CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 – 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Abstract Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

Ultra-structural mapping of sugarcane bagasse after oxalic acid fiber expansion (OAFEX) and ethanol production by Candida shehatae and Saccharomyces cerevisiae

Background Diminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils. Results OAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform–near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g). Conclusions OAFEX treatment revealed marked hemicellulose degradation, improving the cellulases’ ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level.

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1

Abstract Background In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.

Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract Background Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans. Results Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA: GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA:GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose. Conclusions Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

Material embodiment and energy flows as efficiency indicators of soybean (Glycine max) production in Brazil

As the requirement for agriculture to be environmentally suitable there is a necessity to adopt indicators and methodologies approaching sustainability. In Brazil, biodiesel addition into diesel is mandatory and soybean oil is its main source. The material embodiment determines the convergence of inputs into the crop. Moreover, the material flows are necessary for any environmental analysis. This study evaluated distinct production scenarios, and also conventional versus GMO crops, through the material embodiment and energy analysis. GMO crops demanded less indirectly applied inputs. The energy balance showed linearity with yield, whereas for EROI, the increases in input and yield were not affected.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Influence of the use of rice bran extract as a source of nutrients on xylitol production

Xylose-to-xylitol bioconversion using 2.5 or 10% (v/v) rice bran extract was performed to verify the influence of this source of nutrients on Candida guilliermondii metabolism. Semisynthetic medium (SM) and sugarcane bagasse hemicellulosic hydrolysate detoxified with ion-exchange resins (HIE) or with alteration in pH combined with adsorption onto activated charcoal (HAC) were fermented in 125 mL Erlenmeyer flasks at 30 ºC and 200 rpm for 72 hours. Activated charcoal supplemented with 2.5% (v/v) rice bran extract was fermented by C. guilliermondii in a MULTIGEN stirred tank reactor using pH 5.0 and 22.9/hour oxygen transfer volumetric coefficient. Higher values of xylitol productivity (0.70, 0.71, and 0.62 g.Lh-1) and xylose-to-xylitol conversion yield (0.71, 0.69, and 0.63 g.g-1) were obtained with 2.5% (v/v) rice bran in semisynthetic medium, ion-exchange resins, and activated charcoal, respectively. Moreover, during batch fermentation, the xylitol volumetric productivity and fermentation efficiency values obtained were 0.53 g.Lh-1 and 61.1%, respectively.

Spatial variability of soil fertility and its relationship with seed physiological potential in a soybean production area

The correlation of soil fertility x seed physiological potential is very important in the area of seed technology but results published with that theme are contradictory. For this reason, this study to evaluate the correlations between soil chemical properties and physiological potential of soybean seeds. On georeferenced points, both soil and seeds were sampled for analysis of soil fertility and seed physiological potential. Data were assessed by the following analyses: descriptive statistics; Pearson's linear correlation; and geostatistics. The adjusted parameters of the semivariograms were used to produce maps of spatial distribution for each variable. Organic matter content, Mn and Cu showed significant effects on seed germination. Most variables studied presented moderate to high spatial dependence. Germination and accelerated aging of seeds, and P, Ca, Mg, Mn, Cu and Zn showed a better fit to spherical semivariogram: organic matter, pH and K had a better fit to Gaussian model; and V% and Fe showed a better fit to the linear model. The values for range of spatial dependence varied from 89.9 m for P until 651.4 m for Fe. These values should be considered when new samples are collected for assessing soil fertility in this production area.

Evaluating methods for the isolation of marine-derived fungal strains and production of bioactive secondary metabolites

In the present investigation we evaluate methods for the isolation and growth of marine-derived fungal strains in artificial media for the production of secondary metabolites. Inoculation of marine macroorganisms fragments in Petri dishes proved to be the most convenient procedure for the isolation of the largest number of strains. Among the growth media used, 3% malt extract showed the best result for strains isolation and growth, and yielded the largest number of strains from marine macroorganisms. The percentage of strains isolated using each of the growth media which yielded cytotoxic and/or antibiotic extracts was in the range of 23-35%, regardless of the growth media used. Further investigation of extracts obtained from different marine-derived fungal strains yielded several bioactive secondary metabolites, among which (E)-4-methoxy-5-(3-methoxybut-1-enyl)-6-methyl-2H-pyran-2-one is a new metabolite isolated from the Penicillium paxilli strain Ma(G)K.

Effects of microbial inoculants and amino acid production by-product on fermentation and chemical composition of sugarcane silages

The objective of this study was to evaluate the chemical composition, fermentation patterns and aerobic stability of sugarcane silages with addition of amino acid production (monosodium glutamate) by-product (APB) and microbial inoculants. Mature sugarcane was chopped and ensiled in laboratory silos (n = 4/treatment) without additives (control) and with APB (10 g/kg), Pioneer 1174® (PIO, 1.0 mg/kg, Lactobacillus plantarum + Streptoccoccus faecium, Pioneer), Lalsil Cana (2.0 mg/kg, Lactobacillus buchineri, Lallemand) or Mercosil Maís 11C33® (1.0 mg/kg, Lactobacillus buchineri + Lactobacillus plantarum + Streptoccoccus faecium, Timac Agro). Fresh silage and silage liquor samples were obtained to assess pH, chemical composition and organic acid concentrations. Silage temperature was recorded throughout seven days to evaluate aerobic stability. The addition of APB decreased lactic acid levels, increased pH and N-NH3 and did not alter ethanol, acetic and butyric acids concentrations or dry matter (DM) losses. Microbial inoculants enhanced acetic acid levels, although only Pioneer 1174® and Mercosil Maís 11C33® lowered ethanol, butyric acid and DM losses. The addition of APB increased CP content and did not modify DM, soluble carbohydrates contents or in vitro dry matter digestibility. Additives did not alter silage maximum temperature or temperature increasing rate; however, Pioneer 1174® and Mercosil Maís 11C33® increased the time elapsed to reach maximum temperature. Monosodium glutamate production by-product does not alter fermentation patterns or aerobic stability of sugarcane silages, whereas homofermentative bacteria can provide silages of good quality.

Glycerine derived from biodiesel production as a feedstuff for broiler diets

The performance, carcass traits, and litter humidity of broilers fed increasing levels of glycerine derived from biodiesel production were evaluated. In this experiment, 1,575 broilers were distributed according to a completely randomized experimental design into five treatments with seven replicates of 45 birds each. Treatments consisted of a control diet and four diets containing 2.5, 5.0, 7.5, or 10% glycerine. The experimental diets contained equal nutritional levels and were based on corn, soybean meal and soybean oil. The glycerine included in the diets contained 83.4% glycerol, 1.18% sodium, and 208 ppm methanol, and a calculated energy value of 3,422 kcal AMEn/kg. Performance parameters (weight gain, feed intake, feed conversion ratio, live weight, and livability) were monitored when broilers were 7, 21, and 42 days of age. On day 43, litter humidity was determined in each pen, and 14 birds/treatment were sacrificed for the evaluation of carcass traits. During the period of 1 to 7 days, there was a positive linear effect of the treatments on weight gain, feed intake, and live weight gain. Livability linearly decreased during the period of 1 to 21 days. During the entire experimental period, no significant effects were observed on performance parameters or carcass traits, but there was a linear increase in litter humidity. Therefore, the inclusion of up to 5% glycerine in the diet did not affect broiler performance during the total rearing period.

Fresh pasta production enriched with Spirulina platensis biomass

The aim of this work was to study the enrichment of Spirulina platensis in wheat flour to prepare fresh pasta to evaluate the green color and nutritional enrichment in addition to functional properties due to the presence of the bioactive compounds in the cyanobacterium. The pastas were evaluated for the centesimal composition, microbiological contamination, sensorial acceptance and technological characteristics such as cooking time, water absorption, volume displacement and loss of solids. The superior protein contents and the satisfactory technological and sensorial attributes compared with the control with no cyanobacterium showed the usefulness of incorporating S. platensis biomass in the fresh pastas. The microbiological quality was in compliance with the legislation in force. The sensorial quality was considered satisfactory ( liked very much ) and purchase intention high ( probably would buy ).

'Synthetic lipase' production from a newly isolated Sporidiobolus pararoseus strain by submerged fermentation

The lipase produced by a newly isolate Sporidiobolus pararoseus strain has potential catalysis ability for esterification reactions. In order to improve its synthetic activity, this work aimed at optimizing 'synthetic lipase' production by submerged fermentation of a conventional media based on peptone, yeast extract, NaCl and olive oil using experimental design technique. According to the results obtained in the first experimental design (2(4-1)), yeast extract and NaCl concentrations were tested to further optimization by response surface methodology. The maximum 'synthetic lipase' activity obtained was 26.9 U/mL in the optimized media (5.0, 6.8, 7.0 and 1.0% (wt/v) of peptone, yeast extract, NaCl and olive oil, respectively), representing a 6.36-fold increase compared to the initial medium. The time course of 'synthetic lipase' production in the optimized condition was evaluated in terms of synthetic activity, protease activity, biomass and total carbon and the maximum synthetic activity was observed during the stationary phase of growth.