Responses of the Emiliania huxleyi Proteome to Ocean Acidification

Ocean acidification due to rising atmospheric CO2 is expected to affect the physiology of important calcifying marine organisms, but the nature and magnitude of change is yet to be established. In coccolithophores, different species and strains display varying calcification responses to ocean acidification, but the underlying biochemical properties remain unknown. We employed an approach combining tandem mass-spectrometry with isobaric tagging (iTRAQ) and multiple database searching to identify proteins that were differentially expressed in cells of the marine coccolithophore species Emiliania huxleyi (strain NZEH) between two CO2 conditions: 395 (~current day) and ~1340 p.p.m.v. CO2. Cells exposed to the higher CO2 condition contained more cellular particulate inorganic carbon (CaCO3) and particulate organic nitrogen and carbon than those maintained in present-day conditions. These results are linked with the observation that cells grew slower under elevated CO2, indicating cell cycle disruption. Under high CO2 conditions, coccospheres were larger and cells possessed bigger coccoliths that did not show any signs of malformation compared to those from cells grown under present-day CO2 levels. No differences in calcification rate, particulate organic carbon production or cellular organic carbon: nitrogen ratios were observed. Results were not related to nutrient limitation or acclimation status of cells. At least 46 homologous protein groups from a variety of functional processes were quantified in these experiments, of which four (histones H2A, H3, H4 and a chloroplastic 30S ribosomal protein S7) showed down-regulation in all replicates exposed to high CO2, perhaps reflecting the decrease in growth rate. We present evidence of cellular stress responses but proteins associated with many key metabolic processes remained unaltered. Our results therefore suggest that this E. huxleyi strain possesses some acclimation mechanisms to tolerate future CO2 scenarios, although the observed decline in growth rate may be an overriding factor affecting the success of this ecotype in future oceans.

Transport stress in matrinxa, Brycon cephalus (Teleostei : Characidae), at different densities

Matrinxa, Brycon cephalus, is a native teleost fish from the Amazon Basin, and is of economic importance for cultivation for food and sport in Brazil. Mortality losses due to handling and transport of this stenohaline freshwater species are common. The effects of transportation at different densities on the biochemical stress responses of matrinxa (mean weight 1 kg) were examined. Fish were subjected to three different transport densities (100, 200, and 300 kg m(-3)) for four h in water with added salt (0.6%). The fish were bled at departure (baseline level), arrival (immediately after transportation) and at 24 and 96 h after arrival (recovery period). Blood glucose, cortisol, sodium, chloride, potassium and ammonia were used as stress bioindicators. No mortality was observed and no alterations in plasma cortisol were registered. However, blood glucose and ammonia levels increased and serum sodium and plasma chloride decreased on arrival for the fish transported at the highest densities. These stress responses were transient and the concentrations returned to baseline levels within 24 h. This study showed that matrinxa can be transported at densities as high as those tested in the present study, at least under the conditions employed in this study. A recovery period of at least 24 h is strongly recommended.

Sodium chloride added to transport water and physiological responses of Matrinxã Brycon amazonicus (Teleost: Characidae)

A adição de sal à água tem sido utilizada para a mitigação de estresse e aumento da taxa de sobrevivência em peixes. O presente estudo avaliou o efeito do cloreto de sódio (0,0; 1,0; 3,0 e 6.0 g/l) nas concentrações de cortisol plasmático, glicemia, triglicerídios, proteínas total plasmática, hematócrito, hemoglobina, número de eritrócitos, glicogênio e lipídio hepáticos, e lipídio muscular em matrinxã Brycon amazonicum adultos após quatro horas de transporte e durante período de recuperação de 96 h. Amostras foram coletadas antes e depois do transporte, bem como 24 e 96 h após a chegada. O nível de cortisol plasmático estava mais elevado logo após o transporte quando comparado à condição inicial (pré-transporte), exceto para os peixes transportados com sal nas concentrações 3,0 e 6,0 g/l. Comportamento semelhante foi observado para a glicemia, porém os peixes dos tratamentos 0,0, 1,0 e 3,0 g/l necessitaram de período superior a 24 h para recuperar a condição inicial. Foram registrados níveis mais baixos de glicogênio hepático em peixes do tratamento controle (0,0 g/l). Os parâmetros hemoglobina, número de eritrócitos, proteína plasmática total e lipídio hepático não apresentaram alterações durante o período experimental. Os valores de hematócrito diminuíram logo após o transporte em todos os tratamentos, retornando aos níveis iniciais após 24 h. Todos os tratamentos apresentaram redução nos níveis de lipídio muscular e triglicerídios durante o período de recuperação. Os resultados sugerem que a adição de 6,0 g/l de sal na água de transporte reduz as alterações fisiológicas de estresse e que é necessário período de 96 h após o transporte para a recuperação da condição inicial de matrinxãs transportados sem a adição de sal.

A non-classical LysR-type transcriptional regulator PA2206 is required for an effective oxidative stress response in Pseudomonas aeruginosa

LysR-type transcriptional regulators (LTTRs) are emerging as key circuit components in regulating microbial stress responses and are implicated in modulating oxidative stress in the human opportunistic pathogen Pseudomonas aeruginosa. The oxidative stress response encapsulates several strategies to overcome the deleterious effects of reactive oxygen species. However, many of the regulatory components and associated molecular mechanisms underpinning this key adaptive response remain to be characterised. Comparative analysis of publically available transcriptomic datasets led to the identification of a novel LTTR, PA2206, whose expression was altered in response to a range of host signals in addition to oxidative stress. PA2206 was found to be required for tolerance to H2O2 in vitro and lethality in vivo in the Zebrafish embryo model of infection. Transcriptomic analysis in the presence of H2O2 showed that PA2206 altered the expression of 58 genes, including a large repertoire of oxidative stress and iron responsive genes, independent of the master regulator of oxidative stress, OxyR. Contrary to the classic mechanism of LysR regulation, PA2206 did not autoregulate its own expression and did not influence expression of adjacent or divergently transcribed genes. The PA2214-15 operon was identified as a direct target of PA2206 with truncated promoter fragments revealing binding to the 5'-ATTGCCTGGGGTTAT-3' LysR box adjacent to the predicted -35 region. PA2206 also interacted with the pvdS promoter suggesting a global dimension to the PA2206 regulon, and suggests PA2206 is an important regulatory component of P. aeruginosa adaptation during oxidative stress.

The importance of reporting housing and husbandry in rat research

In 1963, the National Institutes of Health (NIH) first issued guidelines for animal housing and husbandry. The most recent 2010 revision emphasizes animal care “in ways judged to be scientifically, technically, and humanely appropriate” (National Institutes of Health, 2010, p. XIII). The goal of these guidelines is to ensure humanitarian treatment of animals and to optimize the quality of research. Although these animal care guidelines cover a substantial amount of information regarding animal housing and husbandry, researchers generally do not report all these variables (see Table ​Table1).1). The importance of housing and husbandry conditions with respect to standardization across different research laboratories has been debated previously (Crabbe et al., 1999; Van Der Staay and Steckler, 2002; Wahlsten et al., 2003; Wolfer et al., 2004; Van Der Staay, 2006; Richter et al., 2010, 2011). This paper focuses on several animal husbandry and housing issues that are particularly relevant to stress responses in rats, including transportation, handling, cage changing, housing conditions, light levels and the light–dark cycle. We argue that these key animal housing and husbandry variables should be reported in greater detail in an effort to raise awareness about extraneous experimental variables, especially those that have the potential to interact with the stress response.

Regrouping unfamiliar animals in the weeks prior to slaughter has few effects on physiology and meat quality in Bos taurus feedlot steers

The response of cattle to alterations in social groupings can lead to physiological changes that affect meat quality. Feedlot practices frequently lead to a proportion of cattle in a pen being drafted for slaughter with the balance retained for a further period until they meet market specifications. An ability to regroup such retained cattle for short periods without consequences for meat quality would facilitate efficient use of feedlot pen space. The current experiment examined the impact on physiological variables and meat quality of regrouped British breed steers 4, 2 or 1 week before dispatch for slaughter. There was little effect of regrouping cattle on physiological variables associated with stress responses. Physical assessment of meat quality indicated that regrouping steers 1 week before slaughter led to higher compression and a tendency for higher peak force values in animals from one genotype than in their respective controls (1.89 v. 1.71 ± 0.05 kg, P = 0.017); however, these assessments were not matched by changes in sensory perception of meat quality. Average daily gain during feedlot finishing was negatively related to the temperament measure and flight time. It was also associated with breed, white cell count, plasma cortisol and haemoglobin at the midpoint of the 70-day finishing period. The results confirm the impact of flight time on growth rate during feedlot finishing and that regrouping cattle less than 2 weeks before slaughter may reduce meat quality.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Modelling the effect of plant water use traits on yield and stay-green expression in sorghum

Post-rainy sorghum (Sorghum bicolor (L.) Moench) production underpins the livelihood of millions in the semiarid tropics, where the crop is affected by drought. Drought scenarios have been classified and quantified using crop simulation. In this report, variation in traits that hypothetically contribute to drought adaptation (plant growth dynamics, canopy and root water conducting capacity, drought stress responses) were virtually introgressed into the most common post-rainy sorghum genotype, and the influence of these traits on plant growth, development, and grain and stover yield were simulated across different scenarios. Limited transpiration rates under high vapour pressure deficit had the highest positive effect on production, especially combined with enhanced water extraction capacity at the root level. Variability in leaf development (smaller canopy size, later plant vigour or increased leaf appearance rate) also increased grain yield under severe drought, although it caused a stover yield trade-off under milder stress. Although the leaf development response to soil drying varied, this trait had only a modest benefit on crop production across all stress scenarios. Closer dissection of the model outputs showed that under water limitation, grain yield was largely determined by the amount of water availability after anthesis, and this relationship became closer with stress severity. All traits investigated increased water availability after anthesis and caused a delay in leaf senescence and led to a ‘stay-green’ phenotype. In conclusion, we showed that breeding success remained highly probabilistic; maximum resilience and economic benefits depended on drought frequency. Maximum potential could be explored by specific combinations of traits.

Thyroid Hormone Control of Cardiac Substrate Metabolism

Thyroid hormone (TH) plays an important role in maintaining a homeostasis in all the cells of our body. It also has significant cardiovascular effects, and abnormalities of its concentration can cause cardiovascular disease and even morbidity. Especially development of heart failure has been connected to low levels of thyroid hormone. A decrease in TH levels or TH-receptor binding adversely effects cardiac function. Although, this occurs in part through alterations in excitation-contraction and transport proteins, recent data from our laboratory indicate that TH also mediates changes in myocardial energy metabolism. Thyroid dysfunction may limit the heart s ability to shift substrate pathways and provide adequate energy supply during stress responses. Our goals of these studies were to determine substrate oxidation pattern in systemic and cardiac specific hypothyroidism at rest and at higher rates of oxygen demand. Additionally we investigated the TH mediated mechanisms in myocardial substrate selection and established the metabolic phenotype caused by a thyroid receptor dysfunction. We measured cardiac metabolism in an isolated heart model using 13Carbon isotopomer analyses with MR spectroscopy to determine function, oxygen consumption, fluxes and fractional contribution of acetyl-CoA to the citric acid cycle (CAC). Molecular pathways for changes in cardiac function and substrate shifts occurring during stress through thyroid receptor abnormalities were determined by protein analyses. Our results show that TH modifies substrate selection through nuclear-mediated and rapid posttranscriptional mechanisms. It modifies substrate selection differentially at rest and at higher rates of oxygen demand. Chronic TH deficiency depresses total CAC flux and selectively fatty acid flux, whereas acute TH supplementation decreases lactate oxidation. Insertion of a dominant negative thyroid receptor (Δ337T) alters metabolic phenotype and contractive efficiency in heart. The capability of the Δ337T heart to increase carbohydrate oxidation in response to stress seems to be limited. These studies provided a clearer understanding of the TH role in heart disease and shed light to identification of the molecular mechanisms that will facilitate in finding targets for heart failure prevention and treatment.

Unstructured N Terminus of the RNA Polymerase II Subunit Rpb4 Contributes to the Interaction of Rpb4·Rpb7 Subcomplex with the Core RNA Polymerase II of Saccharomyces cerevisiae

Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4 center dot Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.

The effects of stall surfaces and milk yield on the lying behavior of dairy cow

The importance of lying behavior to dairy cows and the feasible definition of lying has attracted many studies on the subject. Cattle show both behavioral and physiological stress responses when subjected to thwarting of their lying behavior. If cows are unable to lie down they later compensate for lost lying time when possible. Environmental factors such as housing and bedding systems have been noted to affect the time spent lying, but there is usually large variation in lying time between individuals. Internal factors such as the reproductive stage, age and health of cows affect their lying time and can cause variation. However, the effect of higher milk production on behavior has not previously been illuminated. The objective of this study was to provide data applicable for the improvement of resting conditions of cows. The preference of stall surface material, differences in normal behavior per unit time and various health measures were observed. The aim was to evaluate lying behavior and cow comfort on different stall bedding materials. In addition, the effect of milk yield on behavior was examined in a tie stall experiment. The preferences for surface materials were investigated in 5 experiments using 3 surface materials with bedding manipulations. According to the results, the cows preferred abundant straw bedding and soft rubber mats. However, they showed an aversion to sand bedding. Some individuals even refused to use stalls with sand when no organic bedding material was present. However, this study was unable to determine the reason for the avoidance, as neither the sand particle size nor thermal properties appeared critical. However, previous exposure to particular surface materials increased the preference for them. The amount of straw bedding was found to be an important factor affecting the preferences for stalls, and the lying time in stalls increased when the flooring softness was improved by applying straw or by installing elastic mats. Despite sand being the least preferred flooring material in preference tests, the health of legs improved during exposure to sand-floored stalls. Moreover cows using sand were cleaner than those that used straw stalls. Thus, sand bedding entailed some health benefits despite the contradictory results of preference tests, which more strongly reflected the perceptions of individual animals. Milk yield was observed to affect behavior by reducing the lying time, possibly due to factors other than longer duration of eating. High yielding cows seemed to intensify their lying bouts, as they were observed to lie with the neck muscles relaxed sooner after lying down than lower yielding cows. In conclusion, cows were found to prefer softer stall surface materials and organic bedding material. In addition, the lying time was reduced by a high milk yield, although the lying time seemed to be important for resting. Cows might differ in the needs for their lying environment. The management of dairy cows should eliminate any unnecessary prevention of lying, as even in tie-stalls high yielding cows seem to be affected by time constraints. Adding fresh bedding material to stalls increases the comfort of any stall flooring material.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide-and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Ice induced low temperature fatigue crack propagation in offshore structural steel A131

To investigate the low temperature fatigue crack propagation behavior of offshore structural steel A131 under random ice loading, three ice failure modes that are commonly present in the Bohai Gulf are simulated according to the vibration stress responses induced by real ice loading. The test data are processed by a universal software FCPUSL developed on the basis of the theory of fatigue crack propagation and statistics. The fundamental parameter controlling the fatigue crack propagation induced by random ice loading is determined to be the amplitude root mean square stress intensity factor K-arm. The test results are presented on the crack propagation diagram where the crack growth rate da/dN is described as the function of K-arm. It is evident that the ice failure modes have great influence on the fatigue crack propagation behavior of the steel in ice-induced vibration. However, some of the experimental phenomena and test results are hard to be physically explained at present. The work in this paper is an initial attempt to investigate the cause of collapse of offshore structures due to ice loading.

一种小G蛋白TaRAN1在植物细胞周期和发育过程中的功能研究

　　小G蛋白作为信号转导中重要的分子开关， 进化相当保守，与许多不同的调控因子和效应器分子相互作用，产生细胞功能的多样性。近年来，人们不断发现植物中小G蛋白家族的新成员，也不断揭示小G蛋白的新功能，许多植物特有的信号途径和功能需要小G蛋白这个重要的分子开关来完成，使它越来越成为人们研究的热点问题。但是，有关植物中Ran GTPase及其编码基因的研究工作报道很少，对与之相互作用的调控蛋白研究进展也刚刚开始。 　　TaRAN1 (AF488730) 是小麦来源的Ran同源蛋白编码基因，全长1055 bp, 编码221个氨基酸，它在植物发育过程中的功能还没有任何报道。本论文在验证了它是小G蛋白Ran家族的成员后，从分子水平上还发现它在植物细胞周期调控、对生长素以及胁迫应答信号转导过程中都起着重要作用，这也说明了它可能作为信号转导过程中重要的转换因子，参与了很多细胞的基本生理过程。 　　利用原核表达系统及亲和色谱的方法纯化了TaRAN1融合蛋白，并用放射性标记的GTP和竞争实验证实了它具有特异的GTP结合活性。TaRAN1的转录产物在小麦幼茎和花芽等分生组织活动旺盛的器官表达较多，而在老叶中表达较少。利用洋葱表皮瞬时表达系统分析表现，TaRAN1蛋白主要定位于细胞核，但其没有典型的核定位信号。 　　细胞周期一直是生物学领域中的热门问题，人们虽然在动物细胞中取得了很大进展，但在植物细胞中的研究远落后于动物。裂殖酵母（Schizosaccharomyces pombe）是研究细胞形态和细胞周期的良好系统，利用此系统发现超表达TaRAN1的酵母细胞表现出许多新的细胞学表型，例如G2细胞周期延滞、染色体对紫外线敏感、细胞超长或多隔细胞的出现等;反义表达TaRAN1的酵母细胞呈近圆型、具有高度凝集的核并且生长速度缓慢、核质混合和无核细胞的数目明显增加。流式细胞仪检测实验也证实其细胞周期的异常。这些结果推测TaRAN1蛋白可能参与细胞周期的有丝分裂过程和发育的调控机制，并且在维持染色体结构稳定和完整性方面起着重要的作用。通过免疫荧光实验观察表明，超表达转基因酵母的微管多呈异常的狭小扇形结构，反义表达TaRAN1的酵母微管不能形成丝状结构，推测TaRAN1还可能参与微管（包括纺锤体）的结构形成过程。最后，我们用超表达TaRAN1的转基因拟南芥和水稻也证实了它的功能，其生长点表现出分生组织增多的原基、根生长点的有丝分裂指数有所改变、出现异常的细胞分裂时相等有关细胞周期异常的现象，更进一步说明了TaRAN1确实参与着细胞周期的调控过程，推测其与细胞周期从G2期进入M期的过程有关。 　　TaRAN1基因受IAA的诱导表达，且随着浓度的增加表达量增强。超表达的TaRAN1植株（包括拟南芥和水稻）的根表现出对外源生长素异常敏感，侧根显著变少，地上部分表现出生长素过量的表现型，顶端优势减弱，分蘖增多，生长周期延长等。HPLC测定转基因植物的IAA含量，明显高于对照。所以，TaRAN1可能还参与了复杂的生长素信号转导过程。TaRAN1基因还受各种胁迫处理的诱导表达，并且超表达植株对胁迫的忍受能力有明显提高，这说明TaRAN1还参与了胁迫信号应答的相应机制。Ran蛋白这些新功能目前还未见到其它报道。