The stress-activated protein kinases p38α/β and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest

Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G₂/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.     

Deacetylase activity is required for STAT5-dependent GM-CSF functional activity in macrophages and differentiation to dendritic cells

After interaction with its receptor, GM-CSF induces phosphorylation of the beta-chain in two distinct domains in macrophages. One induces activation of mitogen-activated protein kinases and the PI3K/Akt pathway, and the other induces JAK2-STAT5. In this study we describe how trichostatin A (TSA), which inhibits deacetylase activity, blocks JAK2-STAT5-dependent gene expression but not the expression of genes that depend on the signal transduction induced by the other domain of the receptor. TSA treatment inhibited the GM-CSF-dependent proliferation of macrophages by interfering with c-myc and cyclin D1 expression. However, M-CSF-dependent proliferation, which requires ERK1/2, was unaffected. Protection from apoptosis, which involves Akt phosphorylation and p21(waf-1) expression, was not modified by TSA. GM-CSF-dependent expression of MHC class II molecules was inhibited because CIITA was not induced. The generation of dendritic cells was also impaired by TSA treatment because of the inhibition of IRF4, IRF2, and RelB expression. TSA mediates its effects by preventing the recruitment of RNA polymerase II to the promoter of STAT5 target genes and by inhibiting their expression. However, this drug did not affect STAT5A or STAT5B phosphorylation or DNA binding. These results in GM-CSF-treated macrophages reveal a relationship between histone deacetylase complexes and STAT5 in the regulation of gene expression.     

Whi3, a developmental regulator of budding yeast, binds a large set of mRNAs functionally related to the endoplasmic reticulum

Whi3 is an RNA-binding protein associated with the endoplasmic reticulum (ER) that binds the CLN3 mRNA and plays a key role in the efficient retention of cyclin Cln3 at the ER. In the present work, we have identified new Whi3-associated mRNAs by a genomic approach. A large and significant number of these Whi3 targets encode for membrane and exocytic proteins involved in processes such as transport and cell wall biogenesis. Consistent with the genomic data, we have observed that cell wall integrity is compromised in Whi3-deficient cells and found strong genetic interactions between WHI3 and the cell integrity pathway. Whi3-associated mRNAs are enriched in clusters of the tetranucleotide GCAU, and mutation of the GCAU clusters in the CLN3 mRNA caused a reduction in its association to Whi3, suggesting that these sequences may act as cis-determinants for binding. Our data suggest that Whi3 is involved in the regulation and/or localization of a large subset of mRNAs functionally related to the ER and, since it is important for different molecular processes such as cytoplasmic retention or exocytic traffic of proteins, we propose that Whi3 is a general modulator of protein fate in budding yeast.     

Organic versus conventional citrus. Impact assessment and variability analysis in the Comunitat Valenciana (Spain)

Purpose

Taking into account the large area of citrus in Spain and the impacts generated by agriculture, reducing the environmental impact of this crop represents an important goal. This study attempts to compare the environmental impact of two citrus cropping systems, organic and conventional, in the region of Valencia (Spain), and to assess the variability within both farming systems in order to highlight the influence of management practices on the environmental performance.

Methods

A survey was carried out on citrus farmers, 145 corresponding to organic production and 122 to conventional. Life cycle assessment (LCA) was used to estimate the environmental impacts of farms and the contribution of each production stage to impacts. Two functional units (FUs), mass- and area-based, were chosen. The variability and confidence intervals of the average impact results were assessed by means of a bootstrap technique. Finally, a k-means cluster analysis was performed to identify groups of farms with comparable impact levels.

Results and discussion

The mean impact values of the conventional farm sample were higher than those of the organic farms, when using 1 ha year^?1 as FU, whereas for the FU of 1 kg no differences were found for some impact categories. Most of the impact results were also observed to be highly variable. The distribution of the mean after the bootstrap confirmed both the variability of the impacts and the differences between the farming systems. The cluster analysis determined two groups via their impact categories. Cluster-1, which showed higher impacts, was made up of part of the conventional farms while cluster-2 included the remaining conventional farms and all the organic ones. No difference in yield was found between the conventional farms of both clusters.

Conclusions

Bootstrapped LCA offers the ability to assess the variability of the impacts, regardless of the sample size and the non-normal impact distributions. The cluster analysis shows that conventional farms can attain similar impacts than the organic ones, while maintaining the yield. FU selection is crucial, since a mass-based FU reduces the difference in the environmental performance between conventional and organic farms. To attain a more sustainable citrus farming, a careful selection of the management practices is needed.

     

Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins.

To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn(7)-MT1 and the isolated Zn(4)-alphaMT1 and Zn(3)-betaMT1 fragments. The incorporation of MeHg(+) into Zn(7)-MT and Zn(3)-betaMT entails total displacement of Zn(II) and unfolding of the protein. However, both features are only partial for Zn(4)-alphaMT. The different behavior observed for this fragment, whether isolated or constituting one of the two domains of Zn(7)-MT, indicates interdomain interactions in the whole protein. Overall, the binding properties of Zn(7)-MT, Zn(4)-alphaMT and Zn(3)-betaMT toward MeHg(+) are unprecedented. In addition, the sequestration of MeHg(+) by Zn(7)-MT and the concomitant release of Zn(II) are probably two of the main contributions in the detoxifying role of mammalian MT.     

Enabling large-scale feather mite studies: an Illumina DNA metabarcoding pipeline

Feather mites are among the most common and diverse ectosymbionts of birds, yet basic questions such as the nature of their relationship remain largely unanswered. One reason for feather mites being understudied is that their morphological identification is often virtually impossible when using female or young individuals. Even for adult male specimens this task is tedious and requires advanced taxonomic expertise, thus hampering large-scale studies. In addition, molecular-based methods are challenging because the low DNA amounts usually obtained from these tiny mites do not reach the levels required for high-throughput sequencing. This work aims to overcome these issues by using a DNA metabarcoding approach to accurately identify and quantify the feather mite species present in a sample. DNA metabarcoding is a widely used molecular technique that takes advantage of high-throughput sequencing methodologies to assign the taxonomic identity to all the organisms present in a complex sample (i.e., a sample made up of multiple specimens that are hard or impossible to individualise). We present a high-throughput method for feather mite identification using a fragment of the COI gene as marker and Illumina Miseq technology. We tested this method by performing two experiments plus a field test over a total of 11,861 individual mites (5360 of which were also morphologically identified). In the first experiment, we tested the probability of detecting a single feather mite in a heterogeneous pool of non-conspecific individuals. In the second experiment, we made 2?×?2 combinations of species and studied the relationship between the proportion of individuals of a given species in a sample and the proportion of sequences retrieved to test whether DNA metabarcoding can reliably quantify the relative abundance of mites in a sample. Here we also tested the efficacy of degenerate primers (i.e., a mixture of similar primers that differ in one or several bases that are designed to increase the chance of annealing) and investigated the relationship between the number of mismatches and PCR success. Finally, we applied our DNA metabarcoding pipeline to a total of 6501 unidentified and unsorted feather mite individuals sampled from 380 European passerine birds belonging to 10 bird species (field test). Our results show that this proposed pipeline is suitable for correct identification and quantitative estimation of the relative abundance of feather mite species in complex samples, especially when dealing with a moderate number (>?30) of individuals per sample.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.