Factors associated with the time taken for diagnosis of amyotrophic lateral sclerosis (ALS) in Brazil. An online population-based inquiry. / Factores asociados al tiempo necesario para el diagnóstico de esclerosis lateral amiotrófica (ELA) en Brasil. Una encuesta poblacional en línea.

OBJECTIVE: This study evaluated factors associated with the time, in months, between the onset of symptoms and the diagnosis (time taken for diagnosis) of ALS for patients in Brazil, in the year 2014. PATIENTS AND METHODS: An electronic questionnaire composed of 38 questions was developed and applied through internet-based social networks of patients. From the 210 replies, 194 were considered (86 from women, 108 from men). Most respondents were 51 to 60 years old. The Mann-Whitney test was used to compare the time taken for diagnosis between the strata of the sample. RESULTS: The mean time taken for diagnosis was 14.21 (±16.87) months. There was a statistically significant difference only for higher education conditions (p = 0.009) and low education status (p = 0.042). There was no statistically significant difference between sexes, bulbar onset, age groups, and the presence of spouse, or 'partnership with ALS patients associations or exchange of experiences'. CONCLUSION: These data suggest that the time taken for diagnosis of ALS is influenced by socioeconomic conditions that promote access to information and/or health services.

TITLE: Factores asociados al tiempo necesario para el diagnóstico de esclerosis lateral amiotrófica (ELA) en Brasil. Una encuesta poblacional en línea.Objetivo. Este estudio evaluó los factores asociados con el tiempo, en meses, entre el inicio de los síntomas y el diagnóstico (tiempo necesario para el diagnóstico) de esclerosis lateral amiotrófica (ELA) de los pacientes en Brasil en 2014. Pacientes y métodos. Se elaboró un cuestionario electrónico compuesto por 38 preguntas y se aplicó a través de redes sociales de pacientes basadas en Internet. De las 210 respuestas, se consideraron 194 (86 de mujeres y 108 de hombres). La mayoría de los encuestados tenía entre 51 y 60 años. Se utilizó la prueba de Mann-Whitney para comparar el tiempo transcurrido hasta el diagnóstico entre los estratos de la muestra. Resultados. El tiempo medio transcurrido hasta el diagnóstico fue de 14,21 (±16,87) meses. Hubo una diferencia estadísticamente significativa sólo para las condiciones de educación superior (p = 0,009) y bajo nivel educativo (p = 0,042). No hubo diferencias estadísticamente significativas entre sexos, inicio bulbar, grupos de edad y presencia de cónyuge, o colaboración con asociaciones de pacientes con ELA o intercambio de experiencias. Conclusión. Estos datos sugieren que el tiempo que se tarda en diagnosticar la ELA está influido por las condiciones socioeconómicas que favorecen el acceso a la información y/o a los servicios sanitarios.

Conditioned medium as a strategy for human stem cells chondrogenic differentiation.

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow-derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly-derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three-dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage-related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications.

Assessment of a tannin-based organic polymer to harvest Chlorella vulgaris biomass from swine wastewater digestate phycoremediation.

This study investigated the efficiency of an organic tannin polymer alone or amended with polyacrylamide to harvest Chlorella vulgaris biomass grown in a laboratory-scale photobioreactor treating swine wastewater digestate. The effect of biomass concentration, tannin (TAN) dosages and changes in pH were evaluated in jar test experiments. Among the TAN concentrations tested (11, 22, 44, 89, 178 mg L(-1)), 11 mg L(-1) showed the highest biomass recovery (97%). The highest coagulation/ flocculation efficiencies were obtained at pH 5 to 7. Flocculation efficiency improved from 50 to 97% concomitant with the increasing biomass concentrations from 45 to 165 mg L(-1), respectively. Recovery efficiencies above 95% were achieved with the same TAN dosage (11 mg L(-1)) irrespective of the concentration of organic carbon present (75 to 300 mg TOC L(-1)). Overall, the results suggest that TAN could become an interesting alternative choice of non-toxic organic polymer for harvesting Chlorella sp. from organic-rich wastewater.

Correlating denitrifying catabolic genes with N2O and N2 emissions from swine slurry composting.

This work evaluated N dynamics that occurs over time within swine slurry composting piles. Real-time quantitative PCR (qPCR) analyzes were conducted to estimate concentrations of bacteria community harboring specific catabolic nitrifying-ammonium monooxygenase (amoA), and denitrifying nitrate- (narG), nitrite- (nirS and nirG), nitric oxide- (norB) and nitrous oxide reductases (nosZ) genes. NH3-N, N2O-N, N2-N emissions represented 15.4 ± 1.9%, 5.4 ± 0.9%, and 79.1 ± 2.0% of the total nitrogen losses, respectively. Among the genes tested, temporal distribution of narG, nirS, and nosZ concentration correlated significantly (p<0.05) with the estimated N2 emissions. Denitrifying catabolic gene ratio (cnorB+qnorB)/nosZ ≥ 100 was indicative of N2O emission potential from the compost pile. Considering our current empirical limitations to accurately measure N2 emissions from swine slurry composting at field scale the use of these catabolic genes could represent a promising monitoring tool to aid minimize our uncertainties on biological N mass balances in these systems.

Gene expression and molecular modeling of the HSP104 chaperone of Trypanosoma cruzi.

Heat shock protein (HSP) 104 is a highly conserved molecular chaperone that catalyzes protein unfolding, disaggregation and degradation under stress conditions. We characterized HSP104 gene structure and expression in Trypanosoma cruzi, a protozoan parasite that causes Chagas' disease. The T. cruzi HSP104 is an 869 amino-acid protein encoded by a single-copy gene that has the highest sequence similarity (76%) with that of T. brucei and the lowest (23%) with that of the human protein. HSP104 transcripts were detected at room temperature, and levels increased after incubation at 37° or 40°C. The HSP104 protein was found at low levels in non-heat-shocked cells, and accumulated continuously up to 24 h at elevated temperatures. We developed a predicted structural model of hexameric T. cruzi HSP104, which showed some conserved features.

Chondrogenic differentiation of human bone marrow mesenchymal stem cells in chitosan-based scaffolds using a flow-perfusion bioreactor.

Native articular cartilage is subjected to synovial fluid flow during normal joint function. Thus, it is believed that the morphogenesis of articular cartilage may be positively regulated by the application of similar stimulation in vitro. In the present study, the effect of fluid flow over the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) was investigated. We intended to find out whether the shear stress caused by perfusion of the medium through the constructs was capable of augmenting the differentiation process. Human BMSCs were isolated from bone marrow aspirates and were characterized by flow cytometry. After expansion, hBM-MSCs were seeded statically onto fibre mesh scaffolds, consisting of a blend of 50:50 chitosan:poly(butylene terephthalate adipate) (CPBTA). Constructs were cultured in a flow-perfusion bioreactor for 28 days, using complete medium for chondrogenesis supplemented by TGFß3. An enhanced ECM deposition and collagen type II production was observed in the bioreactor samples when compared to the static controls. Moreover, it was observed that hBM-MSCs, in static cultures, take longer to differentiate. ECM accumulation in these samples is lower than in the bioreactor sections, and there is a significant difference in the expression of collagen type I. We found that the flow-induced shear stress has a beneficial effect on the chondrogenic differentiation of hMSCs.

Cartilage tissue engineering using electrospun PCL nanofiber meshes and MSCs.

Mesenchymal stem cells (MSCs) have been recognized for their ability to differentiate into cells of different tissues such as bone, cartilage, or adipose tissue, and therefore are of great interest for potential therapeutic strategies. Adherent, colony-forming, fibroblastic cells were isolated from human bone marrow aspirates, from patients undergoing knee arthroplasties, and the MSCs phenotype characterized by flow cytometry. Afterward, cells were seeded onto electrospun polycaprolactone nanofiber meshes and cultured in a multichamber flow perfusion bioreactor to determine their ability to produce cartilagineous extracellular matrix. Results indicate that the flow perfusion bioreactor increased the chondrogenic differentiation of hBM-MSCs, as confirmed either by morphological and RT-PCR analysis. Cartilage-related genes such as aggrecan, collagen type II, and Sox9 were expressed. ECM deposition was also detected by histological procedures. Collagen type II was present in the samples, as well as collagen type I. Despite no statistically significant values being obtained for gene expression, the other results support the choice of the bioreactor for this type of culture.

Image analysis of modified cellulose fibers from sugarcane bagasse by zirconium oxychloride.

Surface modification of natural fibers has been made using different methods. In this paper, cellulose fibers from sugarcane bagasse were bleached and modified by zirconium oxychloride in situ. The chemically modified cellulose fibers were compared to those of bleached ones. Cellulose fibers were modified with ZrO(2).nH(2)O nanoparticles through the use of zirconium oxychloride in acidic medium in the presence of cellulose fibers using urea as the precipitating agent. The spatial distribution characterization of hydrous zirconium oxide on cellulose fibers was carried out by combining both processing and image analyses obtained by SEM and statistical methodologies. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TG) were also used to characterize the nanocomposite. Results indicated that ZrO(2).nH(2)O nanoparticles of about 30-80nm diameter deposited on cellulose fibers were heterogeneously dispersed.

Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix formation.

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds' porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold.

Tissue-specific deiodinase regulation during food restriction and low replacement dose of leptin in rats.

The relationship between thyroid function and leptin has been extensively studied; however, the mechanisms underlying the changes in thyroid hormone economy that occur during caloric deprivation remain elusive. Our goal was to evaluate the thyroid function of rats submitted to 40% food restriction after chronic leptin replacement. Caloric restriction for 25 days led to significantly reduced serum leptin, thyroid-stimulating hormone (TSH), thyroxine (T(4)), and triiodothyronine (T(3)) and increased serum corticosterone, while liver, kidney, and thyroid type I deiodinase (D1) and brown adipose tissue (BAT) type II deiodinase (D2) activities were decreased and hypothalamic D2 was significantly increased. Interestingly, thyroid iodide uptake was unchanged by caloric restriction, but thyroperoxidase (TPO) activity was significantly reduced. Leptin replacement for the last 10 days of caloric restriction normalized serum leptin and TSH levels, but serum T(4) and T(3) levels and thyroid D1 and TPO activities were not reestablished. Also, a negative effect of leptin administration on Na(+)-I(-) symporter function was detected. Liver and kidney D1 and hypothalamic and BAT D2 were normalized by leptin, while pituitary D2 was significantly decreased. In conclusion, a tissue-specific modulation of deiodinases might be implicated in the normalization of thyroid function during leptin replacement in food-restricted rats. Although leptin restores the hypothalamus-pituitary axis during food restriction, it exerts a direct negative effect on the thyroid gland; thus normalization of serum thyroid hormones might depend on changes in deiodinase activities and the long-term thyroid stimulation by TSH to counterbalance the direct negative effects of leptin on the thyroid gland.

Autoimmune thrombocytopenia related to chronic hepatitis C virus infection.

Since the identification of hepatitis C virus (HCV) in 1989 as a causative agent for a number of the extrahepatic alterations related to HCV infection an underlying immune mediated pathogenetic mechanism has been postulated. HCV-associated thrombocytopenia may be considered complex and multifactorial in origin, since different mechanisms have been implicated in its pathophysiology. With respect to autoimmune thrombocytopenia in chronic HCV infection, the detection of specific antibodies against platelet glycoproteins have been reported only in a few studies, but no systematic study has been carried out. We examined the clinical, laboratory, and virological characteristics of a case series of 10 patients with autoimmune thrombocytopenia (platelet count <150.0 x 10(9)/L) related to chronic HCV infection. Cases, six males and four females, aged 57.1 +/- 12.6 years, presented high titers of antibodies against platelet glycoprotein (GP) IIb/IIIa, GP Ia/IIa, and/or GP Ib/IX, and no other mechanism involved in the pathogenesis of HCV-associated thrombocytopenia was identified. Furthermore, cases were not associated with particular HCV genotype. Complete platelet response was observed in two patients treated with pegylated interferon plus ribavirin, and partial platelet response was seen in two patients treated with anti-D Ig and one patient treated with corticosteroids. These findings indicate that an autoimmune mechanism may play a role in the pathogenesis of HCV-associated thrombocytopenia in a proportion of these patients.

Melt-based compression-molded scaffolds from chitosan-polyester blends and composites: Morphology and mechanical properties.

Blends of chitosan and synthetic aliphatic polyesters (polybutylene succinate, polybutylene succinate adipate, polycaprolactone, and polybutylene terepthalate adipate) were compounded with and without hydroxyapatite, a bioactive mineral filler known to enhance osteoconduction. The blends and composites were compression molded with two different granulometric salt sizes (63-125 microm and 250-500 microm) having different levels of salt content (60, 70, and 80%) by weight. By leaching the salt particles, it was possible to produce porous scaffolds with distinct morphologies. The relationship between scaffold morphology and mechanical properties was evaluated using scanning electron microscopy, microcomputed tomography, compression testing, differential scanning calorimetry, small-angle X-ray scattering (SAXS), and wide-angle X-ray scattering. The produced scaffolds are characterized by having different morphologies depending on the average particle size and the amount of NaCl used. Specimens with higher porosity level have a less organized pore structure but increased interconnectivity of the pores. The stress-strain curve under compression displayed a linear elasticity followed by a plateau whose characteristics depend on the scaffold polymer composition. A decrease in the salt particle size used to create the porosity caused in general a decrease in the mechanical properties of the foams. Composites with hydroxyapatite had a sharp reduction in yield stress, modulus, and strain at break. The melting temperature decreased with increased chitosan content. SAXS results indicate no preferential crystalline orientation in the scaffolds. Cytotoxicity evaluation were carried out using standard tests (accordingly to ISO/EN 10993 part 5 guidelines), namely MTS test with a 24-h extraction period, revealing that L929 cells had comparable metabolic activities to that obtained for the negative control.

Comparison of bioaugmentation and biostimulation for the enhancement of dense nonaqueous phase liquid source zone bioremediation.

Two 11.7-m(3) experimental controlled release systems (ECRS), packed with sandy model aquifer material and amended with tetrachloroethene (PCE) dense nonaqueous phase liquid (DNAPL) source zone, were operated in parallel with identical flow regimes and electron donor amendments. Hydrogen Releasing Compound (Regenesis Bioremediation Products, Inc., San Clemente, California), and later dissolved lactate, served as electron donors to promote dechlorination. One ECRS was bioaugmented with an anaerobic dechlorinating consortium directly into the source zone, and the other served as a control (biostimulated only) to determine the benefits of bioaugmentation. The presence of halorespiring bacteria in the aquifer matrix before bioaugmentation, shown by nested polymerase chain reaction with phylogenetic primers, suggests that dechlorinating catabolic potential may be somewhat widespread. Results obtained corroborate that source zone reductive dechlorination of PCE is possible at near field scale and that a system bioaugmented with a competent halorespiring consortium can enhance DNAPL dissolution and dechlorination processes at significantly greater rates than in a system that is biostimulated only.

Structural characterization of Botryosphaeran: a (1-->3;1-->6)-beta-D-glucan produced by the ascomyceteous fungus, Botryosphaeria sp.

The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomyceteous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and 13C NMR spectroscopy showed that all the glucosidic linkages were in the beta-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1-->3)-beta-D-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1-->6)-beta-linked glucosyl, and (1-->6)-beta-linked gentiobiosyl residues.

Salt stress response and proline accumulation in Brachiaria humidicola plants with and without mycorrhizal inoculation.

The work was carried out to investigate the effects of different concentrations of NaCl on Brachiaria humidicola Rendle in the presence and absence of Glomus etunicatum Becker and Gerdemann, as well as to evaluate some growth parameters and the accumulation of free proline in the plant leaves. The soil used was a Neosoil Fluvic Eutrophic with pH of 6.5, organic matter, 12.8 g kg-1 and soil texture, sandy loam, in which Brachiaria humidicola Rendle cv. 409 was grown. Five NaCl concentrations were tested 0; 0.22; 1.09; 1.96 and 2.84 g. kg-1 of soil, whose electrical conductivity (EC) were 2.22; 4.00; 8.13; 12.53 and 16.50 dS m-1, respectively. Brachiaria humidicola showed salt tolerance when submitted to an EC of 4 dS m-1. There was a reduction of leaf area, dry matter of shoots and roots for the soil treatments beyond EC at 8 dS m-1. Free proline content in the leaves increased together with the increase in soil salinity (EC at 8 dS m-1) demonstrating that plants submitted to EC of 2 and 4 dS m-1 were less affected by salt stress, and consequently accumulated less proline in the leaves. Root colonization was not affected by the increase of NaCl dosage in the soil.

[Characterization of Rhizobium isolates from acid and alkaline soils in semi-arid regions of Pernambuco]. / Caracterización de aislamientos de rizobios de suelos ácidos y alcalinos en la región semiárida de Pernambuco.

The genetic variety of the Rhizobium isolates from acid and alkaline soils in the semiarid zone of Pernambuco state was evaluated through the use of 17 primers of arbitrary sequence. Amplified products were separated by electrophoresis in agarose gel at 1.4% and visualized by ethidium bromide coloration. The results obtained suggest a high genetic variety of the isolates in relation to the standard strain. Data were analyzed by UPGMA (unweighted pair-group method with arithmetic average), based on Jaccard's coefficient and visualized through dendrograms. The strains isolated from the acid soils were included in one group whereas the strains from alkaline soils were located in other three groups. Meanwhile, one of the groups formed by strain Isol-14, isolated from acid soils is more related to the groups of strains isolated from acid soils than to the remaining groups from alkaline soils.

Caracterización de aislamientos de rizobios de suelos ácidos y alcalinos en la región semiárida de Pernambuco / [Characterization of Rhizobium isolates from acid and alkaline soils in semi-arid regions of Pernambuco]

The genetic variety of the Rhizobium isolates from acid and alkaline soils in the semiarid zone of Pernambuco state was evaluated through the use of 17 primers of arbitrary sequence. Amplified products were separated by electrophoresis in agarose gel at 1.4

and visualized by ethidium bromide coloration. The results obtained suggest a high genetic variety of the isolates in relation to the standard strain. Data were analyzed by UPGMA (unweighted pair-group method with arithmetic average), based on Jaccard’s coefficient and visualized through dendrograms. The strains isolated from the acid soils were included in one group whereas the strains from alkaline soils were located in other three groups. Meanwhile, one of the groups formed by strain Isol-14, isolated from acid soils is more related to the groups of strains isolated from acid soils than to the remaining groups from alkaline soils.

Salt stress response and proline accumulation in Brachiaria humidicola plants with and without mycorrhizal inoculation.

The work was carried out to investigate the effects of different concentrations of NaCl on Brachiaria humidicola Rendle in the presence and absence of Glomus etunicatum Becker and Gerdemann, as well as to evaluate some growth parameters and the accumulation of free proline in the plant leaves. The soil used was a Neosoil Fluvic Eutrophic with pH of 6.5, organic matter, 12.8 g kg-1 and soil texture, sandy loam, in which Brachiaria humidicola Rendle cv. 409 was grown. Five NaCl concentrations were tested 0; 0.22; 1.09; 1.96 and 2.84 g. kg-1 of soil, whose electrical conductivity (EC) were 2.22; 4.00; 8.13; 12.53 and 16.50 dS m-1, respectively. Brachiaria humidicola showed salt tolerance when submitted to an EC of 4 dS m-1. There was a reduction of leaf area, dry matter of shoots and roots for the soil treatments beyond EC at 8 dS m-1. Free proline content in the leaves increased together with the increase in soil salinity (EC at 8 dS m-1) demonstrating that plants submitted to EC of 2 and 4 dS m-1 were less affected by salt stress, and consequently accumulated less proline in the leaves. Root colonization was not affected by the increase of NaCl dosage in the soil.

Caracterización de aislamientos de rizobios de suelos ácidos y alcalinos en la región semiárida de Pernambuco. / [Characterization of Rhizobium isolates from acid and alkaline soils in semi-arid regions of Pernambuco]

The genetic variety of the Rhizobium isolates from acid and alkaline soils in the semiarid zone of Pernambuco state was evaluated through the use of 17 primers of arbitrary sequence. Amplified products were separated by electrophoresis in agarose gel at 1.4

and visualized by ethidium bromide coloration. The results obtained suggest a high genetic variety of the isolates in relation to the standard strain. Data were analyzed by UPGMA (unweighted pair-group method with arithmetic average), based on Jaccards coefficient and visualized through dendrograms. The strains isolated from the acid soils were included in one group whereas the strains from alkaline soils were located in other three groups. Meanwhile, one of the groups formed by strain Isol-14, isolated from acid soils is more related to the groups of strains isolated from acid soils than to the remaining groups from alkaline soils.

Using Shannon entropy on measuring the individual variability in the Rufous-bellied thrush Turdus rufiventris vocal communication.

We applied the information theory concepts to notes repertoire characteristics combined with temporal parameters of the Rufous-bellied thrush Turdus rufiventris song, using this particular case to test a new method of analysing quantitatively complex animal communication systems. Like most Turdus thrushes, Rufous-bellied thrushes are remarkable for their long, varied and melodious songs. For the analysis of the species repertoire, we used recordings of 44 individuals from 24 localities covering its full geographical range. We measured the repertoire size, note duration and rhythm (frequency of note utterance), and combined these parameters with the Shannon entropy values calculated for each individual. Although individuals maintain species-specific recognition capacity, we find a large variation between their song parameters and show that the information theory can be useful to analyse large and varied animal vocal repertoires. We are introducing two new parameters, temporal average entropy (E(t)) and utterance frequency average entropy (E(f)), for measuring such communication systems.