Water conditions influence the detectability of Crossodactylus gaudichaudii (Anura,Hylodidae) in streams of the Atlantic Forest

Stream-dwelling amphibians' occurrence, behaviour and reproductive success are strongly influenced by dynamic abiotic factors, for example, water flow and spray. These factors can disproportionately affect these frogs due to their dependency on specific favourable conditions for development and incapacity to disperse from unfavourable environments. We analysed the influence of environmental covariates on the detectability of Crossodactylus gaudichaudii, an amphibian species endemic to streams in the Brazilian Atlantic Forest. We conducted sampling in the streams of the Duas Bocas Biological Reserve, Brazil, and we measured air and water temperature, air humidity and pH of water on each sampling occasion. We estimated the effects of variables on the detectability of the species using single-season occupancy models. Our results indicated that the detectability of the species increases on occasions with higher water temperatures (24–26°C) and lower pH (5.0–5.5). We investigated the influence of these covariates only on the detectability of adult frogs, but it is likely that the physiochemical properties of stream water are more important to aquatic larvae, and hence, the probability of their detection. Given this, further studies should examine the relevance of covariates on the detectability of adult frogs as well as larvae.     

Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high‐level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.     

Exercise training prevents oxidative stress and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy in heart failure

Background

Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF.

Methods/Principal Findings

Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7^th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects’ levels.

Conclusions

Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.     

Lack of PPARγ in myeloid cells confers resistance to Listeria monocytogenes infection

The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγ(flox/flox)). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6C(hi) monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.     

ZyFISH: a simple, rapid and reliable zygosity assay for transgenic mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.     

In vitro evaluation of co-exposure of arsenium and an organic nanomaterial (fullerene, C₆₀) in zebrafish hepatocytes

Taking into account the concept of the "Trojan Horse", where contaminants may have its entry into specific organs potentiated by its association with nanomaterials, the aim of this study was to analyze the joint toxic effects induced by an organic nanomaterial, fullerene (C(60)) with the metalloid arsenic (As(III)). Hepatocytes of zebrafish Danio rerio were exposed to As(III) (2.5 or 100 μM), C(60) or As+C(60) for 4h, not altering cells viability. Intracellular reactive oxygen species concentration was reduced in cells exposed only to the C(60) (1mg/L) and in the treatment of 100 μM As(III)+C(60). Co-exposure with C(60) abolished the peak of the antioxidant glutathione (GSH) registered in cells exposed to the lowest As(III) concentration (2.5 μM). A similar result was observed in terms of lipid damage (TBARS). Total antioxidant capacity was significantly higher at both As(III) concentrations co-exposed to C(60) when compared with the control group. Activity of glutathione-S-transferase omega, a limiting enzyme in the methylation pathway of As(III), was reduced in the 100 μM As(III)+C(60) treatment. Cells co-exposed to C(60) had a significantly higher accumulation of As(III), showing a "Trojan Horse" effect which did not result in higher cell toxicity. Instead, co-exposure of As(III) with C(60) showed to reduce cellular injury.     

Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase

14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.     

Fine Root Production across a Primary Successional Ecosystem Chronosequence at Mt. Shasta,California

Estimating changes in belowground biomass and production is essential for understanding fundamental patterns and processes during ecosystem development. We examined patterns of fine root production, aboveground litterfall, and forest floor accumulation during forest primary succession at the Mt. Shasta Mudflows ecosystem chronosequence. Fine root production was measured using the root ingrowth cores method over 1 year, and aboveground litterfall was collected over 2 years. Fine root production increased significantly with ecosystem age, but only the youngest ecosystem was significantly different from all of the older ecosystems. Root production was 44.5 ± 13.3, 168.3 ± 20.6, 190.5 ± 33.8, and 236.3 ± 65.4 g m⁻² y⁻¹ in the 77, 255, 616, and >850-year-old ecosystems, respectively. Generally, aboveground litterfall and forest floor accumulation did not follow the same pattern as root production. The relative contribution of fine root production to total fine detrital production increased significantly with ecosystem age, from 14 to 49%, but only the youngest ecosystem was significantly different from all of the older ecosystems. Fine root production was significantly correlated with some measures of soil fertility but was not correlated with leaf or total litterfall, or forest floor accumulation. It was best predicted by soil N concentration alone, but this relationship may not be causal, as soil N concentration was also correlated with ecosystem age. For the oldest ecosystem, fine root production was also measured using the sequential intact cores/compartment-flow model method, and the difference between the two estimates was not significant. Our study suggests that the relative contribution of fine roots to fine detrital production, and hence to soil organic matter accumulation, may increase during forest primary succession.