Carboxilesterase na adaptação do metabolismo de lipídios do pacu à hipoxia

Os peixes são vertebrados que vivem em vários habitats. Adaptações de sua fisiologia e de sua bioquímica são estudadas para entender como conseguem sobreviver aos desafios presentes em cada ambiente. A diminuição da concentração de oxigênio dissolvido na água é um fenômeno natural cíclico em águas do Pantanal e da Amazônia. A crescente poluição antrópica dos rios da Amazônia e do Pantanal pode, somada à hipoxia, oferecer ameaça à permanência de muitas espécies de peixes. Ao estudarmos a carboxilesterase (CarbE), enzima importante para a biotransformação de xenobióticos, observamos que sua atividade diminuía em plasma de pacu, um peixe típico do Pantanal, mantido em hipoxia durante 42 horas. As carboxilesterases participam de inúmeras reações químicas no organismo, incluindo uma transesterificação capaz de produzir ésteres etílicos tóxicos de ácido graxo (FAEE, fatty acid ethyl esters) a partir de etanol e ésteres de ácidos graxos. Já que a diminuição da atividade de CarbE poderia ser uma vantagem, pois que o etanol (um produto da glicólise em peixes sob hipoxia) seria menos esterificado, resolvemos saber mais sobre a bioquímica da CarbE do plasma e do fígado de pacus. Os pacus foram submetidos à hipoxia por diminuição da concentração até 0,5 mg O2/L por meio de borbulhamento de nitrogênio na água. Os animais ficaram nessas condições por 42 horas, quando então coletamos sangue e retiramos seus fígados. A atividade de CarbE ensaiada foi 50% menor no soro e 25% menor nos microssomos de fígado se comparada com a de peixes sob 6 mg O2/L. A CarbE isolada do soro dos pacus em normoxia possui massa molecular relativa de 56.000. A eletroforese em gel desnaturante com a fração purificada rendeu três bandas, mas o gel nativo apresentou só duas bandas com atividades sobre α-naftil acetato. Inferimos que mais do que uma isoforma da enzima está no plasma dos pacus. A CarbE isolada do soro não possui atividade de lipase sobre o Tween 20, mas os microssomos dos fígados dos animais em normoxia e hipoxia possuem. No entanto, a CarbE possui atividade sobre acil-CoA assim como o microssomo de fígado. A enzima pura apresenta Vmáx três vezes maior e uma KM quatro vezes maior do que a atividade presente nos microssomos. Além disso, os microssomos do fígado dos pacus em normoxia podem hidrolisar acil-CoA com o dobro da velocidade daqueles em hipoxia. A atividade clássica de CarbE do soro foi inibida por 4-HNE a 4 mM. A atividade de acil-CoA dos microssomos de fígado também foi sensível a 4-HNE a 4 mM, enquanto 2 mM de 4-HNE inibiu metade desta atividade do soro.

Saline-saturated DMSO-EDTA as a storage medium for microbial DNA analysis from coral mucus swab samples

The mucus surface layer of corals plays a number of integral roles in their overall health and fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier against physical invasions and trauma, and serves as a medium to host a community of microorganisms distinct from the surrounding seawater. In healthy corals the associated microbial communities are known to provide antibiotics that contribute to the coral’s innate immunity and function metabolic activities such as biogeochemical cycling. Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change with changes in the environment and health condition of the coral. These changes may suggest that changes in the microbial associates not only reflect health status but also may assist corals in acclimating to changing environmental conditions. With the increasing availability of molecular biology tools, culture-independent methods are being used more frequently for evaluating the health of the animal host. Although culture-independent methods are able to provide more in-depth insights into the constituents of the coral surface mucus layer’s microbial community, their reliability and reproducibility rely on the initial sample collection maintaining sample integrity. In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry shipper. The samples are then sent to a laboratory for analysis. After the initial collection and preservation of the sample, the duration of the sample voyage to a recipient laboratory is often another critical part of the sampling process, as unanticipated delays may exceed the length of time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust prematurely. In remote areas, service by international shipping companies may be non-existent, which requires the use of an alternative preservation medium. Other methods for preserving environmental samples for microbial DNA analysis include drying on various matrices (DNA cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, however, they add expense and permitting requirements for hazardous liquid components, and the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et al., 2010). A method to preserve coral mucus samples without cold storage or use of hazardous solvents, while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a means of storing tissue of marine invertebrates at ambient temperatures without significant loss of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the coral microbiota DNA would be adversely affected by this storage medium either by degradation of the DNA, or a bias in the DNA recovered during the extraction process created by variations in extraction efficiencies among the various community members. Tests to determine the efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are presented here.

Viscoelastic properties of bone as a function of hydration state determined by nanoindentation

Spherical indentation creep testing was used to examine the effect of hydration state on bone mechanical properties. Analysis of creep data was based on the elastic-viscoelastic correspondence principle and utilized a direct solution for the finite loading-rate experimental conditions. The zero-time shear modulus was computed from the creep compliance function and compared to the indentation modulus obtained via conventional indentation analysis, based on an elastic unloading response. The method was validated using a well-known polymer material under three different loading conditions. The method was applied to bone samples prepared with different water content by partial exchange with ethanol, where 70% ethanol was considered as the baseline condition. A hydration increase was associated with a 43% decrease in stiffness, while a hydration decrease resulted in a 20% increase in bone tissue stiffness.

Nanoindentation measurements of bone viscoelasticity as a function of hydration state

Bone is an anisotropic material, and its mechanical properties are determined by its microstructure as well as its composition. Mechanical properties of bone are a consequence of the proportions of, and the interactions between, mineral, collagen and water. Water plays an important role in maintaining the mechanical integrity of the composite, but the manner in which water interacts within the ultrastructure is unclear. Dentine being an isotropic two-dimensional structure presents a homogenous composite to examine the dehydration effects. Nanoindentation methods for determining the viscoelastic properties have recently been developed and are a subject of great interest. Here, one method based on elastic-viscoelastic correspondence for 'ramp and hold' creep testing (Oyen, J. Mater. Res., 2005) has been used to analyze viscoelastic behavior of polymeric and biological materials. The method of 'ramp and hold' allows the shear modulus at time zero to be determined from fitting of the displacement during the maximum load hold. Changes in the viscoelastic properties of bone and dentine were examined as the material was systematically dehydrated in a series of water:solvent mixes. Samples of equine dentine were sectioned and cryo-polished. Shear modulus was obtained by nanoindentation using spherical indenters with a maximum load hold of 120s. Samples were tested in different solvent concentrations sequentially, 70% ethanol to 50% ethanol, 70 % ethanol to 100% ethanol, 70% ethanol to 70% methanol to 100% methanol, and 70% ethanol to 100% acetone, after storage in each condition for 24h. By selectively removing and then replacing water from the composite, insights in to the ultrastructure of the tissue can be gained from the corresponding changes in the experimentally determined moduli, as well as an understanding of the complete reversibility of the dehydration process. © 2006 Materials Research Society.

Poroelastic indentation analysis for hydrated biological tissues

Indentation techniques are employed for the measurement of mechanical properties of a wide range of materials. In particular, techniques focused at small length-scales, such as nanoindentation and AFM indentation, allow for local characterization of material properties in heterogeneous materials including natural tissues and biomimetic materials. Typical elastic analysis for spherical indentation is applicable in the absence of time-dependent deformation, but is inappropriate for materials with time-dependent responses. Recent analyses for the viscoelastic indentation problem, based on elastic-viscoelastic correspondence, have begun to address the issue of time-dependent deformation during an indentation test. The viscoelastic analysis has been shown to fit experimental indentation data well, and has been demonstrated as useful for characterization of viscoelasticity in polymeric materials and in hydrated mineralized tissues. However, a viscoelastic analysis is not necessarily sufficient for multi-phase materials with fluid flow. In the current work, a poroelastic analysis-based on fluid motion through a porous elastic network-is used to examine spherical indentation creep responses of hydrated biological materials. Both analytical and finite element approaches are considered for the poroelastic Hertzian indentation problem. Modeling results are compared with experimental data from nanoindentation of hydrated bone immersed in water and polar solvents (ethanol, methanol, acetone). Baseline (water-immersed) bone responses are characterized using the poroelastic model and numerical results are compared with altered hydration states due to polar solvents. © 2007 Materials Research Society.

Effect of water on mechanical properties of mineralized tissue composites

In the current study, the effects of polar solvents on tissue volume and mechanical properties are considered. Area shrinkage measurements are conducted for mineralized bone tissue samples soaked in polar solvents. Area shrinkage is used to calculate approximate linear and volume shrinkage. Results are compared with viscoelastic mechanical parameters for bone in the same solvents (as measured previously) and with both shrinkage measurements and mechanical data for nonmineralized tissues, as taken from the existing literature. As expected, the shrinkage of mineralized tissues is minimal when compared with shrinkage of nonmineralized tissues immersed in the same polar solvents. The mechanical changes in bone are also substantially less than in nonmineralized tissues. The largest stiffness values are found in shrunken bone samples (immersed in acetone and ethanol). The mineral phase in bone thus resists tissue shrinkage that would otherwise occur in the pure soft tissue phase. © 2007 Materials Research Society.

Cryopreservation of sperm of common carp, Cyprinus carpio and silver barb, Barbonymus gonionotus for genetically improved seed production

Experiments were conducted to develop and standardize the protocols for cryopreservation of sperm of common carp, Cyprinus carpio and also for using the cryopreserved sperm for fertilization of eggs. Nine extender solutions as Alsever's solution, kurokura-1, kurokura-2, urea egg-yolk, egg-yolk citrate, 0.6% glucose, 0.9% NaCl, Ma and Mb, and five cryoprotectants namely ethanol, methanol, dimethylsulfoxide (DMSO), dimethylamine (DMA) and glycerol were tested. The cryoprotectants were mixed at 10% concentration of the extenders (v/v) to make the cryodiluents. Milt and cryodiluents were mixed at a ratio of 1:9 for Alsever's solution, kurokura-1, kurokura-2, 0.6% glucose and 0.9% NaCl, 1:4 for urea egg-yolk, egg-yolk citrate, Ma and Mb. Among the cryodiluents Alsever's solution mixed with either ethanol or methanol was found to be suitable and it produced more than 90% and 80% spermatozoan motility at equilibrium and post-thaw periods, respectively. Kurokura-1 and kurokura-2 when mixed with the same cryoprotectants showed good spermatozoan motility at equilibrium period (80-90%) but the motility was reduced (30-55%) at post-thaw state. Other extenders did not produce acceptable sperm-motility and in some cases the frozen milt became clotted. Different dilution ratios (1:1, 1:2, 1:4, 1:5, 1:7, 1:9, 1:12, 1:15, 1:20) were formulated for obtaining a suitable milt dilution, the dilution ratio of 1: 9 (milt : cryodiluent) demonstrated the highest post-thaw spermatozoan motility (80%) in Alserver's solution. The optimum concentration of cryoprotectants in the cryodiluents was determined, 10% concentration level was found to be effective to produce the highest number of spermatozoan motility in comparison to the other concentrations (5%, 15%, 20% 30%). Sperm preserved with the cryodiluent Alsever's solution along with either methanol or ethanol was found to be effective to fertilize eggs and produce hatchlings. The hatching rates ranged between 1.48% and 14.76%, compare to control. The fish produced through use of cryopreserved sperm and normal sperm were found to grow well and no significant (P<0.05) growth difference was observed between them. In case of silver barb, Barbonymus gonionotus, sperm tested against six extenders such as egg-yolk citrate, urea-egg-yolk, kurokura-1, kurokura-2, 0.9% NaCl and modified fish ringer (MFR) solution. Cryoprotectants used were the same as those of C. carpio. Milt was diluted with the cryodiluent at a ratio of 1:4 for egg-yolk citrate and urea-egg-yolk, 1:5 for kurokura-1 and 1:9 for 0.9% NaCl, MFR and kurokura-2. The cryoprotectant concentration was maintained at 10% of the extender (v/v) in all the cases. Among the extenders, egg-yolk citrate and urea-egg-yolk mixed with 10% DMSO, methanol and ethanol produced 50% post-thaw spermatozoan motility, whereas DMA and glycerol provided only 10% motility. Trials on milt dilution ratio and cryoprotectant concentration are being conducted. Fertilization trials are also underway.

Novel dual transient temperature modulation technique for multi-vapour detection

Here we present a novel signal processing technique for a square wave thermally-modulated carbon black/polymer composite chemoresistor. The technique consists of only two mathematical operations: summing the off-transient and on-transient conductance signals; and subtracting the steady-state conductance signal. A single carbon black/polyvinylpyrrolidone composite chemo -resistor was fabricated and used to demonstrate the validity of the technique. Classification of water, methanol and ethanol vapours was successfully performed using only the peak time of the resultant curves. Quantification of the different vapours was also possible using the height of the peaks, because it was linearly proportional to concentration. This technique does not require zero-gas calibration and thus is superior to previously reported methods. ©2009 IEEE.

Sperm cryopreservation of Indian major carp, Labeo rohita: cryodiluents, sperm: cryodiluent dilution ratio and cryoprotectan t concentration

Cryogenic preservation trials of spermatozoa of Labeo rohita were carried out. Twenty four cryodiluents (extender + cryoprotectant), with the combination of six extenders such as egg-yolk citrate, urea-egg-yolk, 0.9% NaCl, Kurokura-2, Ma and Mb and four cryoprotectants viz. DMSO, glycerol, methanol and ethanol, were used to screen out the suitable cryodiluents. Sperm was preserved in 0.25ml plastic straw in programmable freezer. Two step freezing method was followed. Sperm preserved with egg-yolk citrate and urea-egg-yolk containing 10% DMSO showed best post-thaw motility (80%) followed by 0.9% NaCl (60%) and Kurokura-2(30%) solutions. Sperm with the extenders M" and Mb clotted at the time of equilibration and also after few days of preservation. Egg-yolk citrate mixed with ethanol and methanol also showed good percentage of motility (80%) but egg-yolk citrate with glycerol showed less sperm motility (>60%). To determine suitable dilution ratio of milt and cryodiluent two best extender eggyolk citrate and urea-egg-yolk with four cryoprotectants such as DMSO, glycerol, methanol and ethanol at different ratio viz 1:2,1:4,1:7,1:10,1:15 and 1:20 were used. Highest post-thaw motility (>80%) was observed when milt was preserved with egg-yolk citrate containing 10% DMSO at 1:2, 1:4, 1:7 and 1:10 dilutions. Meanwhile using glycerol as cryoprotectants provided less post thaw motility at lower dilution ratio but with the increase of its dilution showed good sperm motility compared with other cryoprotectants. Finally, evaluation on the effect of cryoprotectant concentration on post-thaw sperm motility was conducted. Egg-yolk citrate and four cryoprotectant i.e. DMSO, glycerol, methanol and ethanol with six different concentrations namely 5%,7%, 10%, 15%, 20% and 30%.were evaluated. Among the cryoprotectants DMSO, methanol and ethanol showed highest post-thaw motility (about 80%) at 7% and 10% concentrations. Although glycerol was not suitable at low concentration but its 20% and 30% concentration levels provided best post-thaw motility. No post-thaw motility was obtained with DMSO at 30% concentration. The overall analysis on cryoprotectant concentration indicated that below 5% and above 20% cryoprotectant concentrations could not be suitable for effective cryopreservation of spermatozoa.

Velleratretraol, an unusual highly functionalized lactarane sesquiterpene from Lactarius vellereus

An unusual highly functionalized lactarane sesquiterpene, named velleratretraol (1), was isolated from the ethanol extract of the fruiting body of the mushroom Lactarius vellereus. Its structure was determined through spectroscopic analysis and single-cry

Genetic diversity study of Caspian brown trout (Salmo trutta caspius Kessler, 1877) population in five rivers in the southern part of Caspian Sea, Iran

For study the genetic diversity of Caspian brown trout population in five rivers in the southern part of Caspian Sea in Iran 182 number generators in the fall and winter of 1390 were collected in Chalus, Sardab Rud, Cheshmeh Kileh, Kargan Rud and Astara rivers. Then about 3-5 g of soft and fresh tissue from the bottom fin fish removed and were fixed in ethanol 96°. Genomic DNA was extracted by using ammonium acetate, then quantity and quality of the extracted DNA were determined by using spectrophotometry and horizontal electrophoresis in 1% agarose gel. The polymerase chain reaction was performed by using 16 SSR primers and sequencing primers (D-Loop) and the quality of PCR products amplified by SSR method were performed by using horizontal electrophoresis in 2% agarose gel. Alleles and their sizes were determined by using vertical electrophoresis in 6% polyacrylamide gel and silver nitrate staining method. Gel images were recorded by gel documentarian, the bands were scored by using Photo- Capt software and statistical analysis was performed by using Gene Alex and Pop Gene software. Also the PCR sequencing products after quality assessment by usinghorizontal electrophoresis in 1.5% agarose gel were purified and sent to South Korea Bioneer Corporation for sequencing. Sequencing was performed by chain termination method and the statistical analysis was performed by using Bio- Edit, Mega, Arlequin and DNA SP software. The SSR method, 5 pairs of primers produced polymorphic bands and the average real and effective number of alleles were calculated 5.60±1.83 and 3.87±1.46 in the Cheshmeh Kileh river and 7.60±1.75 and 5.48±1.32 in the Karganrud river and the mean observed and expected heterozygosity were calculated 0.44 ±0.15 and 0.52 ±0.16 in the Cheshmeh Kileh river and 0.50 ±0.11 and 0.70±0.13 in the Karganrud river. Analysis of Molecular Variance results showed that significant differences in genetic diversity between and within populations and between and within individuals in the studied rivers (P<0.01). The sequencing method identified 35 different haplotype, the highest number of polymorphic position (251) and haplotype (14) were observed in the Chalus river. The highest mean observed number of alleles (2.24±0.48) was calculated in the Sardabrud river, the highest mean observed heterozygosity (1.00±0.03) was calculated in the Chalus river and the highest mean nucleotide diversity (0.13±0.07) was observed in the Sardabrud river and mean haplotype diversity was obtained (1) in three studied rivers. The overall results show that there are no same population of this fish in the studied rivers and Karganrud and Chalus rivers in the SSR and sequencing methods had the highest levels of genetic diversity.

Implications of local lifecycle analyses and low carbon fuel standard design on gasohol transportation fuels

State and regional policies, such as low carbon fuel standards (LCFSs), increasingly mandate that transportation fuels be examined according to their greenhouse gas (GHG) emissions. We investigate whether such policies benefit from determining fuel carbon intensities (FCIs) locally to account for variations in fuel production and to stimulate improvements in FCI. In this study, we examine the FCI of transportation fuels on a lifecycle basis within a specific state, Minnesota, and compare the results to FCIs using national averages. Using data compiled from 18 refineries over an 11-year period, we find that ethanol production is highly variable, resulting in a 42% difference between carbon intensities. Historical data suggests that lower FCIs are possible through incremental improvements in refining efficiency and the use of biomass for processing heat. Stochastic modeling of the corn ethanol FCI shows that gains in certainty due to knowledge of specific refinery inputs are overwhelmed by uncertainty in parameters external to the refiner, including impacts of fertilization and land use change. The LCA results are incorporated into multiple policy scenarios to demonstrate the effect of policy configurations on the use of alternative fuels. These results provide a contrast between volumetric mandates and LCFSs. © 2011 Elsevier Ltd.

Self-similar breakup of near-inviscid liquids

The final stages of pinchoff and breakup of dripping droplets of near-inviscid Newtonian fluids are studied experimentally for pure water and ethanol. High-speed imaging and image analysis are used to determine the angle and the minimum neck size of the cone-shaped extrema of the ligaments attached to dripping droplets in the final microseconds before pinchoff. The angle is shown to steadily approach the value of 18.0 ±0.4, independently of the initial flow conditions or the type of breakup. The filament thins and necks following a τ2 /3 law in terms of the time remaining until pinchoff, regardless of the initial conditions. The observed behavior confirms theoretical predictions. © 2012 American Physical Society.

Autoignition of monodisperse biodiesel and diesel sprays in turbulent flows

A new experimental configuration has been developed to examine the effects of flow on the autoignition of dilute diesel and biodiesel sprays, where the spray is injected in the form of monodisperse individual droplets at right angles to a hot air turbulent flow. The ignition location has been measured by monitoring the OH * chemiluminescence. A qualitative comparison of the flame behaviour between ethanol, acetone, heptane and biodiesel as fuels has also been carried out. With decreasing volatility of the fuel, the flame showed progressively a higher number of individual droplets burning, with the first autoignition spots appearing at random locations but in general earlier than the intense droplet-flame emission. The time-averaged autoignition length increased with increasing air velocity and with increasing intensity of the turbulence, while it decreased with the temperature and the droplet size. The data can be used for validating models for two-phase turbulent combustion. © 2012 Elsevier Inc.

Self-similar breakup of near-inviscid liquids.

The final stages of pinchoff and breakup of dripping droplets of near-inviscid Newtonian fluids are studied experimentally for pure water and ethanol. High-speed imaging and image analysis are used to determine the angle and the minimum neck size of the cone-shaped extrema of the ligaments attached to dripping droplets in the final microseconds before pinchoff. The angle is shown to steadily approach the value of 18.0 ± 0.4°, independently of the initial flow conditions or the type of breakup. The filament thins and necks following a τ(2/3) law in terms of the time remaining until pinchoff, regardless of the initial conditions. The observed behavior confirms theoretical predictions.