Altered expression of CD1d molecules and lipid accumulation in the human hepatoma cell line HepG2 after iron loading.

Iron overload in the liver may occur in clinical conditions such as hemochromatosis and nonalcoholic steatohepatitis, and may lead to the deterioration of the normal liver architecture by mechanisms not well understood. Although a relationship between the expression of ICAM-1, and classical major histocompatibility complex (MHC) class I molecules, and iron overload has been reported, no relationship has been identified between iron overload and the expression of unconventional MHC class I molecules. Herein, we report that parameters of iron metabolism were regulated in a coordinated-fashion in a human hepatoma cell line (HepG2 cells) after iron loading, leading to increased cellular oxidative stress and growth retardation. Iron loading of HepG2 cells resulted in increased expression of Nor3.2-reactive CD1d molecules at the plasma membrane. Expression of classical MHC class I and II molecules, ICAM-1 and the epithelial CD8 ligand, gp180 was not significantly affected by iron. Considering that intracellular lipids regulate expression of CD1d at the cell surface, we examined parameters of lipid metabolism in iron-loaded HepG2 cells. Interestingly, increased expression of CD1d molecules by iron-loaded HepG2 cells was associated with increased phosphatidylserine expression in the outer leaflet of the plasma membrane and the presence of many intracellular lipid droplets. These data describe a new relationship between iron loading, lipid accumulation and altered expression of CD1d, an unconventional MHC class I molecule reported to monitor intracellular and plasma membrane lipid metabolism, in the human hepatoma cell line HepG2.This work was funded by the Inova Foundation for Medical Research/The American Portuguese Biomedical Research Fund (APBRF, USA). The authors would like to thank L. Mayer and K. Uchida for providing antibodies, A. do Vale, F. Pisarra and M.T. Silva for help and comments on TEM, and M. Santos and R. Hultcrantz for critical reading of the manuscript. We also thank M. de Sousa for mentoring this work. M.C. and C.F.P. were supported by a fellowship from Inova/APBRF. E.M.C. was partially supported by EU grant (QLG1-CT-1999–00665)

Osteosynthesis metal plate system for bone fixation using bicortical screws: numerical–experimental characterization

This study reports the numerical and experimental characterization of a standard immobilization system currently being used to treat simple oblique bone fractures of femoral diaphyses. The procedure focuses on the assessment of the mechanical behavior of a bone stabilized with a dynamic compression plate (DCP) in a neutralization function, associated to a lag screw, fastened with surgical screws. The non-linear behavior of cortical bone tissue was revealed through four-point bending tests, from which damage initiation and propagation occurred. Since screw loosening was visible during the loading process, damage parameters were measured experimentally in independent pull-out tests. A realistic numerical model of the DCP-femur setup was constructed, combining the evaluated damage parameters and contact pairs. A mixed-mode (I+II) trapezoidal damage law was employed to mimic the mechanical behavior of both the screw–bone interface and bone fractures. The numerical model replicated the global behavior observed experimentally, which was visible by the initial stiffness and the ability to preview the first loading peak, and bone crack satisfactorily.This research was funded by the Portuguese Foundation for Science and Technology (FCT), grant numbers SFRH/BD/143736/2019, UIDB/CVT/00772/2020, LA/P/0059/2020, UIDB/04033/2020, PTDC/EME-SIS/28225/2017, UID/EEA/04436/2019 and Laboratório Associado de Energia, Transportes e Aeronáutica (LAETA), grant number UID/EMS/50022/2020

PEDOT:PSS-coated polybenzimidazole electroconductive nanofibers for biomedical applications

Bioelectricity drives several processes in the human body. The development of new materials that can deliver electrical stimuli is gaining increasing attention in the field of tissue engineering. In this work, novel, highly electrically conductive nanofibers made of poly [2,20 - m-(phenylene)-5,50 -bibenzimidazole] (PBI) have been manufactured by electrospinning and then coated with cross-linked poly (3,4-ethylenedioxythiophene) doped with poly (styrene sulfonic acid) (PEDOT:PSS) by spin coating or dip coating. These scaffolds have been characterized by scanning electron microscopy (SEM) imaging and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The electrical conductivity was measured by the four-probe method at values of 28.3 S·m−1 for spin coated fibers and 147 S·m−1 for dip coated samples, which correspond, respectively, to an increase of about 105 and 106 times in relation to the electrical conductivity of PBI fibers. Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) cultured on the produced scaffolds for one week showed high viability, typical morphology and proliferative capacity, as demonstrated by calcein fluorescence staining, 40 ,6-diamidino-2-phenylindole (DAPI)/Phalloidin staining and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay. Therefore, all fiber samples demonstrated biocompatibility. Overall, our findings highlight the great potential of PEDOT:PSS-coated PBI electrospun scaffolds for a wide variety of biomedical applications, including their use as reliable in vitro models to study pathologies and the development of strategies for the regeneration of electroactive tissues or in the design of new electrodes for in vivo electrical stimulation protocols.info:eu-repo/semantics/publishedVersio

Gold nanoparticles functionalised with fast water exchanging Gd3+ chelates: linker effects on the relaxivity.

This is the accepted manuscript. The final version is available at http://dx.doi.org/10.1039/C4DT03210AThe relaxivity displayed by Gd(3+) chelates immobilized onto gold nanoparticles is the result of the complex interplay between the nanoparticle size, the water exchange rate and the chelate structure. In this work we study the effect of the length of ω-thioalkyl linkers, anchoring fast water exchanging Gd(3+) chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd(3+) chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(α-amino)propionate chelator were prepared and studied as potential CA for MRI. High relaxivities per chelate, of the order of magnitude 28-38 mM(-1) s(-1) (30 MHz, 25 °C), were attained thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. Fast local rotational motions of the immobilized chelates around connecting linkers (internal flexibility) still limit the attainable relaxivity. The degree of internal flexibility of the immobilized chelates seems not to be correlated with the length of the connecting linkers. Biodistribution and MRI studies in mice suggest that the in vivo behavior of the gold nanoparticles was determined mainly by size. Small nanoparticles (HD = 3.9 nm) undergo fast renal clearance and avoidance of the RES organs while larger nanoparticles (HD = 4.8 nm) undergo predominantly hepatobiliary excretion. High relaxivities, allied to chelate and nanoparticle stability and fast renal clearance in vivo suggest that functionalized gold nanoparticles hold great potential for further investigation as MRI contrast agents. This study contributes to a better understanding of the effect of linker length on the relaxivity of gold nanoparticles functionalized with Gd(3+) complexes. It is a relevant contribution towards "design rules" for nanostructures functionalized with Gd(3+) chelates as Contrast Agents for MRI and multimodal imaging.This work was financially supported by Fundação para a Ciência e a Tecnologia, Portugal: PhD grant SFRH/BD/63994/2009 to Miguel Ferreira and Sabbatical Grant SFRH/BSAB/1328/2013 to José Martins at Bath University, UK; and Rede Nacional de NMR (REDE/1517/RMN/2005) for the acquisition of the Varian VNMRS 600 NMR spectrometer in Coimbra. T.B.R. was supported by a Marie Curie Fellowship (FP/- PEOPLE-2009-IEF 254380) and an EMBO Fellowship (ALTF 1145-2009). Financial support from Ministerio de Ciencia e Innovación, Spain, projects SAF2011-23622 (S.C.) and CTQ2010-20960-C02-02 (P.L.-L.), and Comunidad de Madrid, Spain, project S2010/BMD-2349 (S.C. and P.L.-L), is also acknowledged. B. Mousavi and L. Helm acknowledge financial support by the Swiss National Science Foundation. This work was carried out in the frame of the COST D38 Action “Metal Based Systems for Molecular Imaging” and COST TD1004 Action “Theranostics Imaging and Therapy”

Valorization of Oleuropein via Tunable Acid-Promoted Methanolysis

The acid-promoted methanolysis of oleuropein was studied using a variety of homogeneous and heterogeneous acid catalysts. Exclusive cleavage of the acetal bond between the glucoside and the monoterpene subunits or further hydrolysis of the hydroxytyrosol ester and subsequent intramolecular rearrangement were observed upon identification of the most efficient catalyst and experimental conditions. Furthermore, selected conditions were tested using oleuropein under continuous flow and using a crude mixture extracted from olive leaves under batch. Formation of (−)-methyl elenolate was also observed in this study, which is a reported precursor for the synthesis of the antihypertensive drug (−)-ajmalicine.Centro de Investigación y Desarrollo en Ciencias Aplicada

Associating the Incest and Paternity Index to discriminate sexual aggression among close blood relatives

 A violência sexual no Brasil, além de um crime hediondo, é uma questão de saúde pública. Entre as crianças, parentes destacam-se como agressores. Quando um crime de natureza sexual resulta em gravidez, o exame de paternidade consiste no meio de prova mais fidedigno. Este exame consiste em verificar o compartilhamento de alelos entre o pretenso filho e o suposto pai, de modo a atribuir termos probabilísticos à transmissão dos alelos por parte do suspeito em relação a um homem qualquer na população, não relacionado, o que se denomina índice de paternidade. Porém, em casos cujo suposto pai possui vínculo biológico com a vítima, devem-se considerar alguns parâmetros matemáticos de modo a evitar a superestimativa dos valores atribuídos aos índices de paternidade. Torna-se preciso distinguir Alelos Idênticos por Descendência daqueles Idênticos por Estado. A partir de análise comparativa de 3 casos incestuosos foram efetuados cálculos de Índice de Paternidade da maneira usual, considerando o suspeito como não relacionado à vítima e considerando o suspeito como relacionado à vítima, atribuindo a este cálculo a denominação Índice de Incesto. O Índice de Paternidade Combinado foi superior ao Índice Combinado de Incesto em até 103 vezes, mostrando que as análises usuais podem superestimar os resultados nos exames de vínculo genético. Assim, propõe-se o uso desta metodologia matemática nos cenários incestuosos a fim de evitar a emissão de laudos equivocados ou estatisticamente sobrevalorizados.Sexual violence in Brazil, as well as being a heinous crime, is a public health issue. In the majority of cases of sexually abused children, close relatives are the most common aggressors. When a crime of a sexual nature results in pregnancy, a paternity test is the most reliable form of evidence. The purpose of this test is to verify the sharing of alleles between the child and alleged father, in order to assign probabilistic terms related to the transmission of alleles from the suspect in relation to an unrelated man in population—this is called the Paternity Index. However, in cases where the alleged father has a biological relationship with the victim, some mathematical parameters should be considered, in order to avoid overestimation of the values assigned to the indices of paternity. It becomes necessary to distinguish alleles identical by descent from those identical by state. From a comparative analysis of 3 incestuous cases, calculations of the Paternity Index were performed in the usual way, both considering the suspect as being unrelated to the victim, and considering the suspect as being related to the victim, and this calculation was named the Incest Index. The Combined Paternity Index was higher than the combined ratio of Incest by up to 103 times, showing that the usual analysis may overestimate the results in the exams of genetic link. Thus, the use of this mathematical methodology is proposed for both incestuous scenarios, in order to avoid issuing overrated or erroneous statistical reports of incest

Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications : scaffold design and its performance when seeded with goat bone marrow stromal cells

Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects. The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects

Aplicação da Calorimetria Exploratória Diferencial (DSC) para Determinação da Pureza de Fármacos

O trabalho aborda um estudo comparativo entre a técnica de Cromatografia a Líquido de Alta Eficiência e a técnica de Calorimetria Exploratória Diferencial, visando avaliar a aplicação desta para a determinação direta da pureza de fármacos candidatos a materiais de referência certificados

Resolvin D2 Attenuates Cardiovascular Damage in Angiotensin II-Induced Hypertension.

Background: Resolution of inflammation is orchestrated by specialized proresolving lipid mediators (SPMs), and this would be impaired in some cardiovascular diseases. Among SPMs, resolvins (Rv) have beneficial effects in cardiovascular pathologies, but little is known about their effect on cardiovascular damage in hypertension. Methods: Aorta, small mesenteric arteries, heart, and peritoneal macrophages were taken from C57BL/6J mice, infused or not with angiotensin II (AngII; 1.44 mg/kg/day, 14 days) in presence or absence of resolvin D2 (RvD2) (100 ng/mice, every second day) starting 1 day before or 7 days after AngII infusion. Results: Enzymes and receptors involved in SPMs biosynthesis and signaling were increased in aorta or heart from AngII-infused mice. We also observed a differential regulation of SPMs in heart from these mice. Preventive treatment with RvD2 partially avoided AngII-induced hypertension and protected the heart and large and small vessels against functional and structural alterations induced by AngII. RvD2 increased the availability of vasoprotective factors, modified SPMs profile, decreased cardiovascular fibrosis, and increased the infiltration of pro-resolving macrophages. When administered in hypertensive animals with established cardiovascular damage, RvD2 partially improved cardiovascular function and structure, decreased fibrosis, reduced the infiltration of neutrophils, and shifted macrophage phenotype toward a pro-resolving phenotype. Conclusions: There is a disbalance between proinflammatory and resolution mediators in hypertension. RvD2 protects cardiovascular function and structure when administered before and after the development of hypertension by modulating vascular factors, fibrosis and inflammation. Activating resolution mechanisms by treatment with RvD2 may represent a novel therapeutic strategy for the treatment of hypertensive cardiovascular disease.pre-print362 K