Plate Adhesion Research Articles

Thermoresponsive poly(N-isopropylacrylamide) hydrogel substrates micropatterned with poly(ethylene glycol) hydrogel for adipose mesenchymal stem cell spheroid formation and retrieval.

Cell spheroid formation is necessary to develop three-dimensional (3D) cellular environments that provide appropriate cell-cell and cell-matrix interactions similar to in vivo environments without additional substrates. Although some methods including stirring culture, low adhesion plate culture, hanging drop, and microfluidics are used to construct cell spheroids, there is no method to fulfill all of the mass production of uniform spheroids, simple media change, and easy retrievability. Here, bulk poly(N-isopropylacrylamide) (PNIPAAm) hydrogel substrate (PHS) was used to fabricate, culture, and retrieve cell spheroids. Adipose-derived stem cells (ASCs) were cultured on bulk PHS to form spheroids. ASCs formed cell spheroids directly on substrates without additional manipulation. These spheroids adhered to the semi-adhesive substrate, while the spheroids fabricated using the nonadhesive surface method floated without getting fixed to the surface. Bulk PHS stiffness was evaluated using the compressive test (compressive modulus: 153±11kPa). A poly(ethylene glycol) (PEG) hydrogel microwell pattern was created on PHS to control the spheroid size, forming uniform ASC spheroids between 100 and 150μm in diameter on 200 and 300μm well-patterned substrates. Cell-cell interactions in the resulting ASC spheroids were evaluated based on fibronectin and laminin expression; fluorescence intensities of fibronectin- and laminin-immunostained images of ASC spheroids were 10.9 and 7.3 times higher than those of ASCs cultured on the tissue culture plate, respectively. ASC spheroids were detached following incubation at 4°C for 10min (retrieval efficiency: 74±19%). Retrieved spheroid cell viability was over 97.5%. The PEG hydrogel microwell-patterned PHS is a convenient spheroid fabrication and retrieval platform that can increase cell spheroid usage.

Immunogenic and inflammatory responses to citrullinated proteins are enhanced following modification with malondialdehyde-acetaldehyde adducts

Background/ObjectiveMalondialdehyde-acetaldehyde adducts (MAA) act as potent immune adjuvants and co-localize with citrullinated antigens in tissues effected by rheumatoid arthritis (RA). We sought to examine the role of MAA-adducts in promoting RA-related autoimmunity and inflammation. MethodsDBA/J1 mice were immunized with human serum albumin (HSA), HSA-MAA, citrullinated HSA (HSA-Cit), or HSA-MAA-Cit with subsequent measurement of serum anti-citrullinated protein antibody (ACPA) and anti-Cit T cell responses. Cellular binding of the same antigens was examined using THP-1 monocytes and Chinese Hamster Ovary (CHO) cells transfected with specific scavenger receptors (SRs: TLR4, SR-B2, SREC-1). The effects of these antigens on THP-1 activation were then examined by quantifying plate adherence, pro-inflammatory (TNFα, IL-1β, IL-10) cytokine release, and SR (CD14, SR-B2)/co-stimulatory molecule (CD80, HLA-DR) expression. Comparisons were completed using one-way ANOVA with Tukey’s post-hoc test. ResultsMice immunized with co-modified HSA produced significantly higher ACPA concentrations than all other groups whereas T cell responses to citrullinated proteins were highest following immunization with HSA-MAA. Both transfected CHO and THP-1 cells demonstrated significantly higher binding of HSA-MAA-Cit vs. HSA or HSA-Cit. THP-1 cells exposed to HSA-MAA-Cit expressed significantly higher concentrations of TNFα, IL-1β, and IL-10 vs. all other groups. Furthermore, THP-1 cells demonstrated significantly increased plate adherence and higher expression of CD14, SR-B2, and HLA-DR following incubation with HSA-MAA-Cit vs. HSA or HSA-Cit. ConclusionThese studies demonstrate that MAA-adduction of citrullinated antigen greatly enhances immune and cellular responses, potentially acting as a key co-factor in RA pathogenesis.

In vitro and in vivo induction of human embryonic stem cells differentiated into rosette neural stem cells and further generation of neuron-like cells

ABSTRACT Nervous system diseases, especially degenerative diseases of the central nervous system (CNS), seriously endanger human health, and the injured adult neurons are difficult to self-renew. Therefore, cell replacement therapy with good application prospects has become a hot spot in biomedical research. Human embryonic stem cells (hESCs) are promising sources of cells with unlimited proliferation ability and multiple differentiation potential. The purpose of this study was to use these characteristics of hESCs to obtain a large number of differentiated and stable neural stem cells (NSCs) and provide potential tissue engineering materials for the treatment of neurological diseases. Cultured on low adhesion plate for 4 days in vitro, the hESCs can differentiate into suspended embryoid bodies (EBs). After further culture of EBs for 4 days, high-purity Nestin-positive NSCs with typical rosette structure can be induced, and these cells can further differentiate into a variety of mature nerve cells. Meanwhile, hESCs could form in vivo typical teratomas that contained rosette structures. Our results indicated that hESCs had the ability to differentiate into rosette NSCs both in vitro and in vivo, and the bioactive rosettes are of great significance to understanding the mechanism of neural development and studying the treatment of neurodegenerative diseases.

A Novel 3D Scaffold for Cell Growth to Asses Electroporation Efficacy.

Tumor electroporation (EP) refers to the permeabilization of the cell membrane by means of short electric pulses thus allowing the potentiation of chemotherapeutic drugs. Standard plate adhesion 2D cell cultures can simulate the in vivo environment only partially due to lack of cell–cell interaction and extracellular matrix (ECM). In this study, we assessed a novel 3D scaffold for cell cultures based on hyaluronic acid and ionic-complementary self-assembling peptides (SAPs), by studying the growth patterns of two different breast carcinoma cell lines (HCC1569 and MDA-MB231). This 3D scaffold modulates cell shape and induces extracellular matrix deposit around cells. In the MDA-MB 231 cell line, it allows three-dimensional growth of structures known as spheroids, while in HCC1569 it achieves a cell organization similar to that observed in vivo. Interestingly, we were able to visualize the electroporation effect on the cells seeded in the new scaffold by means of standard propidium iodide assay and fluorescence microscopy. Thanks to the presence of cell–cell and cell–ECM interactions, the new 3D scaffold may represent a more reliable support for EP studies than 2D cancer cell cultures and may be used to test new EP-delivered drugs and novel EP protocols.

Comparison of a novel adhesive system and plate and screw for facial fracture osteosynthesis

Background: in oral and maxillofacial fractures, plates and screws routinely fix fragments until the completion of healing process, which has its own complications in some critical fractures. To overcome this drawbacks bone adhesives are developed for the immobilization of fractured bones. Objective: In this in-vitro study we compared the bond strength obtained by immobilization of the bone fragments using plates and screw and new adhesive containing BTDMA monomer. Materials and methods: In this experimental in-vitro trial, bone fractures were simulated in bovine’s mandibular bone using an electrical saw. The bone fragments were randomly allocated in different groups and were immobilized with either plate and screws and adhesive containing 0,10, 15% BTDMA. After 24 hours tensile bond strength was calculated using universal testing machine. One-way ANOVA and Tukey post hoe test were used for statistical analysis. Results: Mean (± standard deviation) of tensile bond strength of bone fixation using adhesive containing 15% (W/W) BTDMA monomer were 176.0 (±18.89) N. These values were 149.1 (±23.88) N for adhesive containing 10% BTDMA; 102(±17.99) N for the base adhesive and 278.9 (±24.12) N for the screw and plate technique. Significant differences were found regarding bond strength of bone fixation in 4 groups using bone adhesives or plate technique (P<0.001). The highest bond strength was recorded for the plate group and the least was related to the base adhesive. Significant differences existed between all bone adhesives as declared by paired comparison (p<0.05). Conclusion: Despite the lower bond strength in adhesive groups in comparison with screw and plate, with regards to possible complications of screw and plate technique, it seems bone adhesives containing BTDMA monomer can be used for bone fragment fixation. However, bond strength is just one of the numerous properties that an adhesive should have and more studies must be done on these kinds of adhesives.

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

INVESTIGATIIONT OF CONIC FLOW POINT IN ADHESIVE SYSTEMS BY USING HEPPLER CONSISTOMETER

When restoring wedge-shaped tooth defects, the adhesive system, and in particular, its microhardness provided by an inorganic filler plays a significant role in withstanding the masticatory load. The conic flow point is a measure for the microhardness of spatially-stitched polymer matrices and depends on the nature, structure, density of the polymer chains. The purpose of this work is to compare the properties of analogues: the Conic Flow Point of 5th DC Adhesive NF Adhesive Systems (Dental Central) and the 7th Single Bond (3M ESPE) by using Heppler consistometer under laboratory conditions.
 The study was carried out on adhesive plates manufactured directly in the laboratory before testing. The samples were divided into two groups depending on the adhesive system used: 10 samples of 5 generations (DC Adhesive NF, Dental Center) and 10 samples of 7 generations (Single bond Universal 3M ESPE). To obtain specimens of adhesive systems of 3.0 ± 0.2 mm high, a metallic mould covered with silicone was used. On the back side of the mould we put a sheet of lavsan film, turned the mould upside-down, then put the adhesive into the mould and covered with lavsan film. Then this sample was irradiated with a photopolymerizer for 40 seconds. Without removing the film, the sample was turned over and its other side was irradiated for 40 seconds. Then we peel off the film and take out finished sample from the mould. Prior to testing, samples were kept in distilled water at a temperature of (37 ± 2) ºC for 24 hours.
 According to relevant literature data and research data reported by 3M ESPE and Dental Central firms, no studies for assessing conic flow point by using Heppler consistometer for 5th and 7th generation adhesives were carried put under laboratory conditions. Out test results have demonstrated the regularity that the harder an adhesive is, the higher values of the conical flow point. That is, the adhesive can withstand a load that is positive for retorting cervical defects.
 Thus, the restoration of wedge-shaped defects requires a competent and differentiated approach to the choice of adhesive system in order to obtain positive results and to avert spreading of pathological process that ensured the treatment efficacy. The use of 5th generation AD Adhesive NF promotes the formation of a complete hybrid layer, firm bonding between composite material, dentin and enamel, provides effective sealing at the edge of filling margins with dental tissues and their resistance due to adhesive ductility: all this guarantees the durability of the restorations performed.

Abstract 4685: Cancer associated fibroblasts promote ovarian cancer chemoresistance by inducing cancer stem cells through Wnt signaling

Abstract Ovarian cancer is the most lethal gynecologic malignancy and the 5th leading cause of cancer related deaths among women in the USA. Most patients eventually succumb to chemoresistant disease and the purpose of this study is to understand the mechanism of development of chemoresistance and disease relapse. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are resistant to cytotoxic chemotherapy and cause relapse. The tumor microenvironment can potentially provide an optimal cancer stem cell niche for cancer stem cell growth and maintenance. Cancer associated fibroblasts (CAFs) are one of the main constituents of the tumor microenvironment in ovarian tumors, promoting tumor progression and chemoresistance. We have studied the potential role of CAFs in maintaining CSC population and enhancing chemoresistance with an objective to develop effective approaches to overcome chemoresistance and tumor relapse. Co-culture of high grade serous ovarian cancer cells with CAFs resulted in increased resistance to carboplatin. ALDH1A1 is a well-established marker for ovarian cancer CSCs and the ALDH+ population was significantly increased upon co-culture with CAFs. Similarly, CAFs also enhanced spheroid formation of ovarian cancer cells seeded in ultralow adhesion plates in CSC medium. Interestingly, co-culturing ALDH- ovarian cancer cells with CAFs resulted in the induction of ALDH+ cells within 6 days. Analysis of the signaling pathways activated in ovarian cancer CSCs and the gene expression profiles of ovarian cancer CAFs indicated the potential role of Wnt signaling in the productive cross-talk between CAFs and ovarian cancer CSCs. Treatment with Wnt inhibitors abrogated the induction of CSCs by CAFs. By selectively silencing porcupine, a protein involved in Wnt ligand lipidation and secretion, we further confirmed that CAF derived Wnts are responsible for the induction of CSC. Studies are ongoing to identify the specific Wnt ligand involved in the cross-talk and downstream pathways activated during CSC induction and maintenance by CAFs. Our results indicate that CAF-derived Wnt ligands are instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse. Citation Format: Yiming Fang, Mohamed A. Abd El Aziz, Kartikeya Tiwari, Anirban K. Mitra. Cancer associated fibroblasts promote ovarian cancer chemoresistance by inducing cancer stem cells through Wnt signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4685.

Peristomal plate cut mold: possibilities for applying new products

Introduction: peristomal dermatitis can be caused by leakage between adhesive plate and skin. Objective: to describe the peristomal plate cut mold (PPCM) for simultaneous evaluation of new products for the treatment of dermatitis. Methods: experience report about creation of a cut model, with sufficient size and width to expose part of the skin with dermatitis, to apply another product. The trapezoidal cut, according to positions 3, 6, 9 or 12 hours, corresponding to one of the quadrants, it was performed on the plate of carboxymethylcellulose, pectin, and gelatine coupled to the stoma bag. The entire part of the plate adhered to the skin was adapted to the control product. In the photographic analysis, CorelDRAW 19.0 was used. The lesions were measured in pixels. Result: the cut did not interfere with the adhesion of two parts device board, it made possible the simultaneous application and testing of a new product and the comparison of the healing with the plate. Conclusion: the PPCM is a new strategy for studies of the application of other products for peristomal dermatitis simultaneously in the same patient and demonstrates positive results in its use.

Introduction of a Surface Characterization Parameter Sdrprime for Analysis of Re-entrant Features

Producing components using metal additive manufacturing processes, such as powder bed fusion, presents manufacturing and measurement challenges, but also significant opportunities. The as-built surface may include overhanging (re-entrant) features not intentionally included in the design, but that aid in component functionality. In addition, the additive manufacturing process presents opportunities to design and manufacture re-entrant features intentionally. Re-entrant features increase the specific surface area and, in addition, produce mechanical locking to the surface. These re-entrant features may be intended to improve surface performance in areas such as biological cell attachment, coating adhesion, electrical capacitance and battery plate design, fluid flow and material cooling. Re-entrant features may prove difficult or impossible to measure and characterise using conventional line-of-sight surface metrology instrumentation, however the correct measurement of these surfaces may be vital for functional optimisation. X-ray computed tomography does have the ability to image internal and re-entrant features. This paper reports on the measurement of re-entrant features using X-ray computed tomography and the extraction of actual surface area information (including re-entrant surfaces) from sample additively manufactured surfaces. A proposed new surface texture parameter, Sdrprime, is discussed. This parameter is applicable to true 3D data, including re-entrant features, and is intended to relate directly to the component surface functional performance. The errors produced when using line-of-sight instruments and height map parameter generation per ISO 25178-2 to evaluate surfaces that include re-entrant features are discussed. Measurement results for electron beam melting and selective laser melting additively manufactured components, together with simulated structured surfaces, are presented.

Shear transfer of concrete strengthened using bolted steel plates

Abstract Reinforced concrete (RC) with steel bars set across a shear plane was strengthened using bolted steel plates to transfer shear force. Nineteen Z-series specimens were tested to investigate shear transfer behavior with bar ratios varying from 0.46 to 1.2% and steel plate ratios ranging from 3.57 to 10.42%. Nine D- and B-series specimens were tested to assist to understand shear transfer mechanism by means of a shear component model. In this model, the shear transfer resistance was provided by different sources, i.e., friction, aggregate interlock, adhesion, shear dilation, dowel action, and steel plate resistance. Results found that the ultimate shear transfer capacities increased by 15–57% with a ductile failure mode. The increase of the shear transfer resistance was primarily due to two factors: 1) the increase of the shear components of friction, aggregate interlock, and shear dilation caused by the additional transverse clamping force provided by the steel plates and 2) steel plate resistance. Expressions were developed to predict the ultimate resistance of shear transfer of strengthened RC with approximate accuracy.

In vitro Effects of Antimicrobial Agents on Planktonic and Biofilm Forms of Staphylococcus saprophyticus Isolated From Patients With Urinary Tract Infections.

Bacterial biofilms play an important role in urinary tract infections (UTIs), being responsible for persistent infections that lead to recurrences and relapses. Staphylococcus saprophyticus is one of the main etiological agents of UTIs, however, little is known about biofilm production in this species and especially about its response to the antimicrobial agents used to treat UTIs when a biofilm is present. For this reason, the aim of this work was to evaluate the response of S. saprophyticus biofilms to five antimicrobial agents. Staphylococcus saprophyticus was evaluated for antimicrobial susceptibility in its planktonic form by means of minimum inhibitory concentration (MIC) and in biofilms by means of minimum inhibitory concentration in biofilm (MICB) against the following antimicrobial agents by the microdilution technique: vancomycin, oxacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and norfloxacin. Of the 169 S. saprophyticus studied, 119 produced a biofilm as demonstrated by the polystyrene plate adherence method. Biofilm cells of S. saprophyticus exhibited a considerable increase in MICB when compared to the planktonic forms, with an increase of more than 32 times in the MICB of some drugs. Some isolates switched from the category of susceptible in the planktonic condition to resistant in the biofilm state. Statistical analysis of the results showed a significant increase in MICB (p < 0.0001) for all five drugs tested in the biofilm state compared to the planktonic form. Regarding determination of the minimum bactericidal concentration in biofilm (MBCB), there were isolates for which the minimum bactericidal concentration of all drugs was equal to or higher than the highest concentration tested.

Impact of Hepatic Stellate Cells in Scaffold-Free 3D-Bioprinting of the Liver Model

Background and Hypothesis: Hepatic stellate cells (HSC), which compromise ~15% of liver cells, are vital to hepatocellular function. Scaffold-free 3D-bioprinting (SF3DBP) offers an avenue for the creation of realistic organ models without the use of biomaterials. Therefore, we hypothesized that co-culturing primary hepatocytes with HSC in SF3DBP liver model would uphold hepatocyte function over time, providing us a better 3D-liver model for research. &#x0D; Experimental Design: We used freshly thawed primary pig hepatocytes and immortalized pig HSC to generate spheroids with hepatocytes alone, HSC alone, or a combination of hepatocytes and HSC (2.5:1 ratio). Spheroids were formed using low adhesion plates, then characterized for distance from well center, diameter, roundness, and smoothness. A column of spheroids was printed using a Regenova 3D-bioprinter. Remaining loose spheroids are incubated over two weeks for albumin secretion, mRNA transcription, and histological analysis. &#x0D; Results: Co-cultures of hepatocytes and HSC (2.5:1 ratio) formed spheroids within 48 hours, as did HSC only spheroids (Figure 1). Spheroids composed of only hepatocytes failed to form round spheroids. The combination spheroids increased in roundness and decreased in diameter between characterizations over 6 days. &#x0D; Conclusion and Potential Impact: Spheroids proved too large to print at 48 hours but were successfully recognized and placed by the 3D-bioprinter. SF3DBP of combination spheroids would be viable by day 6. Optimization of spheroid composition using different cell ratios including HSC, hepatocytes, liver sinusoidal endothelial cells and fibroblasts, as well as optimization of spheroid incubation time will allow for production and printing of more advanced liver models.

Експлуатаційні дослідження показників кутового зсуву клейової полімерної пластини корінцевої частини книжкового блоку

The purpose of the work is to study the deformation of the adhesive layer of the root portion of the book block, stitched threads, the influence of the bending angles of the polymeric adhesive plate during the exploitation of book products, which contribute to the creation of zones of variable concentration of destructive stresses of the internal structure of the plate, as well as the influence of the time of the load on these areas, that proportionally influence on the reduction of the elastic properties of the plate and the development of residual plastic deformation phenomena. The amount of deformation of individual sections of a radial adhesive polymer plate, during operational loads when using book blocks stitched together, depends not only on the elastic material properties of the adhesive composition, its isotropy, the thickness of the applied layer, but also on the angles of the plate kinks that contribute to creating variable zones concentration of destructive stresses of the internal structure. Stresses occur at the edges of the joints of the glue plate with the paper of the folds of the backs of the notebooks on the one hand, and the edging paper, on the other hand, in the form of sections of compression and tension in places of discrete kinks. The duration of the loads in these areas, in proportion to the decrease in the elastic properties of the plate and the increase in the phenomena of residual deformation. Taking into account the negative influence of discrete angular bends of rooted glue polymeric plates in any part of it and the heterogeneity of the duration of loads during the exploitation of book products lead to deformation formed during bending of the plate and promotes the accelerated transformation of processes of elastic deformation into residual processes of plastic deformation. The use of high strength and high elastic adhesive compositions, which do not have a negative effect on the stiffness and bending stability of the glued system, can minimize the disadvantages of the angular displacement, which in turn will provide a prolonged operating period and a favorable competitive appearance of book products.

Surface proteins mhp390 (P68) contributes to cilium adherence and mediates inflammation and apoptosis in Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP) and responsible for major economic losses in global swine industry. After colonization of the respiratory epithelium, M. hyopneumoniae elicits a general mucociliary clearance loss, prolonged inflammatory response, host immunosuppression and secondary infections. Until now, the pathogenesis of M. hyopneumoniae is not completely elucidated. This present study explores the pathogenicity of mhp390 (P68, a membrane-associated lipoprotein) by elucidating its multiple functions. Microtitrer plate adherence assay demonstrated that mhp390 is a new cilia adhesin that plays an important role in binding to swine tracheal cilia. Notably, mhp390 could induce significant apoptosis of lymphocytes and monocytes from peripheral blood mononuclear cells (PBMCs), as well as primary alveolar macrophages (PAMs), which might weaken the host immune response. In addition, mhp390 contributes to the production of proinflammatory cytokines, at least partially, via the release of IL-1β and TNF-α. To the best of our knowledge, this is the first report of the multiple functions of M. hyopneumoniae mhp390, which may supplement known virulence genes and further develop our understanding of the pathogenicity of M. hyopneumoniae.

Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program

Hematopoietic stem cell transplantation (HSCT) donor–generated virus-specific T cells (VSTs) can provide effective treatment for viral infection post-HSCT but are not readily accessible to all patients. Off-the-shelf cryopreserved VSTs suitable for treatment of multiple patients are an attractive alternative. We generated a bank of 17 cytomegalovirus (CMV)–, 14 Epstein-Barr virus (EBV)–, and 15 adenovirus (AdV)–specific T cell products from 30 third-party donors. Donors were selected for expression of 6 core HLA antigens expressed at high frequency in the local transplant population. T cells were generated by co-culturing venous blood or mobilized hematopoietic stem cell (HSC)–derived mononuclear cells with monocyte-derived dendritic cells pulsed with overlapping peptides covering CMV pp65, AdV5 hexon, or EBV BZLF1/LMP2A/EBNA1 proteins. Addition of a CD14+ selection step instead of plate adherence to isolate monocytes before culture initiation significantly improved expansion in cultures from HSC material. Phenotyping showed the CD8+ subset to have significantly higher numbers of terminal effector T cells (CD45RA+62L−) and lower numbers of effector memory T cells (CD45RA−62L−) when compared with the CD4+ subset. Increased expression of the immunoinhibitory markers PD-1 and TIM-3 was noted on CD4+ but not CD8+ cells when compared with the control group. VST showed antiviral activity restricted through a variety of common HLAs, and modelling suggested a suitably HLA-matched product would be available for >90% of HSCT patients. Only a small number of carefully selected third-party donors are required to generate a VST bank of broad coverage, indicating the feasibility of local banking integrated into existing allogeneic HSCT programs.

Contact resistivity and adhesion of copper alloy seed layer for copper-plated silicon heterojunction solar cells

To reduce optical loss and contact material cost in silicon heterojunction (SHJ) solar cells, copper plating has been considered as a suitable metallization technique. However, a plated copper contact on indium tin oxide (ITO) generally has low adhesion reliability. For this reason, suitable seed layer materials are required for adhesive copper plating. As a requirement of a suitable seed layer material, contact resistance between the seed and the ITO is also important, as well as the adhesion, since a high series resistance results in a low fill factor. In this work, we applied copper alloy (Cu–X) materials as a seed layer that was deposited by co-evaporating copper with other metals. The contact resistivity (ρc) values of the Cu–X seed layer and copper-plated contact with the Cu–X seed layer (Cu–X/Cu) on the ITO layer were evaluated by the transfer length method (TLM). Also, tape tests were carried out to check the adhesion of the Cu–X/Cu contact.

Isolating and culturing of sputum macrophages: A potential ex vivo/in vitro model

Purpose: This paper aimed to test whether induced sputum samples acquired from human volunteers could be used to isolate and culture airway macrophages for in vitro exposures. This was assessed in terms of the culturing success rate, culture purity, viability and responsiveness of cultured cells. Materials and methods: The isolation and culturing procedure was performed over three days. On Day 1, induced sputum samples were obtained, processed and seeded in culture wells. Differential cell counts and viability tests were performed to allow for calculation of viable macrophage numbers and appropriate sample dilution. After a 1 h rest, seeded wells were washed to remove non-adherent cells, resulting in macrophage isolation. Then, cells rested overnight (Day 1–Day 2), before in vitro exposure for 2–24 h (Day 2–Day 3). The criteria for progressing into the culturing procedure was cell viability >40% and total cell number >106. Successful culturing was evaluated based on cell attachment (N = 40). Culture purity by differential cell analysis and viability was monitored during culturing (N = 4–8). Macrophage responsivity was assessed by measurement of inflammatory cytokine gene expression (N = 4) and cytokine levels (N = 6) following in vitro exposure to lipopolysaccharide (LPS) (2–24 h) and live bacteria (S. aureus) (4h). Results: Overall, 88% (35/40) of the samples acquired were suitable for isolation, and 80% (32/40) were successfully progressed through the 2–3 day culturing protocol. Macrophage purity (88%) and viability (85%) were adequate. Moreover, cultured macrophages were responsive to in vitro stimulation with LPS and viable S. aureus showing positive mRNA responses for TNFα, IL-1β and IL-8 and release of IL-1β, respectively. Conclusion: Sputum macrophage isolation by plate adherence and subsequent culturing of sputum macrophages was successfully performed and represents a promising in vitro model for examination of airway macrophage behavior.

Hindering the formation and promoting the dispersion of medical biofilms: non-lethal effects of seagrass extracts

BackgroundBiofilms have great significance in healthcare-associated infections owing to their inherent tolerance and resistance to antimicrobial therapies. New approaches to prevent and treat unwanted biofilms are urgently required. To this end, three seagrass species (Enhalus acoroides, Halophila ovalis and Halodule pinifolia) collected in Vietnam and in India were investigated for their effects in mediating non-lethal interactions on sessile bacterial (Escherichia coli) and fungal (Candida albicans) cultures. The present study was focused on anti-biofilm activities of seagrass extracts, without killing cells.MethodsMethanolic extracts were characterized, and major compounds were identified by MS/MS analysis. The antibiofilm properties of the seagrass extracts were tested at sub-lethal concentrations by using microtiter plate adhesion assay. The performance of the most promising extract was further investigated in elegant bioreactors to reproduce mature biofilms both at the solid/liquid and the solid/air interfaces. Dispersion and bioluminescent assays were carried out to decipher the mode of action of the bioactive extract.ResultsIt was shown that up to 100 ppm of crude extracts did not adversely affect microbial growth, nor do they act as a carbon and energy source for the selected microorganisms. Seagrass extracts appear to be more effective in deterring microbial adhesion on hydrophobic surfaces than on hydrophilic. The results revealed that non-lethal concentrations of E. acoroides leaf extract: i) reduce bacterial and fungal coverage by 60.9 and 73.9%, respectively; ii) affect bacterial biofilm maturation and promote dispersion, up to 70%, in fungal biofilm; iii) increase luminescence in Vibrio harveyi by 25.8%. The characterization of methanolic extracts showed the unique profile of the E. acoroides leaf extract.ConclusionsE. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extract were found to exert an antibiofilm effect on C. albicans and E. coli biofilm in the first phase of biofilm genesis, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces. The leaf extract also affected the dispersion and maturation steps in C. albicans and E. coli respectively, suggesting an important role in cell signaling processes.

Morphodynamics of the Actin-Rich Cytoskeleton in Entamoeba histolytica.

Entamoeba histolytica is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in Entamoeba histolytica means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, E. histolytica has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in E. histolytica. We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by E. histolytica trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live E. histolytica cells; we observed the cortical cytoskeleton, stress fibers, “dot-like” structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.