COMPOSITION OF FRESHLY HARVESTED BRAZILIAN ROYAL JELLY - IDENTIFICATION OF CARBOHYDRATES FROM THE SUGAR FRACTION

Samples of Brazilian royal jelly from Africanized Apis mellifera were analysed in order to determine the gross composition: crude moisture ranged from 67.80% to 69.40%, crude protein from 15.80% to 16.70%, crude lipid from 2.90% to 3.98% and-total sugars from 11.40% to 11.50%. The sugar fraction was investigated and revealed the presence of the following compounds identified by their retention time during HPLC analysis: ribose, fructose, glucose, sucrose, mannose, trehalose, erythritol, adonitol and mannitol.

CHARACTERIZING AND IMPROVING PRODUCTION OF FERMENTABLE SUGARS AND CO-PRODUCTS FROM A FOREST PRODUCT INDUSTRY WASTEWATER STREAM

Hardboard processing wastewater was evaluated as a feedstock in a bio refinery co-located with the hardboard facility for the production of fuel grade ethanol. A thorough characterization was conducted on the wastewater and the composition changes of which during the process in the bio refinery were tracked. It was determined that the wastewater had a low solid content (1.4%), and hemicellulose was the main component in the solid, accounting for up to 70%. Acid pretreatment alone can hydrolyze the majority of the hemicellulose as well as oligomers, and over 50% of the monomer sugars generated were xylose. The percentage of lignin remained in the liquid increased after acid pretreatment. The characterization results showed that hardboard processing wastewater is a feasible feedstock for the production of ethanol. The optimum conditions to hydrolyze hemicellulose into fermentable sugars were evaluated with a two-stage experiment, which includes acid pretreatment and enzymatic hydrolysis. The experimental data were fitted into second order regression models and Response Surface Methodology (RSM) was employed. The results of the experiment showed that for this type of feedstock enzymatic hydrolysis is not that necessary. In order to reach a comparatively high total sugar concentration (over 45g/l) and low furfural concentration (less than 0.5g/l), the optimum conditions were reached when acid concentration was between 1.41 to 1.81%, and reaction time was 48 to 76 minutes. The two products produced from the bio refinery were compared with traditional products, petroleum gasoline and traditional potassium acetate, in the perspective of sustainability, with greenhouse gas (GHG) emission as an indicator. Three allocation methods, system expansion, mass allocation and market value allocation methods were employed in this assessment. It was determined that the life cycle GHG emissions of ethanol were -27.1, 20.8 and 16 g CO2 eq/MJ, respectively, in the three allocation methods, whereas that of petroleum gasoline is 90 g CO2 eq/MJ. The life cycle GHG emissions of potassium acetate in mass allocation and market value allocation method were 555.7 and 716.0 g CO2 eq/kg, whereas that of traditional potassium acetate is 1020 g CO2/kg.

Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization

BACKGROUND Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms. RESULTS The combination and integration of in silico analyses and in vitro CGH experiments, performed in comparison with the reference microorganisms, allowed establishment of an inter-species hybridization framework with a detection threshold based on a sequence similarity of >or= 70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of the genes are related to ribosomal, sugar metabolism or energy conversion systems. Some of the identified genes, such as als and mycA, could be involved in the pathogenesis of L. garvieae infections. CONCLUSIONS In this study, we identified 267 genes that were potentially present in L. garvieae CECT 4531. Some of the identified genes could be involved in the pathogenesis of L. garvieae infections. These results provide the first insight into the genome content of L. garvieae.

Investigation of gold-based catalysts for liquid phase oxidation of glucose to glucaric acid

Glucaric acid (GA) is one of the building block chemicals derived from sugar biomass with higher added value. Nowadays, GA is produced by oxidation of glucose (Glu) with either stoichiometric oxidants (HNO3), or by means of electrochemical or biochemical synthesis. However, these processes show drawbacks from either the environmental or economic viewpoint. For this reason, gold nanoparticles (Au NPs) supported on activated carbon (AC) have been studied as catalysts for the oxidation of Glu, using O2 as oxidant in the presence of a base. Using sol immobilization technique, Au NPs have been supported on AC following different experimental procedures. UV-Vis spectroscopy, XRD, TEM and TG analysis were utilized in the characterization of the catalysts. The operational conditions were optimized obtaining 24% of yield of GA, 37% to GO and 27% to byproducts in 1 h, 1000 rpm, 10 bar of O2 and Glu:Au:NaOH molar ratio of 1000:1:3000. Under such conditions, catalysts show relatively high Glu conversion (≥82%) with different GA yields. GO+GA yield is around 58-61%. Therefore, the oxidation reaction was performed at 15 min where Au/AC PVA0 reached the highest yield of GA (16%) and Au/AC PVA2.4 gave the lowest (8%). It is evident that the presence of PVA influences to a higher degree the reaction rate than the Au NPs size. Hence, the effect of different heat treatments where applied for the removal of PVA: washing with water at 60℃ or heat treatment (120-250℃) with Air/H2. Washing treatment and heat treatment at 120℃ with Air/H2 may have resulted in the mildest treatments for the removal of PVA. Finally, two different supports have been used in order to study the effect of metal-support interaction in the immobilization of Au NPs: ZrO2 and AC. Au/AC catalyst demonstrated a higher conversion of GO to GA at short reaction times (15.1% yield GA) compared to Au/ZrO2 (2.4% yield GA).

Effects of sugars and lactic acid isomers on Chlamydia trachomatis infectivity, an in vitro study

The thesis investigates two different in vitro aspects of Chlamydia trachomatis (CT). The thesis analyzes the effect of different sugars on CT infectivity. which is investigated on HeLa cells after 2 hour-incubation of elementary bodies (EBs) with glucose, sucrose or mannitol. Sugars effect on EB membrane fluidity is investigated by fluorescence anisotropy measurement, whereas changes in lipopolysaccharide exposure are examined by cytofluorimetric analysis. By Western blot experiments, the phosphorylation state of Focal Adhesion Kinase in cells infected with EBs pre-incubated with sugars it’s explored. Sugar significantly increase infectivity, acting on the EB structure. Sugars induce an increase of EB membrane fluidity, leading to changes in LPS exposure. After incubation with sucrose and mannitol, EBs lead to higher FAK phosphorylation, enhancing activation of anti-apoptotic and proliferative signals in the host. Secondly, the thesis explores the protective effect of different Lactobacilli against CT infection: Lactobacillus crispatus and Lactobacillus reuteri. CT infectivity is evaluated after host cells were treated for 1 hour with diluted supernatant cell-free fraction or with the bacterial cells. Assessed that L.crispatus is more protective than L.reuteri, lactic acid production is evaluated by HPLC. Subsequently Lactate dehydrogenases activity is evaluated by resazurin assay and by LC-MS. Then, D-lactate dehydrogenase specific activity has been investigated by measuring NADH formation. Afterwards, addition of D or L-lactic acid to L.reuteri supernatant has been performed and their effect in promoting protection in the host cells assessed. Then a metabolic analysis has been carried out by real-time measurement of mitochondrial respiration after treatment. Finally, histone acetylation and lactylation, and gene and protein expression of relevant targets, have been investigated. It is shown that the D isomer is more efficient in conferring protection, causing a shift in the host cell metabolic profile and a pattern of histone modifications that changes the expression of important targets.

The Crosslinking Degree Controls The Mechanical, Rheological, And Swelling Properties Of Hyaluronic Acid Microparticles.

Viscosupplements, used for treating joint and cartilage diseases, restore the rheological properties of synovial fluid, regulate joint homeostasis and act as scaffolds for cell growth and tissue regeneration. Most viscosupplements are hydrogels composed of hyaluronic acid (HA) microparticles suspended in fluid HA. These microparticles are crosslinked with chemicals to assure their stability against enzyme degradation and to prolong the action of the viscosupplement. However, the crosslinking also modifies the mechanical, swelling and rheological properties of the HA microparticle hydrogels, with consequences on the effectiveness of the application. The aim of this study is to correlate the crosslinking degree (CD) with these properties to achieve modulation of HA/DVS microparticles through CD control. Because divinyl sulfone (DVS) is the usual crosslinker of HA in viscosupplements, we examined the effects of CD by preparing HA microparticles at 1:1, 2:1, 3:1, and 5:1 HA/DVS mass ratios. The CD was calculated from inductively coupled plasma spectrometry data. HA microparticles were previously sized to a mean diameter of 87.5 µm. Higher CD increased the viscoelasticity and the extrusion force and reduced the swelling of the HA microparticle hydrogels, which also showed Newtonian pseudoplastic behavior and were classified as covalent weak. The hydrogels were not cytotoxic to fibroblasts according to an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.

Geochemistry Of Acid Mine Drainage From A Coal Mining Area And Processes Controlling Metal Attenuation In Stream Waters, Southern Brazil.

Acid drainage influence on the water and sediment quality was investigated in a coal mining area (southern Brazil). Mine drainage showed pH between 3.2 and 4.6 and elevated concentrations of sulfate, As and metals, of which, Fe, Mn and Zn exceeded the limits for the emission of effluents stated in the Brazilian legislation. Arsenic also exceeded the limit, but only slightly. Groundwater monitoring wells from active mines and tailings piles showed pH interval and chemical concentrations similar to those of mine drainage. However, the river and ground water samples of municipal public water supplies revealed a pH range from 7.2 to 7.5 and low chemical concentrations, although Cd concentration slightly exceeded the limit adopted by Brazilian legislation for groundwater. In general, surface waters showed large pH range (6 to 10.8), and changes caused by acid drainage in the chemical composition of these waters were not very significant. Locally, acid drainage seemed to have dissolved carbonate rocks present in the local stratigraphic sequence, attenuating the dispersion of metals and As. Stream sediments presented anomalies of these elements, which were strongly dependent on the proximity of tailings piles and abandoned mines. We found that precipitation processes in sediments and the dilution of dissolved phases were responsible for the attenuation of the concentrations of the metals and As in the acid drainage and river water mixing zone. In general, a larger influence of mining activities on the chemical composition of the surface waters and sediments was observed when enrichment factors in relation to regional background levels were used.

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

A New Extraction Approach To Correct The Effect Of Apparent Increase In The Secoiridoid Content After Filtration Of Virgin Olive Oil.

In the current study, a new approach has been developed for correcting the effect that moisture reduction after virgin olive oil (VOO) filtration exerts on the apparent increase of the secoiridoid content by using an internal standard during extraction. Firstly, two main Spanish varieties (Picual and Hojiblanca) were submitted to industrial filtration of VOOs. Afterwards, the moisture content was determined in unfiltered and filtered VOOs, and liquid-liquid extraction of phenolic compounds was performed using different internal standards. The resulting extracts were analyzed by HPLC-ESI-TOF/MS, in order to gain maximum information concerning the phenolic profiles of the samples under study. The reduction effect of filtration on the moisture content, phenolic alcohols, and flavones was confirmed at the industrial scale. Oleuropein was chosen as internal standard and, for the first time, the apparent increase of secoiridoids in filtered VOO was corrected, using a correction coefficient (Cc) calculated from the variation of internal standard area in filtered and unfiltered VOO during extraction. This approach gave the real concentration of secoiridoids in filtered VOO, and clarified the effect of the filtration step on the phenolic fraction. This finding is of great importance for future studies that seek to quantify phenolic compounds in VOOs.

A Highly Improved Method For Sensitive Determination Of Amitriptyline In Pharmaceutical Formulations Using An Unmodified Carbon Nanotube Electrode In The Presence Of Sulfuric Acid.

The present paper describes a novel, simple and reliable differential pulse voltammetric method for determining amitriptyline (AMT) in pharmaceutical formulations. It has been described for many authors that this antidepressant is electrochemically inactive at carbon electrodes. However, the procedure proposed herein consisted in electrochemically oxidizing AMT at an unmodified carbon nanotube paste electrode in the presence of 0.1 mol L(-1) sulfuric acid used as electrolyte. At such concentration, the acid facilitated the AMT electroxidation through one-electron transfer at 1.33 V vs. Ag/AgCl, as observed by the augmentation of peak current. Concerning optimized conditions (modulation time 5 ms, scan rate 90 mV s(-1), and pulse amplitude 120 mV) a linear calibration curve was constructed in the range of 0.0-30.0 μmol L(-1), with a correlation coefficient of 0.9991 and a limit of detection of 1.61 μmol L(-1). The procedure was successfully validated for intra- and inter-day precision and accuracy. Moreover, its feasibility was assessed through analysis of commercial pharmaceutical formulations and it has been compared to the UV-vis spectrophotometric method used as standard analytical technique recommended by the Brazilian Pharmacopoeia.

Efficacy Of Ethylene-diamine-tetra-acetic Acid Associated With Chlorhexidine On Intracanal Medication Removal: A Scanning Electron Microscopy Study.

The aim of this study was to evaluate the effectiveness of 17% ethylene-diamine-tetra-acetic acid (EDTA) used alone or associated with 2% chlorhexidine gel (CHX) on intracanal medications (ICM) removal. Sixty single-rooted human teeth with fully formed apex were selected. The cervical and middle thirds of each canal were prepared with Gates Glidden drills and rotary files. The apical third was shaped with hand files. The specimens were randomly divided into two groups depending on the ICM used after instrumentation: calcium hydroxide Ca(OH)(2) +CHX or Ca(OH)(2) +sterile saline (SS). After seven days, each group was divided into subgroups according to the protocol used for ICM removal: instrumentation and irrigation either with EDTA, CHX+EDTA, or SS (control groups). All specimens were sectioned and processed for observation of the apical thirds by using scanning electron microscopy. Two calibrated evaluators attributed scores to each specimen. The differences between the protocols for ICM removal were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between the score of debris obtained in each root canal third. Remains of Ca(OH)(2) were found in all specimens independently of the protocol and ICM used (P > 0.05). Seventeen percent EDTA showed the best results in removing ICM when used alone (P < 0.05), particularly in those associated with CHX. It was concluded that the chelating agent 17% EDTA significantly improved the removal of ICM when used alone. Furthermore, the type of the vehicle associated with Ca(OH)(2) also plays a role in the ICM removal.

Polyphenol Extraction Optimisation From Ceylon Gooseberry (dovyalis Hebecarpa) Pulp.

Originally from Asia, Dovyalis hebecarpa is a dark purple/red exotic berry now also produced in Brazil. However, no reports were found in the literature about phenolic extraction or characterisation of this berry. In this study we evaluate the extraction optimisation of anthocyanins and total phenolics in D. hebecarpa berries aiming at the development of a simple and mild analytical technique. Multivariate analysis was used to optimise the extraction variables (ethanol:water:acetone solvent proportions, times, and acid concentrations) at different levels. Acetone/water (20/80 v/v) gave the highest anthocyanin extraction yield, but pure water and different proportions of acetone/water or acetone/ethanol/water (with >50% of water) were also effective. Neither acid concentration nor time had a significant effect on extraction efficiency allowing to fix the recommended parameters at the lowest values tested (0.35% formic acid v/v, and 17.6 min). Under optimised conditions, extraction efficiencies were increased by 31.5% and 11% for anthocyanin and total phenolics, respectively as compared to traditional methods that use more solvent and time. Thus, the optimised methodology increased yields being less hazardous and time consuming than traditional methods. Finally, freeze-dried D. hebecarpa showed high content of target phytochemicals (319 mg/100g and 1,421 mg/100g of total anthocyanin and total phenolic content, respectively).

Petroleomics By Electrospray Ionization Ft-icr Mass Spectrometry Coupled To Partial Least Squares With Variable Selection Methods: Prediction Of The Total Acid Number Of Crude Oils.

Negative-ion mode electrospray ionization, ESI(-), with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was coupled to a Partial Least Squares (PLS) regression and variable selection methods to estimate the total acid number (TAN) of Brazilian crude oil samples. Generally, ESI(-)-FT-ICR mass spectra present a power of resolution of ca. 500,000 and a mass accuracy less than 1 ppm, producing a data matrix containing over 5700 variables per sample. These variables correspond to heteroatom-containing species detected as deprotonated molecules, [M - H](-) ions, which are identified primarily as naphthenic acids, phenols and carbazole analog species. The TAN values for all samples ranged from 0.06 to 3.61 mg of KOH g(-1). To facilitate the spectral interpretation, three methods of variable selection were studied: variable importance in the projection (VIP), interval partial least squares (iPLS) and elimination of uninformative variables (UVE). The UVE method seems to be more appropriate for selecting important variables, reducing the dimension of the variables to 183 and producing a root mean square error of prediction of 0.32 mg of KOH g(-1). By reducing the size of the data, it was possible to relate the selected variables with their corresponding molecular formulas, thus identifying the main chemical species responsible for the TAN values.

Albumin Is Synthesized In Epididymis And Aggregates In A High Molecular Mass Glycoprotein Complex Involved In Sperm-egg Fertilization.

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.