Establishment of a Brazilian Line of Human Embryonic Stem Cells in Defined Medium: Implications for Cell Therapy in an Ethnically Diverse Population

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research, and a potential source of different tissues for transplantation. However, one important challenge for the clinical use of these cells is the issue of immunocompatibility, which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population, named BR-I, in commercial defined medium. In contrast to the other hES cell lines established in defined medium, BR-I maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge, this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-I and another 22 hES cell lines established elsewhere with those of the Brazilian population, finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture.

Comparing methods for sampling large- and medium-sized mammals: camera traps and track plots

Activities involving fauna monitoring are usually limited by the lack of resources; therefore, the choice of a proper and efficient methodology is fundamental to maximize the cost-benefit ratio. Both direct and indirect methods can be used to survey mammals, but the latter are preferred due to the difficulty to come in sight of and/or to capture the individuals, besides being cheaper. We compared the performance of two methods to survey medium and large-sized mammal: track plot recording and camera trapping, and their costs were assessed. At Jatai Ecological Station (S21 degrees 31`15 ``- W47 degrees 34`42 ``-Brazil) we installed ten camera traps along a dirt road directly in front of ten track plots, and monitored them for 10 days. We cleaned the plots, adjusted the cameras, and noted down the recorded species daily. Records taken by both methods showed they sample the local richness in different ways (Wilcoxon, T=231; p;;0.01). The track plot method performed better on registering individuals whereas camera trapping provided records which permitted more accurate species identification. The type of infra-red sensor camera used showed a strong bias towards individual body mass (R(2)=0.70; p=0.017), and the variable expenses of this method in a 10-day survey were estimated about 2.04 times higher compared to track plot method; however, in a long run camera trapping becomes cheaper than track plot recording. Concluding, track plot recording is good enough for quick surveys under a limited budget, and camera trapping is best for precise species identification and the investigation of species details, performing better for large animals. When used together, these methods can be complementary.

Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes

The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simulate the cyclic characteristics of the daily melatonin profile, isolated rat adipocytes were exposed in a circadian-like pattern to melatonin added to the incubating medium for 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day) combined or not with Ins. This intermittent incubation was interrupted when four and a half 24-hr cycles were fulfilled. At the end, either during the induced night (melatonin present) or the induced day (melatonin absent), the rates of lipolysis and D-[U-(14)C]-glucose incorporation into lipids were estimated, in addition to the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and clock gene (Bmal-1b, Clock, Per-1 and Cry-1) mRNA expression. The leptin release was also measured. During the induced night, the following effects were observed: an increase in the mRNA expression of Clock, Per-1 and FAS; a rise in lipogenic response and leptin secretion; and a decrease in the lipolytic activity. The intermittent exposure of adipocytes to melatonin temporally and rhythmically synchronized their metabolic and hormonal function in a circadian fashion, mimicking what is observed in vivo in animals during the daily light-dark cycle. Therefore, this work helps to clarify the physiological relevance of the circadian pattern of melatonin secretion and its interactions with Ins, contributing to a better understanding of the adipocyte biology.

Effect of medium/omega-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

Lipid emulsion (LE) containing medium/omega-6 long chain triglyceride-based emulsion (MCT/omega-6 LCT LE) has been recommended in the place of omega-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/omega-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/omega-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/omega-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/omega-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription.

Culture medium for isolating chitinolytic bacteria from seawater and plankton

Chitin degradation is a key step in the cycling of nutrients in marine ecosystems and chitinolytic bacteria are the primary agents of this process. Chitinases, produced by bacteria, have potential applications in agriculture, medicine and in a wide range of biotechnological processes. We utilized a simple, rapid and cost-effective method of colloidal chitin preparation and a culture medium, in which colloidal chitin is the sole carbon source for the purpose of counting and isolating chitinolytic bacteria from seawater and plankton. This culture medium could be useful to isolate bacteria with the ability to produce extracellular chitinases for biotechnological applications.

Catalytic hydrodechlorination of chlorobenzene over supported palladium catalyst in buffered medium

The catalytic hydrodechlorination (HDC) reaction, which is an attractive abatement process for chlorinated organic wastes, was studied over a magnetically recoverable supported Pd(0) catalyst. We investigated the most favorable reaction conditions under which to obtain the highest substrate conversion rates while preserving the catalyst properties and morphology. Sodium hydroxide, triethylamine and buffered solutions were used as proton scavengers in the HDC of chlorobenzene under mild conditions. It was observed that sodium hydroxide caused corrosion of the silica support, triethylamine in 2-propanol preserved the morphology of the catalyst which could be recycled for up to five successive H DC reactions, and aqueous buffer solutions preserved the catalyst morphology and the catalytic activity for up to four successive HDC reactions. The use of buffer solutions to neutralize the HCl formed during the HDC reaction is an interesting, less aggressive, alternative approach to HDC reactions. (C) 2010 Elsevier B.V. All rights reserved.

CLUSTER MODEL FOR REACTIONS INDUCED BY WEAKLY BOUND AND/OR EXOTIC HALO NUCLEI WITH MEDIUM-MASS TARGETS

An experimental overview of reactions induced by the stable, but weakly-bound nuclei (6)Li, (7)Li and (9)Be, and by the exotic, halo nuclei (6)He, (8)B, (11)Be and (17)F On medium-mass targets, such as (58)Ni, (59)Co or (64)Zn, is presented. Existing data on elastic scattering, total reaction cross sections, fusion, breakup and transfer channels are discussed in the framework of a CDCC approach taking into account the breakup degree of freedom.

Reaction mechanisms for weakly-bound, stable nuclei and unstable, halo nuclei on medium-mass targets

An experimental overview of reactions induced by the stable, but weakly-bound nuclei (6)Li, (7)Li and (9)Be, and by the exotic, halo nuclei (6)He, (8)B, (11)Be and (17)F on medium-mass targets, such as (58)Ni, (59)Co or (64)Zn, is presented. Existing data on elastic scattering, total reaction cross sections, fusion processes, breakup and transfer channels are discussed in the framework of a CDCC approach taking into account the breakup degree of freedom.

Chromium ions phytoaccumulation by three floating aquatic macrophytes from a nutrient medium

In the present work, the trivalent and hexavalent chromium phytoaccumulation by three living free floating aquatic macrophytes Salvinia auriculata, Pistia stratiotes, and Eicchornia crassipes was investigated in greenhouse. These plants were grown in hydroponic solutions supplied with non-toxic Cr3+ and Cr6+ chromium concentrations, performing six collections of nutrient media and plants in time from a batch system. The total chromium concentrations into Cr-doped hydroponic media and dry roots and aerial parts were assayed, by using the Synchrotron radiation X-ray fluorescence technique. The aquatic plant-based chromium removal data were described by using a nonstructural kinetic model, obtaining different bioaccumulation rate, ranging from 0.015 to 0.837 1 mg(-1) d(-1). The Cr3+ removal efficiency was about 90%, 50%, and 90% for the E. crassipes, P. stratiotes, and S. auriculata, respectively; while it was rather different for Cr6+ one, with values about 50%, 70%, and 90% for the E. crassipes, P. stratiotes, and S. auriculata.

Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent

The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at -20 degrees C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human P-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully. (c) 2007 Elsevier B.V. All rights reserved.

On the motion under focal attraction in a rotating medium

New results are established here on the phase portraits and bifurcations of the kinematic model in system (1), first presented by H.K. Wilson in [3], and by him attributed to L. Markus (unpublished). A new, self-sufficient, study which extends that of [3] and allows an essential conclusion for the applicability of the model is reported here.

Peroxymonocarbonate and Carbonate Radical Displace the Hydroxyl-like Oxidant in the Sod1 Peroxidase Activity under Physiological Conditions

Despite being one of the most important antioxidant defenses, Cu,Zn-superoxide dismutase (Sod1) has been frequently associated with harmful effects, including neurotoxicity. This toxicity has been attributed to immature forms of Sod1 and extraneous catalytic activities. Among these, the ability of Sod1 to function as a peroxidase may be particularly relevant because it is increased in bicarbonate buffer and produces the reactive carbonate radical. Despite many studies, how this radical forms remains unknown. To address this question, we systematically studied hSod1 peroxidase activity in the presence of nitrite, formate, and bicarbonate-carbon dioxide. Kinetic analyses of hydrogen peroxide consumption and of nitrite, formate, and bicarbonate-carbon dioxide oxidation showed that the Sod1-bound hydroxyl-like oxidant functions in the presence of nitrite and formate. In the presence of bicarbonate-carbon dioxide, this oxidant is replaced by peroxymonocarbonate, which is then reduced to the carbonate radical. Peroxymonocarbonate intermediacy was evidenced by (13)C NMR experiments showing line broadening of its peak in the presence of Zn,ZnSod1. In agreement, peroxymonocarbonate was docked into the hSod1 active site, where it interacted with the conserved Arg(143). Also, a reaction between peroxymonocarbonate and Cu(I)Sod1 was demonstrated by stopped-flow experiments. Kinetic simulations indicated that peroxymonocarbonate is produced during Sod1 turnover and not in bulk solution. In the presence of bicarbonate-carbon dioxide, sustained hSod1-mediated oxidations occurred with low steady-state concentrations of hydrogen peroxide (4-10 mu M). Thus, carbonate radical formation through peroxymonocarbonate may be a key event in Sod1-induced toxicity.

Radical acetylation of 2`-deoxyguanosine and L-histidine coupled to the reaction of diacetyl with peroxynitrite in aerated medium

Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant (k(2) = 1.0 X 10(4) M-1 s(-1)) when compared with monocarbonyl substrates (k(2) < 10(3) M-1 s(-1)). Phosphate ions (100-500 MM) do not affect the rate of spontaneous peroxynitrite decay, but the H2PO4- anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum (a(N) = a(H) = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either L-histidine or 2`-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS"". These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.

New material for orthopedic implants: Electrochemical study of nickel free P558 stainless steel in minimum essential medium

Nickel, a component of stainless steels (SS) applied in orthopedic implants may cause allergic processes in human tissues P558 nickel free SS was studied to verify its viability as a substitute for stainless steel containing nickel Its performance is compared to ISO 5832-9 and F138 most used nowadays grades in implants fabrications, in minimum essential medium. MEM, at 37 degrees C. Potentiodynamic polarization curves, electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and ""in vitro"" cytotoxicity were used as techniques. From the electrochemical point of view P558 SS is comparable to ISO 5832-9 SS in MEM It remains passivated until the transpassivation potential, above which generalized corrosion occurs F138 presents pitting corrosion at 370 mV/SCE. The cytotoxicity results showed that P558. ISO 5832-9 and F138 do not present cytotoxic character Therefore, these results suggest that P558 SS can be applied in orthopedic implants (C) 2010 Elsevier BV All rights reserved

EVALUATION OF METAL TOXICITY BY A MODIFIED METHOD BASED ON THE FUNGUS GERRONEMA VIRIDILUCENS BIOLUMINESCENCE IN AGAR MEDIUM

Metal cation toxicity to basidiomycete fungi is poorly understood, despite its well-known importance in terrestrial ecosystems. Moreover, there is no reported methodology for the routine evaluation of metal toxicity to basidiomycetes. In the present study, we describe the development of a procedure to assess the acute toxicity of metal cations (Na(+), K(+), Li(+), Ca(2+), Mg(2+), Co(2+), Zn(2+), Ni(2+), Mn(2+), Cd(2+), and Cu(2+)) to the bioluminescent basidiomycete fungus Gerronema viridilucens. The method is based on the decrease in the intensity of bioluminescence resulting from injuries sustained by the fungus mycelium exposed to either essential or nonessential metal toxicants. The assay described herein enables LIS to propose a metal toxicity series to Gerronenia viridilucens based on data obtained from the bioluminescence intensity (median effective concentration [EC50] values) versus metal concentration: Cd(2+) > Cu(2+) > Mn(2+) approximate to Ni(2+) approximate to Co(2+) > Zn(2+) > Mg(2+) > Li(+) > K(+) approximate to Na(+) > Ca(2+), and to shed some li-ht on the mechanism of toxic action of metal cations to basidiomycete fungi. Environ. Toxicol. Chem. 2010;29:320-326. (C) 2009 SETAC