Acoustic detection of cell adhesion to a coated quartz crystal microbalance – implications for studying the biocompatibility of polymers

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D -lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.     

Evaluating Purifying Selection in the Mitochondrial DNA of Various Mammalian Species

Mitochondrial DNA (mtDNA), the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this selection is fundamental to evolutionary studies and to the identification of deleterious mutations.     

Anti‐HSV‐1 and HSV‐2 Flavonoids and a New Kaempferol Triglycoside from the Medicinal Plant Kalanchoe daigremontiana

Kalanchoe daigremontiana (Crassulaceae) is a medicinal plant native to Madagascar. The aim of this study was to investigate the flavonoid content of an aqueous leaf extract from K. daigremontiana (Kd), and assess its antiherpetic potential. The major flavonoid, kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 1 ), was isolated from the AcOEt fraction (Kd‐AC). The BuOH‐soluble fraction afforded quercetin 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 2 ) and the new kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside‐7‐O‐β‐d ‐glucopyranoside ( 3 ), named daigremontrioside. The crude extract, Kd‐AC fraction, flavonoids 1 and 2 were evaluated using acyclovir‐sensitive strains of HSV‐1 and HSV‐2. Kd‐AC was highly active against HSV‐1 (EC₅₀ = 0.97 μg/ml, SI > 206.1) and HSV‐2 (EC₅₀ = 0.72 μg/ml, SI > 277.7). Flavonoids 1 and 2 showed anti‐HSV‐1 (EC₅₀ = 7.4 μg/ml; SI > 27 and EC₅₀ = 5.8 μg/ml; SI > 8.6, respectively) and anti‐HSV‐2 (EC₅₀ = 9.0 μg/ml; SI > 22.2 and EC₅₀ = 36.2 μg/ml; SI > 5.5, respectively) activities, suggesting the contribution of additional substances to the antiviral activity.     

Age-Dependent Changes in Geometry,Tissue Composition and Mechanical Properties of Fetal to Adult Cryopreserved Human Heart Valves

There is limited information about age-specific structural and functional properties of human heart valves, while this information is key to the development and evaluation of living valve replacements for pediatric and adolescent patients. Here, we present an extended data set of structure-function properties of cryopreserved human pulmonary and aortic heart valves, providing age-specific information for living valve replacements. Tissue composition, morphology, mechanical properties, and maturation of leaflets from 16 pairs of structurally unaffected aortic and pulmonary valves of human donors (fetal-53 years) were analyzed. Interestingly, no major differences were observed between the aortic and pulmonary valves. Valve annulus and leaflet dimensions increase throughout life. The typical three-layered leaflet structure is present before birth, but becomes more distinct with age. After birth, cell numbers decrease rapidly, while remaining cells obtain a quiescent phenotype and reside in the ventricularis and spongiosa. With age and maturation–but more pronounced in aortic valves–the matrix shows an increasing amount of collagen and collagen cross-links and a reduction in glycosaminoglycans. These matrix changes correlate with increasing leaflet stiffness with age. Our data provide a new and comprehensive overview of the changes of structure-function properties of fetal to adult human semilunar heart valves that can be used to evaluate and optimize future therapies, such as tissue engineering of heart valves. Changing hemodynamic conditions with age can explain initial changes in matrix composition and consequent mechanical properties, but cannot explain the ongoing changes in valve dimensions and matrix composition at older age.     

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO₄ backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.     

Culture medium influence on growth,fatty acid,and pigment composition of <Emphasis Type="Italic">Choricystis minor</Emphasis> var. <Emphasis Type="Italic">minor</Emphasis>: a suitable microalga for biodiesel production

The purposes of this study were to assess the influence of culture medium on biomass production, fatty acid, and pigment composition of Choricystis minor var. minor and to evaluate the use of this microalga as a source of fatty raw material for biodiesel production. Cultures of C. minor var. minor were grown using WC (Wright’s cryptophyte) and BBM (Bold’s Basal medium) media. BBM medium produced more biomass (984.3 mg L^?1) compared to the WC medium (525.7 mg L^?1). Despite this result, WC medium produced a higher methyl ester yield for biodiesel production than the BBM medium (170.0 and 90.2 mg g^?1 of biomass, respectively). The average percentage of fatty acids obtained using the WC medium (17.0 %) was similar to soybean (18.0 %) and with similar biomass fatty acid profile. However, for pigment production, carotenoids and chlorophyll concentrations were twice as high when using the BBM medium.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.