A simple and rapid method for lithium acetate-mediated transformation of Kluyveromyces marxianus cells

Efficient plasmid transformation of Kluyveromyces marxianus cells of 1.9 × 10³ transformant μg⁻¹ DNA with an episomal plasmid was achieved by the use of a simple lithium acetate method with the addition of 10 mM DTT and an increased heat shock temperature of 47 °C. This method is shown to be also efficient for replicative plasmids. Therefore, we suggest its use as a routine method to transform K. marxianus cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Recent advances in plasmid-based tools for establishing novel microbial chassis

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.     

Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

The effect of metal diffusion and supply limitations on conditional stability constants determined for durum wheat roots

Conditional stability constants (log K), and binding site densities (Γ_max) for dissolved metals and biota are important input parameters for the Biotic Ligand Model. However, determination of these binding parameters is likely to be influenced by solution kinetics because roots have a large metal-binding capacity and can accumulate metals rapidly. The aim of this study was to determine if the rate of free metal ion diffusion to the root surface, and amount of metal in the bulk solution, is sufficient to accommodate the maximum root–metal accumulation capacity. The extent to which these kinetic limitations affect the magnitude of log K and Γ_max values was also assessed. Seven day old hydroponically grown durum wheat (Triticum turgidum L. var durum, cv ‘Arcola’) were exposed to solutions with p{Cu²⁺}s ranging from 10.54 to 2.26 (~20 °C, pH = 6.0, ionic strength = 0.03 M). Exposure solutions were prepared with and without the metal buffer nitrilotriacetic acid (NTA) so that the total amount of metal in the exposure solution, and net flux of metal to the root, could be varied. The results demonstrate that NTA enhances Cu accumulation at exposure p{Cu²⁺}s between 10 and 6. Comparison of the diffusive flux to the root with the metal flux into the root, for (−NTA) and (+NTA) Cu exposures, showed that the flux of the un-buffered free metal ion to the root was not large enough to accommodate the maximum Cu binding capacity between 10 and 6 p{Cu²⁺} in solution. The total amount of Cu in solution may have limited uptake for exposure p{Cu²⁺}s of 10.01 and 9.01, but the background concentrations of Cu in the control plants prevented definitive conclusions from being made within this exposure range. Similar results were found for Mn and Ni. For Cd, which had lower background concentrations in the roots, the amount of metal in solution did not limit uptake until a p{Cd²⁺} of 10.01. Limiting the supply of Cu²⁺ to the root (i.e. low {Cu²⁺}s with no NTA) caused only a moderate bias in Γ_max values, but suppressed the log K value by 3.44 log units. The log K values for Cd, Mn and Ni, in the absence of NTA, were more similar than expected, which suggests that the kinetics of free ion re-supply to the root surface limited metal uptake, as it did for Cu. Section Editor: T. B. Kinraide     

Ria de Alvor as a spawning place and a nursery ground

Ria de Alvor is a small estuary between Lagos and Portimão on the south coast of Portugal, 37°7'–37°10'N, 8°35'–8°38'W.
Samples were collected monthly in the period April 1985–March 1986 in an ichthyoplankton and ichthyofauna survey of the estuary. A WP-2 plankton net was used for catching ichthyoplankton, and a beam trawl and a beach seine were used for catching ichthyofauna.
Scaldfish, Arnoglossus laterna (Walbaum, 1792), even though the most abundant species in ichthyoplankton samples, was never found in ichthyofauna samples.
From the results obtained, it is concluded that a great part of the fish species are temporary marine visitors using the Ria as a nursery ground, although this system appears to provide a spawning area for species with benthic eggs. No evidence was found of pelagic spawning.     

Genomic prediction ability for beef fatty acid profile in Nelore cattle using different pseudo-phenotypes

The aim of the present study was to compare the predictive ability of SNP-BLUP model using different pseudo-phenotypes such as phenotype adjusted for fixed effects, estimated breeding value, and genomic estimated breeding value, using simulated and real data for beef FA profile of Nelore cattle finished in feedlot. A pedigree with phenotypes and genotypes of 10,000 animals were simulated, considering 50% of multiple sires in the pedigree. Regarding to phenotypes, two traits were simulated, one with high heritability (0.58), another with low heritability (0.13). Ten replicates were performed for each trait and results were averaged among replicates. A historical population was created from generation zero to 2020, with a constant size of 2000 animals (from generation zero to 1000) to produce different levels of linkage disequilibrium (LD). Therefore, there was a gradual reduction in the number of animals (from 2000 to 600), producing a “bottleneck effect” and consequently, genetic drift and LD starting in the generation 1001 to 2020. A total of 335,000 markers (with MAF greater or equal to 0.02) and 1000 QTL were randomly selected from the last generation of the historical population to generate genotypic data for the test population. The phenotypes were computed as the sum of the QTL effects and an error term sampled from a normal distribution with zero mean and variance equal to 0.88. For simulated data, 4000 animals of the generations 7, 8, and 9 (with genotype and phenotype) were used as training population, and 1000 animals of the last generation (10) were used as validation population. A total of 937 Nelore bulls with phenotype for fatty acid profiles (Sum of saturated, monounsaturated, omega 3, omega 6, ratio of polyunsaturated and saturated and polyunsaturated fatty acid profile) were genotyped using the Illumina BovineHD BeadChip (Illumina, San Diego, CA) with 777,962 SNP. To compare the accuracy and bias of direct genomic value (DGV) for different pseudo-phenotypes, the correlation between true breeding value (TBV) or DGV with pseudo-phenotypes and linear regression coefficient of the pseudo-phenotypes on TBV for simulated data or DGV for real data, respectively. For simulated data, the correlations between DGV and TBV for high heritability traits were higher than obtained with low heritability traits. For simulated and real data, the prediction ability was higher for GEBV than for Yc and EBV. For simulated data, the regression coefficient estimates (b_(Yc,DGV)), were on average lower than 1 for high and low heritability traits, being inflated. The results were more biased for Yc and EBV than for GEBV. For real data, the GEBV displayed less biased results compared to Yc and EBV for SFA, MUFA, n-3, n-6, and PUFA/SFA. Despite the less biased results for PUFA using the EBV as pseudo-phenotype, the b_(Yi,DGV estimates obtained for the different pseudo-phenotypes (Yc, EBV and GEBV) were very close. Genomic information can assist in improving beef fatty acid profile in Zebu cattle, since the use of genomic information yielded genomic values for fatty acid profile with accuracies ranging from low to moderate. Considering both simulated and real data, the ssGBLUP model is an appropriate alternative to obtain more reliable and less biased GEBVs as pseudo-phenotype in situations of missing pedigree, due to high proportion of multiple sires, being more adequate than EBV and Yc to predict direct genomic value for beef fatty acid profile.     

Structural and physiological responses of <Emphasis Type="Italic">Halodule wrightii</Emphasis> to ocean acidification

Coastal areas face high variability of seawater pH. Ocean acidification (OA) and local stressors are enhancing this variability, which poses a threat to marine life. However, these organisms present potential phenotypic plasticity that can offer physiological and structural tools to survive in these extreme conditions. In this study, we evaluated the effects of elevated CO₂ levels and consequent pH reduction on the physiology, anatomy and ultrastructure of the seagrass Halodule wrightii. A mesocosm study was conducted in an open system during a 30-day experiment, where different concentrations of CO₂ were simulated following the natural variability observed in coastal reef systems. This resulted in four experimental conditions simulating the (i) environmental pH (control condition, without CO₂ addition) and (ii) reduced pH by ? 0.3 units, (iii) ? 0.6 units and (iv) ? 0.9 units, in relation to the field condition. The evaluated population only suffered reduced optimum quantum yield (Y(II)), leaf width and cross-section area under the lowest CO₂ addition (? 0.3 pH units) after 30 days of experiment. This fitness commitment should be related to carbon concentration mechanisms present in the evaluated species. For the highest CO₂ level, H. wrightii demonstrated a capacity to compensate any negative effect of the lowest pH. Our results suggest that the physiological behaviour of this primary producer is driven by the interactions among OA and environmental factors, like irradiance and nutrient availability. The observed behaviour highlights that high-frequency pH variability and multifactorial approaches should be applied, and when investigating the impact of OA, factors like irradiance, nutrient availability and temperature must be considered as well.     

Ticks (Acari: Ixodidae) in the Serra da Canastra National Park in Minas Gerais,Brazil: species,abundance, ecological and seasonal aspects with notes on rickettsial infection

The Cerrado Biome is the second largest in Brazil covering roughly 2 million km², with varying features throughout its area. The Biome is endangered but it is also source of animal species for rural, green urban and degraded rainforest areas. Ticks are among Cerrado species that establish at anthropogenic sites and although information about them is steadily increasing, several features are unknown. We herein report tick species, abundance and some ecological relationships within natural areas of the Cerrado at higher altitudes (800–1500 m) within and around Serra da Canastra National Park, in Minas Gerais State Brazil. In total of 1196 ticks were collected in the environment along 10 campaigns held in 3 years (2007–2009). Amblyomma sculptum was the most numerous species followed by Amblyomma dubitatum and Amblyomma brasiliense. Distribution of these species was very uneven and an established population of A. brasiliense in the Cerrado is reported for the first time. Other tick species (Amblyomma ovale, Amblyomma nodosum, Amblyomma parvum, Ixodes schulzei and Haemaphysalis leporispalustris) were found in lesser numbers. Domestic animals displayed tick infestations of both rural and urban origin as well as from natural areas (Rhipicephalus sanguineus sensu lato, Rhipicephalus microplus, Dermacentor nitens, A. sculptum, A. ovale, Amblyomma tigrinum, Argas miniatus). Amblyomma sculptum had the widest domestic host spectrum among all tick species. DNA of only one Rickettsia species, R. bellii, was found in an A. dubitatum tick. Several biological and ecological features of ticks of the studied areas are discussed.