Correlations between the biochemistry and mechanical states of a sea-urchin ligament: a mutable collagenous structure

Mutable collagenous tissues (MCTs) of echinoderms can be regarded as intelligent and dynamic biomaterials, due to their ability to reversibly change their mechanical properties in a short physiological time span. This mutability phenomenon is nervously mediated and involves secreted factors of the specialized 'juxtaligamental' cells, which, when released into the extracellular matrix (ECM), change the cohesive forces between collagen fibrils. MCTs exist in nature in several forms, including some associated with echinoderm autotomy mechanisms. Since the molecular mechanism of mutability is still incompletely understood, the aim of this work was to provide a detailed biochemical analysis of a typical mutable collagenous structure and to identify possible correlations between its biochemistry and mechanical states. A better understanding of the mutability phenomena is likely to provide a unique opportunity to develop new concepts that can be applied in the design of dynamic biomaterial for tissue regeneration, leading to new strategies in regenerative medicine. The MCT model used was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was analyzed in different mechanical states, mimicking the mutability phenomenon. Spectroscopic techniques, namely Fourier transform infrared (FT-IR) and confocal Raman microscopy, were used to identify the specific molecular components that contribute to the CDL biochemical microenvironment and to investigate the possibility that remodelling/synthesis of new ECM components occurs during the mutability phenomenon by analogy with events during pregnancy in the uterine cervix of mammals (which also consists mainly of mechanically adaptable connective tissues). The results demonstrate that CDL ECM includes collagen with biochemical similarities to mammalian type I collagen, as well as sulphated glycosaminoglycans (GAGs). CDL mutability seems to involve a molecular rearrangement of the ECM, without synthesis of new ECM components. Although there were no significant biochemical differences between CDLs in the various mechanical states were observed. However, subtle adjustments in tissue hydration seemed to occur, particularly during stiffening.     

Effect of matrigel on function and morphology of human endometrial epithelial cell in vitro

INTRODUCTION: The importance of extra cellular matrix (ECM) in development and function of different cells has been reported but little is known about its role in human endometrial epithelial cells. The aim of the present study was to examine effects of artificial ECM (Matrigel) and progesterone on the function and morphology of human endometrial epithelial cells in vitro. METHODS: Endometrial samples were removed, with informed patients consent and Ethics Committee approval, from 17 previously fertile women undergoing total abdominal hysterectomy. The tissue was dissociated and centrifuged to provide an epithelial rich suspension which was cultured either on plastic or seeded into Matrigel to produce polarized cells and then supplemented with or without progesterone (10(-6) M). The amount of nucleic acid content of the cells in both in vitro model systems was examined by DNA, RNA extraction methods. The DNA and RNA content were later measured by spectrophotometry. RESULTS: The amount of total RNA in cells grown on Matrigel (23 +/- 1.5 pg/cell) was more than double that in cells grown on pllastic (9.1 +/- 1.4 pg/cell). Cells cultured on both in vitro model systems had RNA induced by steroid hormones, but the extent of induction was greater in cells grown on Matrigel (30 +/- 2 pg/cell) than those on plastic (12 +/- 1.9 pg/cell). Cells cultured on Matrigel were differentiated and became polarized but cells grown on plastic proliferated to full confluency. Cells grown on Matrigel with progesterone supplementation were highly polarized, euchromatic and had greater mitochondria and accumulation of glycogen, when compared to unsupplemented cultures. CONCLUSION: These results suggest that ECM plays an important role in gene expression, polarization and differentiation of human endometrial epithelial cells in vitro. Endometrial cells grown on ECM responded to steroid hormone in a manner to that reported in endometrial cells in vivo.     

Improvement of thermo-mechanical properties of basalt fiber using heat resistant polymeric coatings

Heat resistant coatings of textile materials are required so that they can fulfill the high security demand in the case of resistance to fire and improve thermo-mechanical properties. These coatings also enhance the interface properties of textiles in the composites. Liquid phase coatings, based on polysilazane and polysiloxane polymers were deposited onto the basalt fiber (BF) yarn using impregnation coating method. Tensile testing under thermal stress was conducted to examine the thermo-mechanical properties of both coated and uncoated yarns. The thermo-mechanical study indicated that the heat resistant coatings enhanced 40–70 % tensile strength and 25–40 % stiffness of original BF yarns up to 400 °C. BF yarn retained 65–90 % of its tensile strength at 500 °C due to coatings, whereas the uncoated BF yarn lost 85 % strength at this temperature. Thermo gravimetric analysis (TGA) showed that the coatings have good thermal stability. In addition, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, optical microscopy and fourier transform infrared (FTIR) spectroscopy analyses were executed in order to evaluate the surface microstructure as well as surface chemical compositions of the BF yarns.     

SiO2-kaolinite affecting the surface properties of ternary poly(vinyl chloride)/silica/kaolinite nanocomposites

The products from the dispersion of nanoscale particulates such as the layered clays or the spherical inorganic minerals within the polymeric matrices are called polymeric nanocomposites. In this paper, we prepared poly(vinyl chloride) (PVC) based nanocomposites containing SiO₂-kaolinite by melt compounding. The influence of SiO₂-kaolinite on the surface properties of PVC was investigated by the use of various surface analysis techniques including a ttenuated total reflectance spectroscopy (ATR), wide angle X-ray diffractometry (WAXD), atomic force microscopy (AFM), scanning electron microscopy (SEM), electron dispersive X-ray spectrometer (EDX), contact angle measurement (CAM), and reflectance spectroscopy (RS). ATR spectroscopy showed possible interaction between layered kaolinite and PVC at surface. Microscopic methods illustrated an increased surface roughness compared to the pure PVC. Contact angle measurements of the resultant PVC nanocomposites demonstrated that the wettability of substrates depends on the surface interactions between kaolinite layers and PVC matrix. Optical properties of nanocomposite films were finally measured by the aid of reflectance spectrophotometer. It can be seen from optical studies that reflectance values were increased after incorporation of SiO₂-kaolinite in nanocomposite.     

Ultrastructural changes in tubers of potato (Solanum tuberosum L.) affected by rust spot

Summary The medullary tissues of potato tubers affected by rust spot were embedded in resin, sectioned and stained for optical and electron microscopy. Under the optical microscope, the damaged zones were seen to have thickened and suberized cell walls, with cells partially or totally lacking starch. These features were confirmed by electron microscopy which revealed in relief the cell wall deformation and the characteristic presence, in the vacuole, of osmiophilic granules with a spongy appearance attached to the tonoplast, probable catabolic products of a disturbed metabolism. Separation and degeneration of the plasmalemma and the death of the cells were observed in tissues in the final stages of damage.     

Fluorescence microscopy investigations of ligand propagation and accessibility under adherent cells

Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex?, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex? (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.     

Soy protein films with the hydrophobic surface created through non-covalent interactions

Different plasticizers (P) such as glycerol, 1,2 propanediol, trimethylol propane, thiodiglycol, and formamide (30% by weight w.r.t. soy protein) were used to prepare soy protein films (coded as SP) by compression molding at 140 °C and 20 MPa. The SP films were immersed in 0.5% (w/v) benzilic acid solution for 26 h to get arylated soy protein films (SP-B) with the evolution of CO₂, leading to the formation of diphenylhydroxymethane (DPHM) on the surface. Lotus leaf like structure, on the surface of SP-B film, with high hydrophobicity has been created, supported by the existence of nano/micro spheres. The SP-B and SP films were characterized by Fourier transformed infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, tensile testing, optical transmittance, contact angle, and biodegradation experiments. The mechanical properties, transmittance, and contact angle of SP-B films increased significantly, compared with SP. The SP-B films exhibited high water resistance in the range of 67–82%. These results indicated attractive interactions (often termed as π-interactions) of DPHM aromatic groups took place in the SP-B films resulting in the formation of nanospheres or microspheres. This work provided a good way for increasing the surface hydrophobicity and the mechanical properties of the protein materials through non-covalent interactions.     

Pattern of laminin expression during kidney morphogenesis in Balb/c mice

Basement membrane of glomerular mesangium (BMG) is one of important components which play a key role to support of the capillary loops in a renal glomerulus and completeness of BMG due to interaction of ureteric bud and metanephric mesenchyme during glomerulogenesis. As laminin contribute in extra cellular matrix and especially in basement membrane, the aim of the present study was to demonstrate the distribution of this molecule so, in this investigation specific antibody against laminin have been used in light microscopy to study development of BMG of fetal and postnatal mouse glomerular mesangium. Female inbred Balb/c mice were selected and were kept under normal condition and finding vaginal plug was assumed as day zero of pregnancy. Two pregnant mice were sacrificed by cervical dislocation in one of gestational days 13-18, respectively and their fetuses were fixed, serially sectioned and by using antibody against laminin in BMG were carried out. The same process was used for kidneys preparation at 15 postnatal days. Present data revealed that laminin showed weak reaction on day 14 of gestation. The amount of laminin increased continuously until next days of fetal life and primary of 10 days postnatal in BMG. After this period, laminin reaction did not show significant change in newborns. These data indicate that laminin appears just during the glomerulogenesis and because of continuity with vasculature which is required for Extra Cellular Matrix (ECM) and glomerular endothelial cell differentiation, laminin, is the one of major structural proteins in BMG.     

Reporter Dyes Demonstrate Functional Expression of Multidrug Resistance Proteins in the Marine Flatworm Macrostomum lignano: The Sponge-Derived Dye Ageladine A Is Not a Substrate of These Transporters

The marine plathyhelminth Macrostomum lignano was recently isolated from Adriatic shore sediments where it experiences a wide variety of environmental challenges, ranging from hypoxia and reoxygenation, feeding on toxic algae, to exposure to anthropogenic contaminants. As multidrug resistance transporters constitute the first line of defense against toxins and toxicants we have studied the presence of such transporters in M. lignano in living animals by applying optical methods and pharmacological inhibitors that had been developed for mammalian cells. Application of the MDR1 inhibitor Verapamil or of the MRP1 inhibitors MK571 or Probenecid increased the intracellular fluorescence of the reporter dyes Fura-2 am, Calcein am, Fluo-3 am in the worms, but did not affect their staining with the dyes Rhodamine B, CMFDA or Ageladine A. The marine sponge alkaloid Ageladine A remained intracellularly trapped for several days in the worms, suggesting that it does not serve as substrate of multidrug resistance exporters. In addition, Ageladine A did not affect multidrug resistance-associated protein (MRP)-mediated dye export from M. lignano or the MRP1-mediated glutathione (GSH) export from cultured rat brain astrocytes. The data obtained demonstrate that life-imaging is a useful tool to address physiological drug export from intact marine transparent flatworms by using multiphoton scanning microscopy.     

Induction of apoptosis and non-apoptosis in human breast cancer cell line (MCF-7) by cisplatin and caffeine

Background: Molecular targeted therapy by different cell death inducers are recently considered in cancer therapy. The aim of this study was to compare the effect of cisplatin and inositol trisphosphate kinase inhibitor (caffeine) on human breast cancer cell line (MCF-7). The pattern of cell death in MCF-7 cells following the exposure to cisplatin and caffeine in individual and combination forms was characterized. Methods: MCF-7cells at late exponential phase were divided into two groups: control and experimental groups. Experimental group was exposed to cisplatin, caffeine and combination of them and control group was treated by vehicle. Forty-eight hours after incubation, floating and attached cells were collected separately. Flowcytometry analysis and electron microscopy were carried out on both attached and floating cells. Results: Two types of apoptotic and non-apoptotic cells were observed in the floating cells as well as in sub G1 cells of both experimental and control groups by electron microscopy. Both early and late stages of apoptosis were characterized and the attached cells remained unaffected. Conclusion: Although two different forms of cell death (apoptosis and non-apoptosis) were appeared in MCF-7 following exposure to cisplatin and caffeine, apoptosis was the major mechanism of cell death. The combination form of anti-cancer drugs with different mechanisms could decrease the dosage of employed anti-cancer drugs.Key Words: Cisplatin, Caffeine, Apoptosis     

Microstructure and crystallinity of polyolefins oriented via solid-state stretching at an elevated temperature

Different polyolefines (low density polyethylene (LDPE), linear low density polyethylene (LLDPE), high density polyethylene (HDPE) and isotactic polypropylene (iPP)) were oriented via solid-state stretching at an elevated temperature. In order to investigate orientation-induced changes in microstructure and crystallinity, optical microscopy (OM), scanning electron microscopy (SEM), wide angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC) were employed. To quantify the degree of molecular orientation WAXS data were used to calculate Herman’s orientation function (f _c ); the results reinforce the morphological picture obtained from OM and SEM confirming Peterlin’s molecular model of drawing. Furthermore, orientation-induced changes in the crystalline phase, especially in its volume and perfection, were observed by DSC and WAXS. Comparison between these data showed that the biggest change in the degree of crystallinity was achieved during the transformation of the initial lamellar into fibrillar structure. After completion of this transformation further orientation, which occurs through plastic deformation of fibre structure, introduces only minor changes in crystallinity. The overall orientation-induced behaviour hasn’t been considerably influenced by the structural differences amongst polyolefins.     

Fibroblast adhesion on unidirectional polymeric nanofilms

Nanotextured polymeric surfaces with inclined rods reveal highly anisotropic properties concerning wetting and adhesion. In this work, we report on the interaction of fibroblast cells with these highly anisotropic materials. The authors quantified removal of adherent cells from such surfaces by a laminar flow. The critical shear force needed for cell removal from the surface depends on the inclination direction. Based on electron microscopy cross sections we deduce that interactions of cellular filopodia extending into the nanotextured surface are causing the direction depending removal.     

A 3D-Printed Polycaprolactone/Marine Collagen Scaffold Reinforced with Carbonated Hydroxyapatite from Fish Bones for Bone Regeneration

In bone tissue regeneration, extracellular matrix (ECM) and bioceramics are important factors, because of their osteogenic potential and cell–matrix interactions. Surface modifications with hydrophilic material including proteins show significant potential in tissue engineering applications, because scaffolds are generally fabricated using synthetic polymers and bioceramics. In the present study, carbonated hydroxyapatite (CHA) and marine atelocollagen (MC) were extracted from the bones and skins, respectively, of Paralichthys olivaceus. The extracted CHA was characterized using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis, while MC was characterized using FTIR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The scaffolds consisting of polycaprolactone (PCL), and different compositions of CHA (2.5%, 5%, and 10%) were fabricated using a three-axis plotting system and coated with 2% MC. Then, the MC3T3-E1 cells were seeded on the scaffolds to evaluate the osteogenic differentiation in vitro, and in vivo calvarial implantation of the scaffolds was performed to study bone tissue regeneration. The results of mineralization confirmed that the MC/PCL, 2.5% CHA/MC/PCL, 5% CHA/MC/PCL, and 10% CHA/MC/PCL scaffolds increased osteogenic differentiation by 302%, 858%, 970%, and 1044%, respectively, compared with pure PCL scaffolds. Consequently, these results suggest that CHA and MC obtained from byproducts of P. olivaceus are superior alternatives for land animal-derived substances.     

Electrospinning and mechanical properties of polymeric fibers using a novel gap-spinning collector

Tissue engineering is an interdisciplinary field which combines the basic principles of life sciences and engineering. One promising idea is the combination of scaffolds and living cells in order to produce new functional tissue. The scaffolds play the role of a microenvironment that guides the cells towards tissue formation and regeneration. One of the most frequently used techniques to produce scaffolds is electrospinning. Tissue engineered constructs have to exhibit physiological and mechanical properties comparable to the native tissue they are intended to replace. To create polymeric fibers with controlled orientation, a cylindrical collector that rotates at a certain speed could be used, creating fibers that run longitudinally. The process of gap-spinning enables the production of specifically aligned fibers. Aim of this study was to develop a novel setup capable of producing multilayered structures with controlled fiber angle. The structural, morphological and mechanical characteristics of the fibers were accessed using scanning electron microscopy and uniaxial tensile tests. Longer pre-stretching led to thinner (in the sub-micron scale), more brittle and less elastic fibers. In a nutshell, the results indicated that fiber mats of desired orientation, fiber diameter and mechanical properties could be produced by controlled gap-spinning with a translational collector.     

Investigation of Host Responses of Different Potato Genotypes at Tissue, Cellular and Subcellular Levels After Infection with Phytophthora infestans

In histological and cytological investigations, the infection process of Phytophthora infestans, the late blight pathogen, was comparatively studied in several potato cultivars and somatic hybrid genotypes and their parents using fluorescence microscopy and electron microscopy methods. The results showed that germination of zoospores of P. infestans and frequency of invading by infection hyphae did not differ among the cultivar-pathogen interactions, but, extension of hyphae in host cells markedly differed among these genotypes. In the susceptible genotypes the pathogen grew rapidly inter- and intracellularly, 12 h after inoculation (hai), and some digital like haustoria were formed and the cytoplasm of the host cells became disorganized. In the resistance genotypes, the pathogen was restricted to the site of initial penetration, although some hyphae could penetrate the epidermal cell, however, the host cells produced resistance responses, such as formed wall appositions when in contact with hyphae, and no haustoria like structures were found. In the somatic hybride genotypes, the host response was different according to their parents as shown by transmission electron microscopy. In the hybrid genotype 1508/2, like in the wild species S. bulbocastanum, no hyphae were found in host cells. In the other genotypes, hyphae of P. infestans spread intercellularly and formed haustoria, but the cytoplasm of hyphae and haustoria was disorganized and host cell resistant responses often appeared, such as, host cells were disorganized and necrotic and cell wall apposition were observed.     

CLSM study of layers in laminated dough: Roll out of layers and elastic recoil

It has been thought that all fat and dough layers in laminated dough had the same thickness at the end of lamination. Moreover dough rheology (taking into account elastic recoil) is too complex to generalize calculation of the final thickness of the dough after lamination. Confocal laser scanning microscopy can differentiate the fat from the dough when combined with fluorophore staining (Nile Red and Alexa), and an algorithm was applied to quantify their respective thicknesses. When fat consistency was close to that of dough, the median thickness of fat was fairly close to theoretical values. Successive sheeting steps resulted in homogenization of layer thicknesses. The surface area of fat in relation to paste was greater than expected, especially for low numbers of sheeting steps, and also for samples with higher numbers of sheeting steps, presenting an exceptionally thick layer of fat (including that produced by back flow). Elastic recoil was between 20 and 90% higher than the targeted roll out; no significant effect of a harder fat consistency was found. Elastic recoil was less when the amount of fat in the sample was greater.     

Effect of growth parameters on structural and optical properties of CdS nanowires prepared by polymer controlled solvothermal route

Cadmium sulfide nanowires were successfully obtained via a poly (ethylene glycol)-assisted solvothermal route. In this procedure, cadmium nitrate and thiourea were used as Cd and S sources, respectively, and polyethylene glycol 400 was used as an inducing soft template to control the one-dimensional growth of CdS nanostructures. The effects of different growth parameters in the solvothermal process such as type of the solvents, reaction time, and temperature on the morphology, structural and optical properties of the products were investigated. The provided structures were characterized by X-ray diffractometery, scanning electron microscopy, and UV-visible absorption spectroscopy. The results show that the as-prepared samples have hexagonal phase and grow into long nanowire shape with increasing the reaction time, temperature and volume ratios of ethylenediamine (en) to H₂O. Uniform sized nanowires with the average diameter of 75 nm and the average length of 2.5 µm were obtained using ethylenediamine solvent at 170 °C for 3 days.     

Various nano-silica particles affecting dyeability of poly(ethylene terephthalate)/silica nanocomposite films

Poly(ethylene terephthalate) [PET] based nanocomposites containing three differently modified silica particles were prepared by melt compounding. The influence of type of nano-silica on dispersibility, thermal and dyeing properties of the resultant nanocomposite was investigated by various analytic techniques, namely, polarized optical microscopy (POM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), reflectance spectroscopy (RS), and light fastness. Optical microscopy images illustrated that nano-silica particles tended to increase the number of spherulites in the PET matrix which were dependent on nano-silica type and content. Thermal studies of the resultant nanocomposites showed a slight decrease in the melting temperature compared to a pristine PET. Silica nanocomposites were finally dyed with a disperse dye and their reflectances were determined by the aid of reflectance spectrophotometer. Such reflectances were converted to the corresponding color coordinate values which are indicative of dyeability of such nanocomposites.     

菠萝蜜叶绿素缺失突变体幼苗茎的解剖结构

木本植物茎的发育在木材形成中具有重要意义。以菠萝蜜叶绿素缺失突变体幼苗（Chlorophyll deficient mutant, CDM）为研究材料,选取正常幼苗作为对照（Control check, CK）,观察并测量其株高和基部茎粗。使用石蜡切片法和光学显微技术对茎各节间进行观察,测量木质部、韧皮部、表皮和周皮的厚度,比较CDM与CK之间的差异。结果显示,CDM的株高和基部茎粗的增长受到一定的抑制,各节间木质部、韧皮部以及周皮的厚度均减小;相比次生木质部,次生韧皮部的抑制作用更显著。