Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

Heat-shock protein70 gene expression in four hatchery Pacific Abalone Haliotis discus hannai Ino populations using for marker-assisted selection

Heat shock proteins (Hsps) are molecular chaperones that help organisms cope with stressful conditions. Here, we report on the growth rates and Hsp70 expressions in inbred and hybrid populations of abalone Haliotis discus hannai Ino. In abalone, inbred populations expressed more Hsp70 than hybrid populations at all temperatures, except at very high temperatures close to the physiological limit. At benign temperatures, there was a clear trend towards higher Hsp70 expression in inbred than hybrid populations, whereas at higher temperatures, a trend in the opposite direction was observed. The temperature of maximal Hsp70 expression (T-peak) varied with the population type. The T-peak of inbred populations (26 degrees C) was lower than that of the hybrid populations (28 degrees C). The maximal inducible Hsp70 of inbred populations was higher than that of hybrid populations. The results showed a trend towards higher expression in inbred population at a lower temperature. These results provide direct experimental evidence that hybrids can cope with the intrinsic stress even at non-stressful temperatures. The constitutive Hsp70 may therefore be used for marker-assisted selection in a breeding programme.

Proteomic approach to oxidative stress in Daphnia magna

The keystone aquatic organism Daphnia magna is extensively used to assess the toxicity of chemicals. This has recently lead to an increase in the omics literature focusing on daphnids, an increase fuelled by the sequencing of the Daphnia pulex genome. Yet, no omics study has looked directly at oxidative stress (OS) in daphnids, even though OS is of primary importance in the response of aquatic organisms to their changing environment and is often induced by anthropogenic xenobiotics. This thesis thus focuses on the application of redox-proteomics, the study of the oxidative modification of proteins, to D. magna Specifically, daphnids were exposed to copper or paraquat, two well studied prooxidants, and protein carbonyls were labelled with fluorescein-5-thiosemicarbazide prior to twodimensional electrophoresis (2DE). This showed clearly that both compounds affect a different portion of the proteome. The identified proteins indicated that energy metabolism was affected by paraquat, while copper induced a reduction of the heat shock response (heat shock proteins, proteases and chaperones) a counterintuitive result which may be adaptative to metal toxicity in arthropods. The same approach was then applied to the study of the toxicity mechanism of silver nanoparticles (AgNP), an increasingly utilised form of silver with expected environmental toxicity, and its comparison to silver nitrate. The results demonstrate that, although less toxic than silver ions, AgNP toxicity functions through a different mechanism. AgNP toxicity is thus not a product of silver dissolution and increased protein carbonylation indicates that AgNP cause OS. Interestingly three of the four tested compounds altered vitellogenin levels and oxidation. Vitellogenins could thus represent an interesting subproteome for the detection of stress in daphnids. Finally, an experiment with oxidised BSA demonstrates the applicability of solid phase hydrazide in the enrichment of undigested carbonylated proteins.

Transgenerational Effects of Early Life Starvation on Growth, Reproduction, and Stress Resistance in Caenorhabditis elegans.

Starvation during early development can have lasting effects that influence organismal fitness and disease risk. We characterized the long-term phenotypic consequences of starvation during early larval development in Caenorhabditis elegans to determine potential fitness effects and develop it as a model for mechanistic studies. We varied the amount of time that larvae were developmentally arrested by starvation after hatching ("L1 arrest"). Worms recovering from extended starvation grew slowly, taking longer to become reproductive, and were smaller as adults. Fecundity was also reduced, with the smallest individuals most severely affected. Feeding behavior was impaired, possibly contributing to deficits in growth and reproduction. Previously starved larvae were more sensitive to subsequent starvation, suggesting decreased fitness even in poor conditions. We discovered that smaller larvae are more resistant to heat, but this correlation does not require passage through L1 arrest. The progeny of starved animals were also adversely affected: Embryo quality was diminished, incidence of males was increased, progeny were smaller, and their brood size was reduced. However, the progeny and grandprogeny of starved larvae were more resistant to starvation. In addition, the progeny, grandprogeny, and great-grandprogeny were more resistant to heat, suggesting epigenetic inheritance of acquired resistance to starvation and heat. Notably, such resistance was inherited exclusively from individuals most severely affected by starvation in the first generation, suggesting an evolutionary bet-hedging strategy. In summary, our results demonstrate that starvation affects a variety of life-history traits in the exposed animals and their descendants, some presumably reflecting fitness costs but others potentially adaptive.

Investigation into the performance of turbulence models for fluid flow and heat transfer phenomena in electronic applications

Heat is extracted away from an electronic package by convection, conduction, and/or radiation. The amount of heat extracted by forced convection using air is highly dependent on the characteristics of the airflow around the package which includes its velocity and direction. Turbulence in the air is also important and is required to be modeled accurately in thermal design codes that use computational fluid dynamics (CFD). During air cooling the flow can be classified as laminar, transitional, or turbulent. In electronics systems, the flow around the packages is usually in the transition region, which lies between laminar and turbulent flow. This requires a low-Reynolds number numerical model to fully capture the impact of turbulence on the fluid flow calculations. This paper provides comparisons between a number of turbulence models with experimental data. These models included the distance from the nearest wall and the local velocity (LVEL), Wolfshtein, Norris and Reynolds, k-��, k-��, shear-stress transport (SST), and k��/kl models. Results show that in terms of the fluid flow calculations most of the models capture the difficult wake recirculation region behind the package reasonably well, although for packages whose heights cause a high degree of recirculation behind the package the SST model appears to struggle. The paper also demonstrates the sensitivity of the models to changes in the mesh density; this study is aimed specifically at thermal design engineers as mesh independent simulations are rarely conducted in an industrial environment.

Microstructure evolution of a 10Cr heat-resistant steel during high temperature creep

The microstructure evolution of a 10Cr ferritic/martensitic heat-resistant steel during creep at 600��C was investigated in this work. Creep tests demonstrated that the 10Cr steel had higher creep strength than conventional ASME-P92 steel at 600��C. The microstructure after creep was studied by transmission electron microscopy, scanning electron microscopy and electron probe microanalysis. It was revealed that the martensitic laths were coarsened with time and eventually developed into subgrains after 8354 h. Laves phase was observed to grow and cluster along the prior austenite grain boundaries during creep and caused the fluctuation of solution and precipitation strengthening effects, which was responsible for the two slope changes on the creep rupture strength vs rupture time curve. It was also revealed that the microstructure evolution could be accelerated by stress, which resulted in the lower hardness in the deformed part of the creep specimen, compared with the aging part.

Transient Heat Conduction of Variable Stiffness Composite Laminate

Recent research on Variable Stiffness (VS) laminates, which are constructed by steering the fiber orientation as a spatial function of location, have shown to improve laminate performance under mechanical loads. Two distinct cases of stiffness variation can be achieved either by variation of the fiber orientation in the direction of the global x-axis, or perpendicular to it. In the present paper, thermal analysis of a VS laminate is performed to study the effect of steering fibers on transient heat conduction under uniform heat flux using finite element method. The goal of the present paper is a parametric study of the effect of variable stiffness properties on transient response including time to reach steady state and temperature profile. Also, stress resultants and maximum stress location are investigated under different boundary conditions. A FEM algorithm is applied to exactly incorporate the boundary conditions for stress resultant analysis.

Transient Heat Conduction of Variable Stiffness Composite Laminate

Recent research on Variable Stiffness (VS) laminates, which are constructed by steering the fiber orientation as a spatial function of location, have shown to improve laminate performance under mechanical loads. Two distinct cases of stiffness variation can be achieved either by variation of the fiber orientation in the direction of the global x-axis, or perpendicular to it. In the present paper, thermal analysis of VS laminate is performed to study the effect of steering fibers on transient heat conduction under uniform heat flux using finite element method. The goal of the present paper is a parametric study of the<br/>effect of variable stiffness properties on transient response including time to reach steady state and temperature profile. Also, stress resultants and maximum stress location are investigated under different boundary conditions. A FEM algorithm is applied to exactly incorporate the boundary conditions.

Susceptibility to stress corrosion cracking of NiTi laser weldment in Hanks��� solution

In this study, the susceptibility to stress corrosion cracking (SCC) of laser-welded NiTi wires in Hanks��� solution at 37.5 ��C was studied by the slow strain-rate test (SSRT) at open-circuit potential and at different applied anodic potentials. The weldment shows high susceptibility to SCC when the applied potential is near to the pitting potential of the heat-affected zone (HAZ). The pits formed in the HAZ become sites of crack initiation when stress is applied, and cracks propagate in an intergranular mode under the combined effect of corrosion and stress. In contrast, the base-metal is immune to SCC under similar conditions. The increase in susceptibility to SCC in the weldment could be attributed to the poor corrosion resistance in the coarse-grained HAZ.

Hot deformation characteristics of a nitride strengthened martensitic heat resistant steel

Constitutive equations including an Arrhenius term have been applied to analyze the hot deformation behavior of a nitride-strengthened (NS) martensitic heat resistant steel in temperature range of 900���1200 ��C and strain rate range of 0.001���10 /s. On the basis of analysis of the deformation data, the stress���strain curves up to the peak were divided into four regions, in sequence, representing four processes, namely hardening, dynamic recovery (DRV), dynamic strain induced transformation (DSIT), and dynamic recrystallization (DRX), according to the inflection points in �����/����������/����� and ���(�����/�����)/��������(�����/�����)/����� curves. Some of the inflection points have their own meanings. For examples, the minimum of �����/����������/����� locates the start of DRV and the maximum of it indicates the start of DRX. The results also showed that the critical strain of DRX was sensitive to ln(Z) below 40, while the critical stress of DRX was sensitive to it above 40. The final microstructures under different deformation conditions were analyzed in terms of softening processes including DRV, DRX, metadynamic crystallization (MDRX) and DSIT.

Analysis of deformation behavior and workability of advanced 9Cr-Nb-V ferritic heat resistant steels

Hot compression tests were carried out on 9Cr���Nb���V heat resistant steels in the temperature range of 600���1200 ��C and the strain rate range of 10���2���100 s���1 to study their deformation characteristics. The full recrystallization temperature and the carbon-free bainite phase transformation temperature were determined by the slope-change points in the curve of mean flow stress versus the inverse of temperature. The parameters of the constitutive equation for the experimental steels were calculated, including the stress exponent and the activation energy. The lower carbon content in steel would increase the fraction of precipitates by increasing the volume of dynamic strain-induced (DSIT) ferrite during deformation. The ln(��c) versus ln(Z) and the ln(��c) versus ln(Z) plots for both steels have similar trends. The efficiency of power dissipation maps with instability maps merged together show excellent workability from the strain of 0.05 to 0.6. The microstructure of the experimental steels was fully recrystallized upon deformation at low Z value owing to the dynamic recrystallization (DRX), and exhibited a necklace structure under the condition of 1050 ��C/0.1 s���1 due to the suppression of the secondary flow of DRX. However, there were barely any DRX grains but elongated pancake grains under the condition of 1000 ��C/1 s���1 because of the suppression of the metadynamic recrystallization (MDRX).

Constitutive modeling, microstructure evolution, and processing map for a nitride-strengthened heat-resistant steel

A constitutive equation was established to describe the deformation behavior of a nitride-strengthened (NS) steel through isothermal compression simulation test. All the parameters in the constitutive equation including the constant and the activation energy were precisely calculated for the NS steel. The result also showed that from the stress-strain curves, there existed two different linear relationships between critical stress and critical strain in the NS steel due to the augmentation of auxiliary softening effect of the dynamic strain-induced transformation. In the calculation of processing maps, with the change of Zener-Hollomon value, three domains of different levels of workability were found, namely excellent workability region with equiaxed-grain microstructure, good workability region with ���stripe��� microstructure, and the poor workability region with martensitic-ferritic blend microstructure. With the increase of strain, the poor workability region first expanded, then shrank to barely existing, but appeared again at the strain of 0.6.

Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation

<p>The virulence to insects and tolerance to heat and UV-B radiation of conidia of entomopathogenic fungi are greatly influenced by physical, chemical, and nutritional conditions during mycelial growth. This is evidenced, for example, by the stress phenotypes of Metarhizium robertsii produced on various substrates. Conidia from minimal medium (Czapek's medium without sucrose), complex medium, and insect (Lepidoptera and Coleoptera) cadavers had high, moderate, and poor tolerance to UV-B radiation, respectively. Furthermore, conidia from minimal medium germinated faster and had increased heat tolerance and were more virulent to insects than those from complex medium. Low water-activity or alkaline culture conditions also resulted in production of conidia with high tolerance to heat or UV-B radiation. Conidia produced on complex media exhibited lower stress tolerance, whereas those from complex media supplemented with NaCl or KCl (to reduce water activity) were more tolerant to heat and UV-B than those from the unmodified complex medium. Osmotic and nutritive stresses resulted in production of conidia with a robust stress phenotype, but also were associated with low conidial yield. Physical conditions such as growth under illumination, hypoxic conditions, and heat shock before conidial production also induced both higher UV-B and heat tolerance; but conidial production was not decreased. In conclusion, physical and chemical parameters, as well as nutrition source, can induce great variability in conidial tolerance to stress for entomopathogenic fungi. Implications are discussed in relation to the ecology of entomopathogenic fungi in the field, and to their use for biological control. This review will cover recent technologies on improving stress tolerance of entomopathogenic fungi for biological control of insects.</p>

The role of environmental stress on protein synthesis fidelity

Estudos recentes estabelecem uma liga����o entre erros na tradu����o do mRNA e cancro, envelhecimento e neurodegenera����o. RNAs de transfer��ncia mutantes que introduzem amino��cidos em locais errados nas prote��nas aumentam a produ����o de esp��cies reactivas de oxig��nio e a express��o de genes que regulam autofagia, ribofagia, degrada����o de prote��nas n��o-funcionais e protec����o contra o stress oxidativo. Erros na tradu����o do mRNA est��o portanto relacionados com stress proteot��xico. Sabe-se agora que o mecanismo de toxicidade do cr��mio est�� associado �� diminui����o da fidelidade de tradu����o e �� agrega����o de prote��nas com malforma����es que destabilizam a sua estrutura terci��ria. Desta forma, �� poss��vel que os efeitos do stress ambiental ao n��vel da degenera����o celular possam estar relacionados com a altera����o da integridade da maquinaria da tradu����o. Neste estudo procedeu-se a uma avalia����o alargada do impacto do stress ambiental na fidelidade da s��ntese de prote��nas, utilizando S. cerevisiae como um sistema modelo. Para isso recorreu-se a rep��rteres policistr��nicos de luciferase que permitiram quantificar especificamente a supress��o de cod��es de termina����o e o erro na leitura do cod��o AUG em c��lulas exposta a concenta����es n��o letais de metais pesados, etanol, cafe��na e H2O2. Os resultados sugerem que a maquinaria de tradu����o �� na generalidade muito resistente ao stress ambiental, devido a uma conjuga����o de mecanismos de homeostase que muito eficientemente antagonizam o impacto negativo dos erros de tradu����o. A nossa abordagem quantitativa permitiu-nos a identificar genes regulados por uma resposta programada ao stress ambiental que s��o tamb��m essenciais para mitigar a ocorr��ncia de erros de tradu����o, nomeadamente, HSP12, HSP104 e RPN4. A exposi����o prolongada ao stress ambiental conduz �� satura����o dos mecanismos de homeostase, contribuindo para a acumula����o de prote��nas contendo erros de tradu����o e diminuindo a disponibilidade de prote��nas funcionais directamente envolvidas na manuten����o da fidelidade de tradu����o e integridade celular. Ao contr��rio de outras Hsps, a Hsp12p adopta normalmente uma localiza����o membranar em condi����es de stress, que pode modular a fluidez e estabilidade membranar, sugerindo que a membrana plasm��tica �� um alvo preferencial da perda de fidelidade da tradu����o. Para melhor compreender as respostas celulares aos erros de tradu����o, c��lulas contendo dele����es em genes codificadores das Hsps foram transformadas com tRNAs mutantes que introduzem altera����es no proteoma. Os nossos resultados demonstram que para al��m da resposta geral ao stress, estes tRNAs induzem altera����es a n��vel do metabolismo celular e um aumento de aminoacila����o com Metionina em v��rios tRNAs, sugerindo um mecanismo de protec����o contra esp��cies reactivas de oxig��nio. Em conclus��o, este estudo sugere um papel para os erros de tradu����o na gest��o de recursos energ��ticos e na adapta����o das c��lulas a ambientes desfavor��veis.

Clams from Tagus estuary : microbiological, physiological and chemical responses to depuration, transpot and environmental stress

Tese de doutoramento, Biologia (Biologia Marinha e Aquacultuta), Universidade de Lisboa, Faculdade de Ci��ncias, 2014