Properties of heart fibroblasts of adult rats in culture

Summary In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.     

Pierce into the Native Structure of Ata,a Trimeric Autotransporter of Acinetobacter baumannii ATCC 17978

International Journal of Peptide Research and Therapeutics - Acinetobacter baumannii is an important pathogen responsible for nosocomial infections worldwide. Trimeric autotransporters, the...     

Characteristics of Quantum Plasmonic Waves Guided by a Symmetric Metal–Gap–Dielectric Nano-system

Plasmonics - The guiding properties of a symmetric conductor–gap–dielectric system consists of a metal film symmetrically surrounded by media of two dielectrics, and is theoretically...     

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.     

Optimizing culture medium ingredients and micrografting devices can promote in vitro micrografting of cut roses on different rootstocks

Plant Cell, Tissue and Organ Culture (PCTOC) - Tissue culture and micrografting techniques are known as effective procedures to produce healthy rose rootstocks and high quality scions. In order to...     

Exploring dengue proteome to design an effective epitope-based vaccine against dengue virus

Dengue, a mosquito-borne disease, is caused by four known dengue serotypes. This infection causes a range of symptoms from a mild fever to a sever homorganic fever and death. It is a serious public health problem in subtropical and tropical countries. There is no specific vaccine currently available for clinical use and study on this issue is ongoing. In this study, bioinformatics approaches were used to predict antigenic, immunogenic, non-allergenic, and conserved B and T-cell epitopes as promising targets to design an effective peptide-based vaccine against dengue virus. Molecular docking analysis indicated the deep binding of the identified epitopes in the binding groove of the most popular human MHC I allele (human leukocyte antigens [HLA] A*0201). The final vaccine construct was created by conjugating the B and T-cell identified epitopes using proper linkers and adding an appropriate adjuvant at the N-terminal. The characteristics of the new subunit vaccine demonstrated that the epitope-based vaccine was antigenic, non-toxic, stable, and soluble. Other physicochemical properties of the new designed construct including isoelectric point value, aliphatic index, and grand average of hydropathicity were biologically considerable. Molecular docking of the engineered vaccine with Toll-like receptor 2 (TLR2) model revealed the hydrophobic interaction between the adjuvant and the ligand binding regions in the hydrophobic channel of TLR2. The study results indicated the high potential capability of the new multi-epitope vaccine to induce cellular and humoral immune responses against the dengue virus. Further experimental tests are required to investigate the immune protection capacity of the new vaccine construct in animal models.

Communicated by Ramaswamy H. Sarma     

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm²) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm² format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.     

Fabrication of a novel nanocomposite based on sol-gel process for hollow fiber-solid phase microextraction of aflatoxins: B1 and B2, in cereals combined with high performane liquid chromatography-diode array detection

The new pre-concentration technique, hollow fiber-solid phase microextraction based on carbon nanotube reinforced sol-gel and liquid chromatography-photodiode array detection was applied to determination of aflatoxins B(1), B(2) (AFB(1), AFB(2)) in rice, peanut and wheat samples. This research provides an overview of trends related to synthesis of solid phase microextraction (SPME) sorbnents that improves the assay of aflatoxins as the semi-polar compounds in several real samples. It mainly includes summary and a list of the results for a simple carbon nanotube reinforced sol-gel in-fiber device. This device was used for extraction, pre-concentration and determination of aflatoxins B1, B2 in real samples. In this technique carbon nanotube reinforced sol was prepared by the sol-gel method via the reaction of phenyl trimethoxysilane (PTMS) with a basic catalyst (tris hydroxymethyl aminomethan). The influences of microextraction parameters such as pH, ageing time, carbon nanotube contents, desorption conditions, desorption solvent and agitation speed were investigated. Optimal HPLC conditions were: C(18) reversed phase column for separation, water-acetonitril-methanol (35:10:55) as the mobile phase and maximum wavelength for detection was 370 nm. The method was evaluated statistically and under optimized conditions, the detection limits for the analytes were 0.074 and 0.061 ng/mL for B1 and B2 respectively. Limit of quantification for B1 and B2 was 0.1 ng/mL too (n=7). The precisions were in the range of 2.829-2.976% (n=3), and linear ranges were within 0.1 and 400 ng/mL. The method was successfully applied to the analysis of cereals (peanut, wheat, rice) with the relative recoveries from 47.43% to 106.83%.     

Characterization of adenosine receptor in its native environment: insights from molecular dynamics simulations of palmitoylated/glycosylated, membrane-integrated human A2B adenosine receptor

Selective A(2B) receptor antagonists and agonists may play a role in important pathologies such as gastrointestinal, neurological (i.e., Alzheimer disease and dementia) and hypersensitive disorders (i.e., asthma), diabetes, atherosclerosis, restenosis and cancer. Hence, it is regarded as a good target for the development of clinically useful agents. In this study, the effects of lipid bilayer, N-acetylglucosamine and S-palmitoyl on the dynamic behavior of A(2B)AR model is explored. Homology modeling, molecular docking and molecular dynamics simulations were performed to explore structural features of A(2B)AR in the presence of lipid bilayer. Twenty ns MD simulation was performed on the constructed model inserted in a hydrated lipid bilayer to examine stability of the best model. OSIP339391 as the most potent antagonist was docked in the active site of the model. Another MD simulation was performed on the ligand-protein complex to explore effects of the bilayer on this complex. A similar procedure was performed for the modified protein with N-acetylglucosamine and S-palmitoyl moieties in its structure. Phe173 and Glu174 located in EL2 were determined to be involved in ligand-receptor interactions through π-π stacking and hydrogen bonding. Asn254 was crucial to form hydrogen-bonding. The reliability of the model was assessed through docking using both commercial and synthetic antagonists and an r(2) of 0.70 was achieved. Our results show that molecular dynamics simulations of palmitoylated/glycosylated, membrane-integrated human A(2B)AR in its native environment is a possible approach and this model can be used for designing potent and selective A(2B)AR antagonists.     

A simple method for preparation of cultured cardiac fibroblasts from adult human ventricular tissue

Cardiac fibroblasts constitute greater than 90% of non-myocyte cells in the heart. Because they are responsible for synthesis of components of the extracellular matrix, growth factors and cytokines in the myocardium, they play an important role in normal and pathologic performance of the heart. An understanding of their biology requires in depth studies in a stable and reliable system in which the biological responses of cardiac fibroblasts to various stimuli can be determined. With the exception of few, all studies have been performed on cardiac fibroblasts obtained from rodent hearts. We present a method for isolation and subsequent culture of viable cardiac fibroblasts from ventricular tissue of adult human. This method allows rapid and reliable isolation and subsequent culture of cardiac fibroblasts from adult heart tissue without the need for cumbersome isolation techniques and complex nutrient-enriched and hormone-supplemented culture media for maintenance.