Comparative chondrogenesis of human cells in a 3D integrated experimental/computational mechanobiology model

We present an integrated experimental–computational mechanobiology model of chondrogenesis. The response of human articular chondrocytes to culture medium perfusion, versus perfusion associated with cyclic pressurisation, versus non-perfused culture, was compared in a pellet culture model, and multiphysic computation was used to quantify oxygen transport and flow dynamics in the various culture conditions. At 2 weeks of culture, the measured cell metabolic activity and the matrix content in collagen type II and aggrecan were greatest in the perfused+ pressurised pellets. The main effects of perfusion alone, relative to static controls, were to suppress collagen type I and GAG contents, which were greatest in the non-perfused pellets. All pellets showed a peripheral layer of proliferating cells, which was thickest in the perfused pellets, and most pellets showed internal gradients in cell density and matrix composition. In perfused pellets, the computed lowest oxygen concentration was 0.075mM (7.5% tension), the maximal oxygen flux was 477.5 nmol/m2/s and the maximal fluid shear stress, acting on the pellet surface, was 1.8mPa (0.018 dyn/cm2). In the non-perfused pellets, the lowest oxygen concentration was 0.003mM (0.3% tension) and the maximal oxygen flux was 102.4nmol/m2/s.Alocal correlationwas observed, between the gradients in pellet properties obtained from histology, and the oxygen fields calculated with multiphysic simulation. Our results showup-regulation of hyalinematrix protein production by human chondrocytes in response to perfusion associated with cyclic pressurisation. These results could be favourably exploited in tissue engineering applications

Chitosan-Graft-Branched Polyethylenimine Copolymers: Influence of Degree of Grafting on Transfection Behavior

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity

Bioreducible Liposomes for Gene Delivery: From the Formulation to the Mechanism of Action

BACKGROUND: A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25:50:25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro. CONCLUSIONS/SIGNIFICANCE: The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors

Expression of Distal-less, dachshund, and optomotor blind in Neanthes arenaceodentata (Annelida, Nereididae) does not support homology of appendage-forming mechanisms across the Bilateria

The similarity in the genetic regulation of arthropod and vertebrate appendage formation has been interpreted as the product of a plesiomorphic gene network that was primitively involved in bilaterian appendage development and co-opted to build appendages (in modern phyla) that are not historically related as structures. Data from lophotrochozoans are needed to clarify the pervasiveness of plesiomorphic appendage forming mechanisms. We assayed the expression of three arthropod and vertebrate limb gene orthologs, Distal-less (Dll), dachshund (dac), and optomotor blind (omb), in direct-developing juveniles of the polychaete Neanthes arenaceodentata. Parapodial Dll expression marks premorphogenetic notopodia and neuropodia, becoming restricted to the bases of notopodial cirri and to ventral portions of neuropodia. In outgrowing cephalic appendages, Dll activity is primarily restricted to proximal domains. Dll expression is also prominent in the brain. dac expression occurs in the brain, nerve cord ganglia, a pair of pharyngeal ganglia, presumed interneurons linking a pair of segmental nerves, and in newly differentiating mesoderm. Domains of omb expression include the brain, nerve cord ganglia, one pair of anterior cirri, presumed precursors of dorsal musculature, and the same pharyngeal ganglia and presumed interneurons that express dac. Contrary to their roles in outgrowing arthropod and vertebrate appendages, Dll, dac, and omb lack comparable expression in Neanthes appendages, implying independent evolution of annelid appendage development. We infer that parapodia and arthropodia are not structurally or mechanistically homologous (but their primordia might be), that Dll’s ancestral bilaterian function was in sensory and central nervous system differentiation, and that locomotory appendages possibly evolved from sensory outgrowths

Opportunistic illnesses in Brazilian children with AIDS: results from two national cohort studies, 1983-2007

<p>Abstract</p> <p>Background</p> <p>HAART has significantly reduced AIDS-related morbidity in children. However, limited evidence is available from developing countries regarding patterns of opportunistic illnesses. We describe these events and their associated factors in children with AIDS in Brazil.</p> <p>Methods</p> <p>This study is based on two representative retrospective multi-center cohorts including a total 1,859 children with AIDS, infected via mother-to-child transmission (MTCT), between 1983-2002. Opportunistic illnesses were described and analyzed over time. The association of demographic, clinical and operational data with the occurrence of opportunistic diseases was assessed.</p> <p>Results</p> <p>In total, 1,218 (65.5%) had at least one event of an opportunistic disease. Variables significantly associated with occurrence of these events included: region of residence (OR 2.68-11.33, as compared to the Northern region), age < 1 year at diagnosis (OR 2.56, 95% CI 1.81-3.61, p < 0.001), and non-performance of MTCT prevention measures (OR 1.58, 95% CI 1.21-2.07, p < 0.001). Protective factors included year of HIV diagnosis in the HAART era (OR 0.34, 95% CI 0.15-0.76, p = 0.009) and ART use (OR 0.58, 95% CI 0.44-0.77, p < 0.001). In both periods bacterial infections represented the most common opportunistic events (58.6 vs. 34.7%; p < 0.001), followed by <it>Pneumocystis jirovecii </it>pneumonia (21.9 vs. 13.2%; p < 0.001), and bacterial meningitis/sepsis (16.8 vs. 7.4%; p < 0.001).</p> <p>Conclusions</p> <p>Despite the significant reduction in recent years, opportunistic illnesses are still common in Brazilian children with AIDS in the HAART era, especially bacterial diseases. The data reinforce the need for scaling up prevention of MTCT, early diagnosis of infection, and improvement of comprehensive pediatric care.</p

Socioeconomic position, lifestyle habits and biomarkers of epigenetic aging: A multi-cohort analysis

Differences in health status by socioeconomic position (SEP) tend to be more evident at older ages, suggesting the involvement of a biological mechanism responsive to the accumulation of deleterious exposures across the lifespan. DNA methylation (DNAm) has been proposed as a biomarker of biological aging that conserves memory of endogenous and exogenous stress during life. We examined the association of education level, as an indicator of SEP, and lifestyle-related variables with four biomarkers of age-dependent DNAm dysregulation: the total number of stochastic epigenetic mutations (SEMs) and three epigenetic clocks (Horvath, Hannum and Levine), in 18 cohorts spanning 12 countries. The four biological aging biomarkers were associated with education and different sets of risk factors independently, and the magnitude of the effects differed depending on the biomarker and the predictor. On average, the effect of low education on epigenetic aging was comparable with those of other lifestyle-related risk factors (obesity, alcohol intake), with the exception of smoking, which had a significantly stronger effect. Our study shows that low education is an independent predictor of accelerated biological (epigenetic) aging and that epigenetic clocks appear to be good candidates for disentangling the biological pathways underlying social inequalities in healthy aging and longevity

British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy Bacilli

<p>Eurasian red squirrels (Sciurus vulgaris) in the British Isles are the most recently discovered animal reservoir for the leprosy bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Initial data suggest that prevalence of leprosy infection is variable and often low in different squirrel populations. Nothing is known about the presence of leprosy bacilli in other wild squirrel species despite two others (Siberian chipmunk [Tamias sibiricus], and Thirteen-lined ground squirrel [Ictidomys tridecemlineatus]) having been reported to be susceptible to experimental infection with M. leprae. Rats, a food-source in some countries where human leprosy occurs, have been suggested as potential reservoirs for leprosy bacilli, but no evidence supporting this hypothesis is currently available. We screened 301 squirrel samples covering four species [96 Eurasian red squirrels, 67 Eastern gray squirrels (Sciurus carolinensis), 35 Siberian chipmunks, and 103 Pallas's squirrels (Callosciurus erythraeus)] from Europe and 72 Mexican white-throated woodrats (Neotoma albigula) for the presence of M. leprae and M. lepromatosis using validated PCR protocols. No DNA from leprosy bacilli was detected in any of the samples tested. Given our sample-size, the pathogen should have been detected if the prevalence and/or bacillary load in the populations investigated were similar to those found for British red squirrels.</p

Socioeconomic position, lifestyle habits and biomarkers of epigenetic aging: A multi-cohort analysis

Differences in health status by socioeconomic position (SEP) tend to be more evident at older ages, suggesting the involvement of a biological mechanism responsive to the accumulation of deleterious exposures across the lifespan. DNA methylation (DNAm) has been proposed as a biomarker of biological aging that conserves memory of endogenous and exogenous stress during life.We examined the association of education level, as an indicator of SEP, and lifestyle-related variables with four biomarkers of age-dependent DNAm dysregulation: the total number of stochastic epigenetic mutations (SEMs) and three epigenetic clocks (Horvath, Hannum and Levine), in 18 cohorts spanning 12 countries.The four biological aging biomarkers were associated with education and different sets of risk factors independently, and the magnitude of the effects differed depending on the biomarker and the predictor. On average, the effect of low education on epigenetic aging was comparable with those of other lifestyle-related risk factors (obesity, alcohol intake), with the exception of smoking, which had a significantly stronger effect.Our study shows that low education is an independent predictor of accelerated biological (epigenetic) aging and that epigenetic clocks appear to be good candidates for disentangling the biological pathways underlying social inequalities in healthy aging and longevity

Patterns, trends and sex differences in HIV/AIDS reported mortality in Latin American countries: 1996-2007

<p>Abstract</p> <p>Background</p> <p>International cohort studies have shown that antiretroviral treatment (ART) has improved survival of HIV-infected individuals. National population based studies of HIV mortality exist in industrialized settings but few have been presented from developing countries. Our objective was to investigate on a population basis, the regional situation regarding HIV mortality and trends in Latin America (LA) in the context of adoption of public ART policies and gender differences.</p> <p>Methods</p> <p>Cause of death data from vital statistics registries from 1996 to 2007 with "good" or "average" quality of mortality data were examined. Standardized mortality rates and Poisson regression models by country were developed and differences among countries assessed to identify patterns of HIV mortality over time occurring in Latin America.</p> <p>Results</p> <p>Standardized HIV mortality following the adoption of public ART policies was highest in Panama and El Salvador and lowest in Chile. During the study period, three overall patterns were identified in HIV mortality trends- following the adoption of the free ART public policies; a remarkable decrement, a remarkable increment and a slight increment. HIV mortality was consistently higher in males compared to females. Mean age of death attributable to HIV increased in the majority of countries over the study period.</p> <p>Conclusions</p> <p>Vital statistics registries provide valuable information on HIV mortality in LA. While the introduction of national policies for free ART provision has coincided with declines in population-level HIV mortality and increasing age of death in some countries, in others HIV mortality has increased. Barriers to effective ART implementation and uptake in the context of free ART public provision policies should be further investigated.</p