mL), ZOI of acetonitrile extracts of leaves and stem, ethyl acetate extract of pseudobulb was 12±0.0 mm, 12±01.4 mm, 12±2.8 mm against K. pneumoniae (MIC 1.8 mg/mL, 0.68 mg/mL and 3 mg/mL). Acetonitrile extract of pseudobulbs exhibited the highest Minimum Biofilm Inhibition concentration (MBIC) at 0.25 mg/mL against S. aureus, water root extract inhibited attachment of K. pneumoniae with lowest MBIC value 0.093 mg/mL, water and acetone extract of leaves inhibited cell attachment of P. mirabilis at lowest MBIC 0.117 and 0.171 mg/mL. The UV-VIS absorption band of P. maculata extracts ranges from 204-665 nm indicating the presence of phenolic and flavonoid compounds. The study indicates the potentiality of P. maculata as a rich source of medicinal active compounds as an antibiofilm agent against antibiotic-resistant clinical isolates.

Development of Thermally Responsive PolyNIPAm Microcarrier for Application of Cell Culturing-Part I: A Feasibility Study.

Poly(N-isopropylacrylamide) (polyNIPAm) microspheres were synthesized via the suspension polymerization technique. Thermal and redox initiators were compared for the polymerization, in order to study the effect of initiator type on the surface charge and particle size of polyNIPAm microspheres. The successful polymerization of NIPAm was confirmed by FTIR analysis. Microspheres of diameter >50 µm were synthesized when a pair of ammonium persulfate (APS) and N,N,N’,N’-tetramethylene-diamine (TEMED) redox initiators was used, whilst relatively small microspheres of ~1 µm diameter were produced using an Azobis-isobutyronitrile (AIBN) thermal initiator. Hence, suspension polymerization using a redox initiator pair was found to be more appropriate for the synthesis of polyNIPAm microspheres of a size suitable for human embryonic kidney (HEK) cell culturing. However, the zeta potential of polyNIPAm microspheres prepared using an APS/TEMED redox initiator was significantly more negative than AIBN thermal initiator prepared microspheres and acted to inhibit cell attachment. Conversely, strong cell attachment was observed in the case of polyNIPAm microspheres of diameter ~90 µm, prepared using an APS/TEMED redox initiator in the presence of a cetyl trimethyl ammonium bromide (CTAB) cationic surfactant; demonstrating that surface charge modified polyNIPAm microspheres have great potential for use in cell culturing.

Protein phosphatase 1 regulatory subunit 18 suppresses the transcriptional activity of NFATc1 via regulation of c-fos

The transcription factor NFATc1 and its binding partner AP-1 (a complex containing c-fos and c-Jun) play a central role in osteoclast differentiation. NFATc1 and AP-1 promote the expression of target genes such as Acp5, Ctsk and also auto-regulate NFATc1 expression as well. We previously reported that protein phosphatase 1 regulatory subunit 18 (PPP1r18) is a negative regulator of osteoclast bone resorption by inhibiting cell attachment to bone matrix. We also reported that PPP1r18 potentially regulates NFATc1 expression during osteoclast differentiation. To further explore this, in this study we have examined the effect of PPP1r18 on NFATc1 expression and activity by overexpressing PPP1r18 during the early stage of osteoclast differentiation. We found that PPP1r18 suppressed NFATc1 expression through inhibition of the transcriptional activity of NFATc1. Since PPP1r18 does not regulate NFATc1 directly, we next explored the involvement of AP-1. Our data showed that c-fos phosphorylation and nuclear localization were reduced by PPP1r18 overexpression. Further experiments showed that overexpression of c-fos together with PPP1r18 rescued NFATc1 expression and transcriptional activity. Moreover, c-fos activity inhibition by PPP1r18 was canceled by mutation of the phosphatase binding site of PPP1r18. Taken together, PPP1r18-regulated phosphatase activity targets c-fos phosphorylation and suppresses subsequent NFATc1 expression and activity.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells.

Objectives Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons.Methodology After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied.Results Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs.Conclusion These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Biomimetic phosphorylcholine-modified bacterial cellulose membranes with cell fouling resistance

Novel biomimetic zwitterionic phosphorylcholine (PC) modified bacterial cellulose (PCBC) membranes were prepared based on the Pudovik reaction of oxidized BC nanofiber clusters and then solution casting technology. Their chemical structure, morphology, hydrophilicity, mechanical and biocompatible properties were characterized and compared with non-modified BC membranes. The composition and morphology analysis of PCBC membranes confirmed the existence of zwitterionic PC groups on the membrane surfaces, and their nano-fibrous structure was kept. Compared with BC membranes, PCBC membranes exhibited better hydrophilicity, similar tensile strength, but decreased rupture strain. And PCBC membranes were non-hemolytic and non-cytotoxic like BC membranes. The anti-biofouling performance investigation revealed that PCBC membranes with zwitterionic surface showed significantly reduced protein adsorption for bovine serum albumin, as well as inhibited cell attachment for both L929 cells and macrophage RAW 264.7. The results strongly suggested that PCBC membranes with excellent cell fouling resistance are expected to be a promising anti-adhesion biomaterial.

Lipid-Bicelle-Coated Microfluidics for Intracellular Delivery with Reduced Fouling.

Innovative technologies for intracellular delivery are ushering in a new era for gene editing, enabling the utilization of a patient's own cells for stem cell and immunotherapies. In particular, cell-squeezing platforms provide unconventional forms of intracellular delivery, deforming cells through microfluidic constrictions to generate transient pores and to enable effective diffusion of biomolecular cargo. While these devices are promising gene-editing platforms, they require frequent maintenance due to the accumulation of cellular debris, limiting their potential for reaching the throughputs necessary for scalable cellular therapies. As these cell-squeezing technologies are improved, there is a need to develop next-generation platforms with higher throughput and longer lifespan, importantly, avoiding the buildup of cell debris and thus channel clogging. Here, we report a versatile strategy to coat the channels of microfluidic devices with lipid bilayers based on noncovalent lipid bicelle technology, which led to substantial improvements in reducing cell adhesion and protein adsorption. The antifouling properties of the lipid bilayer coating were evaluated, including membrane uniformity, passivation against nonspecific protein adsorption, and inhibition of cell attachment against multiple cell types. This surface functionalization approach was applied to coat constricted microfluidic channels for the intracellular delivery of fluorescently labeled dextran and plasmid DNA, demonstrating significant reductions in the accumulation of cell debris. Taken together, our work demonstrates that lipid bicelles are a useful tool to fabricate antifouling lipid bilayer coatings in cell-squeezing devices, resulting in reduced nonspecific fouling and cell clogging to improve performance.

Phosphatidylinositol 4,5-bisphosphate is localized in the plasma membrane outer leaflet and regulates cell adhesion and motility

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.

Biological Mechanisms for Cartilage Repair Using a BioCartilage Scaffold: Cellular Adhesion/Migration and Bioactive Proteins.

Objective. BioCartilage is a desiccated, particulated cartilage allograft used for repair of focal cartilage defects. It is mixed with a biologic such as bone marrow concentrate (BMC), pressed into a contained defect, and sealed with fibrin glue. The objective of this study was to assess if BioCartilage could serve as a bioactive scaffold by affecting cellular adhesion, cellular migration, or the release interleukin-1 receptor antagonist protein (IL-1RA), and to identify its full proteomic makeup. Design. Cartilage explants were used to model confined defects. BioCartilage was mixed with BMC, grafted into defects, and sealed with 1 of 5 fibrin glues. Constructs were cultured for 24 or 48 hours and then processed for live/dead microscopy. Chondrocyte and mesenchymal stem cell (MSC) adhesion on BioCartilage was assessed using scanning electron microscopy. Conditioned medium from cultures and the biologics used in the study were assayed for IL-1RA. The protein footprint of BioCartilage was determined using bottom-up proteomics. Results. BioCartilage supported chondrocyte and MSC attachment within 24 hours, and cell viability was retained in all constructs at 24 and 48 hours. Fibrin glue did not inhibit cell attachment. BMC had the highest concentration of IL-1RA. Proteomics yielded 254 proteins, including collagens, proteoglycans, and several bioactive proteins with known anabolic roles including cartilage oligomeric matrix protein. Conclusions. This study suggests that BioCartilage has the chemical composition and architecture to support cell adherence and migration and to provide bioactive proteins, which together should have biologics advantages in cartilage repair beyond its role as a scaffold.

Cell Adhesion, Morphology, and Metabolism Variation via Acoustic Exposure within Microfluidic Cell Handling Systems.

Acoustic fields are capable of manipulating biological samples contained within the enclosed and highly controlled environment of a microfluidic chip in a versatile manner. The use of acoustic streaming to alter fluid flows and radiation forces to control cell locations has important clinical and life science applications. While there have been significant advances in the fundamental implementation of these acoustic mechanisms, there is a considerable lack of understanding of the associated biological effects on cells. Typically a single, simple viability assay is used to demonstrate a high proportion of living cells. However, the findings of this study demonstrate that acoustic exposure can inhibit cell attachment, decrease cell spreading, and most intriguingly increase cellular metabolic activity, all without any impact upon viability rates. This has important implications by showing that mortality studies alone are inadequate for the assessment of biocompatibility, but further demonstrates that physical manipulation of cells can also be used to influence their biological activity.

Assessment of Toxic Effects of Ochratoxin A in Human Embryonic Stem Cells.

Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species, and it is considered a common contaminant in food and animal feed worldwide. On the other hand, human embryonic stem cells (hESCs) have been suggested as a valuable model for evaluating drug embryotoxicity. In this study, we have evaluated potentially toxic effects of OTA in hESCs. By using in vitro culture techniques, specific cellular markers, and molecular biology procedures, we found that OTA produces mild cytotoxic effects in hESCs by inhibiting cell attachment, survival, and proliferation in a dose-dependent manner. Thus, we suggest that hESCs provide a valuable human and cellular model for toxicological studies regarding preimplantation stage of human fetal development.

Surface stabilization treatment enhances initial cell viability and adhesion for biodegradable zinc alloys

Zinc-based alloys hold promise as next generation biodegradable implants. Although zinc implants exhibit excellent biocompatibility in vivo, they have been found to inhibit cell attachment and viability under in vitro conditions. In order to clarify the cell response disparity and improve the biocompatibility of zinc implant materials, Zn-1Mg and Zn-1Mg-0.5Ca alloys underwent surface stabilization treatment in cell culture medium. Surface processing resulted in a stable surface oxide film of ∼300 nm thickness. The stability of the oxide layer substantially increased the viability of cells in both direct and indirect contact assays. It is consequently believed that the surface film characteristics of zinc implants may be an important determinant of biocompatibility.

Mesenchymal Cells Utilize Integrins to Attach to Titanium Implant Surfaces

ObjectivesTitanium implants have been used for decades to effectively replace missing teeth. Recently, surface modifications have been used to enhance implant osteointegration. Despite these innovations, little is known about how cells interact with modified titanium surfaces. This study aims to examine how mesenchymal cells attached to modified titanium surfaces.MethodsHuman periodontal ligament (PDL) and gingival fibroblasts were evaluated for their ability to survive and attach to three different commercially available implants; Dual thermo‐etched titanium (DTET), hydroxyapatite coated dual transition surface (HADTS) and moderately rough titanium oxide layer with high crystallinity and a high phosphorus content (MRTOP). Subsequently, the cell attachments to modified titanium surfaces were examined for enhancement using serum proteins and inhibition using anti‐integrin as well as anti‐extracellular matrix molecule (ECM) antibodies.ResultsCells attached to modified titanium implant surfaces in a time dependent manner over 24 hours, with no significant difference between the surface modifications examined. Cell attachment was dependent upon the presence of serum in the growth media. Serum is known to contain several ECM proteins, including; fibronection, fibrinogen, and vitronectin. Integrin β1 antibodies significantly inhibited cell attachment, while αVβ3 antibodies did not. Fibronectin/RGD dependent integrin receptor antibodies (αV and α5) inhibited early attachment (before 8 hours). In contrast, collagen dependent integrin receptor antibodies (α1 and α2) inhibited only later attachment (after 8 hours). Finally, laminin dependent integrin receptor antibodies (α3 and α6) failed to significantly inhibit cell attachment. In addition, fibronectin (and to a lesser extent fibrinogen) antibodies inhibited early cell attachment, while collagen Type I antibodies inhibited later attachment.ConclusionsCell attachments to titanium surfaces are at least partially modulated by integrin receptors via ECM proteins coating the titanium surfaces. Cell attachment can be broken down into 2 phases: (1) mediated by serum fibronectin/fibrinogen for the first 8 hours, and (2) mediated by collagens produced by the mesenchymal cells between 8 and 24 hours. Further studies are needed to determine if any of these integrin mediated attachment events effect osteoinduction or osteointegration.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

2-Methacryloyloxyethyl phosphorylcholine (MPC)-polymer suppresses an increase of oral bacteria: a single-blind, crossover clinical trial

ObjectivesThe biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymers, which mimic a biomembrane, reduce protein adsorption and bacterial adhesion and inhibit cell attachment. The aim of this study is to clarify whether MPC-polymer can suppress the bacterial adherence in oral cavity by a crossover design. We also investigated the number of Fusobacterium nucleatum, which is the key bacterium forming dental plaque, in clinical samples.Materials and methodsThis study was a randomized, placebo-controlled, single-blind, crossover study, with two treatment periods separated by a 2-week washout period. We conducted clinical trial with 20 healthy subjects to evaluate the effect of 5% MPC-polymer mouthwash after 5 h on oral microflora. PBS was used as a control. The bacterial number in the gargling sample before and after intervention was counted by an electronic bacterial counter and a culture method. DNA amounts of total bacteria and F. nucleatum were examined by q-PCR.ResultsThe numbers of total bacteria and oral streptcocci after 5 h of 5% MPC-polymer treatment significantly decreased, compared to the control group. Moreover, the DNA amounts of total bacteria and F. nucleatum significantly decreased by 5% MPC-polymer mouthwash.ConclusionsWe suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adherence.Clinical relevanceMPC-polymer can be a potent compound for the control of oral microflora to prevent oral infection.

Water Extract Activity of Papaya Leaf as Antibiofilm against <i>Escherichia coli</i>

Biofilms are significant hazards in the food industry. The presence of Escherichia coli biofilms in food industry is potentially causing food spoilage that will shorten the shelf life as well as lead to the spread of disease through food. The purpose of this study is to determine the phytochemical compound of aqueous extract of papaya leaves and to determine its activity on inhibition of cell attachment and growth, and on degradation of the biofilm using Micro-titter Plate Biofilm Assay. The results phytochemical test showed that papaya leave extract contains alkaloid, tanin, flavonoid, and steroid and have anti-biofilm activity. Papaya leave extract with highest activity caused biofilm degradation of 48,99%. The optimum temperature of biofilm degradation (>52,5%) by papaya leaves extract was around 35oC - 40oC. The optimum contact time and extract concentration were 57-60 minutes and 75-100%, respectively.

The P2X7 receptor is an upstream regulator of dynamic blebbing and a pluripotency marker in human embryonic stem cells

New methods are needed to reduce dynamic blebbing which inhibits cell attachment and survival during passaging of pluripotent stem cells. We tested the hypothesis that activation of the P2X7 receptor by extracellular ATP during passaging initiates dynamic blebbing. The P2X7 receptor was present in human embryonic stem cells (hESC), but not in differentiating cells. Extracellular ATP concentrations were 14× higher in medium during passaging. Addition of ATP to culture medium prolonged dynamic blebbing and inhibited attachment. Inhibition of P2X7 by specific drugs or by siRNA significantly reduced dynamic blebbing and improved cell attachment. When cells were incubated in calcium chelators (EGTA or BAPTA), blebbing decreased and attachment improved. Calcium influx was observed using Fura-4 when ATP was added to culture medium and inhibited in the presence of the P2X7 inhibitor. Over-expressing activated Rac in hESC reduced blebbing and promoted cell attachment, while a Rac inhibitor prolonged blebbing and reduced attachment. These data identify a pathway involving P2X7 that initiates and prolongs dynamic blebbing during hESC passaging. This pathway provides new insight into factors that increase dynamic blebbing and identifies new targets, such as P2X7, that can be used to improve the culture of cells with therapeutic potential.