Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40A degrees C for 72 h and 96 h, respectively. Addition of glucose or fructose (a parts per thousand yen1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH4)(2)HPO4 inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102 kDa estimated by SDS-PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60A degrees C and 4.0-4.5, respectively. Both invertases were stable for 1 h at 60A degrees C with half-lives of 10 min at 70A degrees C. Mg2+, Ba2+ and Mn2+ activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters K-m and V-max for the purified extracellular enzyme corresponded to 2.5 mM and 481 U/mg prot(-1), respectively.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2 degrees C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows (R). Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 +/- 5.94% and 9.43 +/- 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 +/- 3.37 and 8.67 +/- 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2 degrees C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from MO were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5 degrees C under an atmosphere of 5% CO2 with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 +/- 4.8 and 57.3 +/- 4.8, respectively, mean +/- SEM), or among MO (62.3 +/- 5.7%), M3 (56.9 +/- 6.0%), and M6 (66.5 +/- 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture. (C) 2012 Elsevier Inc. All rights reserved.

Determination of labile barium in petroleum-produced formation water using paper-based DGT samplers

A polyacrylamide hydrogel containing the Chelex-100 resin has traditionally been used as the binding agent for the diffusion gradients in thin films (DGT) technique. The Chelex-100 resin, although important for the determination of various transition metals, is unsatisfactory for the determination of alkaline earth metals, particularly Ba. In this paper, a cellulose membrane, treated with phosphate (P81 membrane), was evaluated as a binding agent for DGT devices for the determination of Ba in produced formation water (PEW) samples. In addition, diffusive layers of filter paper (cellulose) were tested to diffuse Ba through the DGT devices. Experiments to evaluate the key variables of the technique (pH, deployment time, and ionic strength/salinity) were performed. The Ba sampled by these DGT devices was measured using inductively coupled plasma optical emission spectrometry. Aiming to generate information (related to bioavailability of Ba) on the reuse of PEW for irrigation, the determination of Ba in onshore and offshore samples was performed. The new approach was effective for determination of Ba in onshore samples. To determine Ba in offshore samples, it was necessary to use an alternative calibration procedure due to the high NaCl concentration in these samples. (C) 2012 Elsevier B.V. All rights reserved.

Aberrant transcript produced by a splice donor site deletion in the TECTA gene is associated with autosomal dominant deafness in a Brazilian family

We ascertained a Brazilian family with nine individuals affected by autosomal dominant nonsyndromic sensorineural hearing loss. The bilateral hearing loss affected mainly mid-high frequencies, was apparently stable with an early onset. Microsatellites close to the DFNA8/DFNA12 locus, which harbors the TECTA gene, showed significant multipoint lod scores (32) close to marker D11S4107. Sequencing of the exons and exon-intron boundaries of the TECTA gene in one affected subject revealed the deletion c.5383 + 5delGTGA in the 5' end of intron 16, that includes the last two bases of the donor splice site consensus sequence. This mutation segregates with deafness within the family. To date, 33 different TECTA mutations associated with autossomal dominant hearing loss have been described. Among them is the mutation reported herein, first described by Hildebrand et al. (2011) in a UK family. The audioprofiles from the UK and Brazilian families were similar. In order to investigate the transcripts produced by the mutated allele, we performed cDNA analysis of a lymphoblastoid cell line from an affected heterozygote with the c.5383 + 5delGTGA and a noncarrier from the same family. The analysis allowed us to identify an aberrant transcript with skipping of exon 16, without affecting the reading frame. One of the dominant TECTA mutations already described, a synonymous substitution in exon 16 (c.5331 G<A), was also shown to affect splicing resulting in an aberrant transcript lacking exon 16. Despite the difference in the DNA level, both the synonymous substitution in exon 16 (c.5331 G<A) and the mutation described herein affect splicing of exon 16, leading to its skipping. At the protein level they would have the same effect, an in-frame deletion of 37 amino-acids (p.S1758Y/G1759_N1795del) probably leading to an impaired function of the ZP domain. Thus, like the TECTA missense mutations associated with dominant hearing loss, the c5383 + 5delGTGA mutation does not have an inactivating effect on the protein. (C) 2012 Elsevier B.V. All rights reserved.

Mixed colonies of two species of congeneric stingless bees (Hymenoptera: Apinae, Meliponini) display environmentally-acquired and endogenously-produced recognition signals

Nestmate recognition is fundamental for the maintenance of social organization in insect nests. It is becoming well recognized that cuticle hydrocarbons mediate the recognition process, although the origin of recognition cues in stingless bees remains poorly explored. The present study investigates the effects of endogenously-produced and environmentally-acquired components in cuticular hydrocarbons in stingless bees. The tests are conducted using colonies of Plebeia droryana Friese and Plebeia remota Holmberg. Recognition tests are performed with four different groups: conspecific nestmates, conspecific non-nestmates, heterospecifics and conspecific, genetically-related individuals that emerge in a heterospecific nest. This last group is produced by introducing brood cells of P. droryana into a P. remota colony, and the resulting adult bees are tested for acceptance 10 days after emergence. For all groups, 15 individuals are sampled for chemical analysis. The results show the acceptance of all conspecific nestmates, and the rejection of almost every conspecific non-nestmate and every heterospecific bee. Genetically-related individuals emerging from heterospecific nests present intermediate rejection (66.7% rejection). Chemical analysis shows that P. droryana individuals emerging in a P. remota nest have small amounts of alkene and diene isomers found in P. remota cuticle that are not found in workers from the natal nest. The data clearly show that the majority of the compounds present in P. droryana cuticle are endogenously produced, although a few unsaturated compounds are acquired from the environment, increasing the chemical differences and, consequently, the rejection percentages.

Efficient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNai (TM)). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80 degrees C. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAi (TM) was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Quality and quantity of milk produced by Saanen goats submitted to stress during three sequential days

The experiment aimed to study the effect of physiological stress on cortisol levels, quality and quantity of milk through punctual administration of ACTH. Twelve Saanen goats were divided in two experimental groups: ACTH group (0,5 mu g of ACTH/Kg.L.W); Placebo group (placebo solution). Milk production, and percentages of protein, fat, lactose and SCC (somatic cells counting) of the milk were analyzed before, during and after the administration of ACTH/placebo. Simultaneously to the ACTH/placebo administration and during three sequential days, blood was collected to evaluate cortisol concentrations. At times -30 and zero, both groups presented basal concentrations of cortisol. The increase of cortisol contents was significant at times 60 (group ACTH: 59.00 +/- 5.70 and groups placebo: 5.23 +/- 1.37ng/mL) and 120 (group ACTH: 47.96 +/- 9.72 and group placebo: 4.38 +/- 1,14ng/mL) since the cortisol content was higher on the ACTH group. The values returned to the basal level at 300 minutes. Concerning milk production, no differences were found between ACTH and placebo groups. Milk, protein, fat, lactose and SCC did not distinguish one group from another. The results indicated that the physiological stress induced during three days was not harmful to milk production and milk quality of Saanen goats.

THERMOSTABILITY OF XYLANOLYTIC ENZYMES PRODUCED BY LENTINULA EDODES UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65 degrees C for 21 hours, losing only 15-25% activity. Fungus incubation at 50 degrees C for 12 hours and at 65 degrees C for 24 hours increased the amount of xylose produced.

Photoluminescence in silicon/silicon oxide films produced by the Pulsed Electron Beam Ablation technique

In this work we report studies of the photoluminescence emission in samples based on Si/SiOx films deposited by the Pulsed Electron Beam Ablation (PEBA) technique. The samples were prepared at room temperature using targets with different Si/SiO2 concentrations. The samples were characterized using X-ray Absorption Edge Spectroscopy (XANES) at the Si-K edge, Raman spectroscopy, Photoluminescence (PL) and X-ray Photoelectron Spectroscopy (XPS). The concentration of a-Si and nc-Si in the film was dependent on the silicon concentration in the target. It was also observed that the PL is strongly dependent on the structural amorphous/crystalline arrangement. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Functional properties of gelatin-based films containing Yucca schidigera extract produced via casting, extrusion and blown extrusion processes: A preliminary study

Gelatin-based films containing both Yucca schidigera extract and low concentrations of glycerol (0.25-8.75 g per 100 g protein) were produced by extrusion (EF) and characterized in relation to their mechanical properties and moisture content. The formulations that resulted in either larger or smaller elongation values were used to produce films via both blown extrusion (EBF) and casting (CF) and were characterized with respect to their mechanical properties, water vapor permeability, moisture content, solubility, morphology and infrared spectroscopy. The elongation of the EF films was significantly higher than that of the CF and EBF films. The transversal section possessed a compact, homogeneous structure for all of the films studied. The solubility of the films (36-40%) did not differ significantly between the different processes evaluated. The EBF films demonstrated lower water vapor permeability (0.12 g mm m-(2) h(-1) kPa(-1)) than the CF and EF films. The infrared spectra did not indicate any strong interactions between the added compounds. Thermoplastic processing of the gelatin films can significantly increase their elongation; however, a more detailed assessment and optimization of the extrusion conditions is necessary, along with the addition of partially hydrophobic compounds, such as surfactants. (C) 2012 Elsevier Ltd. All rights reserved.

CHARACTERIZATION OF LYOPHILIZED LIPOSOMES PRODUCED WITH NON-PURIFIED SOY LECITHIN: A CASE STUDY OF CASEIN HYDROLYSATE MICROENCAPSULATION

A commercial casein hydrolysate was microencapsulated in liposomes produced with non-purified soy lecithin, cryoprotected with two different disaccharides and lyophilized. The encapsulation efficiency of casein hydrolysate ranged from 30 to 40%. The powders were analyzed by differential scanning calorimetry (DSC), scanning electron micrography (SEM), infrared spectroscopy (FTIR) and wide angle X-ray diffraction (WAXD). DSC data revealed the presence of an exothermal transition in empty lyophilized liposomes, which was ascribed to the presence of a quasicrystalline lamellar phase (intermediary characteristics between the L-beta and L-c phases). The addition of peptides to the liposomal system caused the disappearance of this exothermic phenomenon, as they were located in the polar headgroup portion of the bilayer, causing disorder and preventing the formation of the quasicrystalline phase. Infrared data indicated the presence of the peptides in the lyophilized formulations and showed that the cryoprotectants interacted effectively with the polar heads of phospholipids in the bilayer.

Effects of Black Leaf Streak Disease and Sigatoka Disease on fruit quality and maturation process of bananas produced in the subtropical conditions of southern Brazil

Banana fruits are harvested at the green-mature stage (pre-climacteric) in order to allow sufficient time for transport and marketing. The time between the harvest and the initiation of the natural ripening process is called green life (GL), which is closely correlated to physiological age. Sigatoka Disease (SD: also called yellow Sigatoka) and Black Leaf Streak Disease (BLSD; also called black Sigatoka) are the main foliar diseases affecting banana production. The aim of this work was to investigate the influence of these diseases on banana GL and postharvest behavior in subtropical conditions (southeastern Brazil). The results showed that both diseases shortened the banana's GL when compared to control bananas of the same physiological age. Moreover, fruits from infested plots showed higher values of CO2 (+100% for SD and +300% for BLSD) and C2H4 production (+30% for SD and +60% for BLSD) at the climacteric peak. BLSD caused 40% reduction in fruit weight. Fruits from plants with a high degree of SD or BLSD undergo an altered maturation process. (C) 2011 Elsevier Ltd. All rights reserved.

Evaluation of the Concentration of Nonessential and Essential Elements in Chicken, Pork, and Beef Samples Produced in Brazil

Food safety is a global concern. Meat represents the most important protein source for humans. Thus, contamination of meat products by nonessential elements is a ready source of human exposure. In addition, knowledge of the concentration of essential elements is also relevant with respect to human nutrition. The aim of the present study was to determine the concentration of 17 elements in pork, beef, and chicken produced in Brazil. Meat samples were analyzed by inductively coupled plasma mass spectrometry. The estimated daily intake for nonessential elements including arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg), and antimony (Sb) through meat consumption is below the toxicological reference values. However, high levels were detected for the nonessential element cesium (Cs), mainly in beef samples, an observation that deserves future studies to identify the source of contamination and potential adverse consequences.

Acaricidal action of destruxins produced by a marine-derived Beauveria felina on the bovine tick Rhipicephalus (Boophilus) microplus

The increasing resistance of Rhipicephalus (Boophilus) microplus tick to commercial insecticides requires alternative methods for the control of this cattle plague. The enthomopathogenic fungus Beauveria feline produces destruxins in culture media, cyclic depsipeptides which display an array of biological activities. The present investigation aimed to evaluate the acaricide action of destruxins isolated from B. felina culture media on R. (B.) microplus engorged females. B. felina was grown in MF medium under 19 different growth conditions. HPLC-PDA analysis of chromatographic fractions obtained from the 19 different growth media extracts indicated the presence of destruxins in all lipophylic fractions. Such fractions were combined and subjected to separation by HPLC. Fractions containing distinct destruxins composition were tested against R. (B.) micro plus. Two fractions, composed of destruxin Ed(1) and pseudodestruxin B and/or pseudodestruxin C (fraction P1) as well as by hydroxyhomodestruxin B and/or destruxin D and/or roseotoxin C (fraction P7), displayed 30% and 28.7% acaricidal efficacy, respectively. This activity profile in such low concentration is adequate to consider destruxins as potential leading compounds to be developed for tick biological control. (C) 2012 Elsevier Inc. All rights reserved.