Menin links the stress response to genome stability in Drosophila melanogaster

Background

The multiple endocrine neoplasia type I gene functions as a tumor suppressor gene in humans and mouse models. In Drosophila melanogaster, mutants of the menin gene (Mnn1) are hypersensitive to mutagens or gamma irradiation and have profound defects in the response to several stresses including heat shock, hypoxia, hyperosmolarity and oxidative stress. However, it is not known if the function of menin in the stress response contributes to genome stability. The objective of this study was to examine the role of menin in the control of the stress response and genome stability.

Methodology/Principal Findings

Using a test of loss-of-heterozygosity, we show that Drosophila strains lacking a functional Mnn1 gene or expressing a Mnn1 dsRNA display increased genome instability in response to non-lethal heat shock or hypoxia treatments. This is also true for strains lacking all Hsp70 genes, implying that a precise control of the stress response is required for genome stability. While menin is required for Hsp70 expression, the results of epistatic studies indicate that the increase in genome instability observed in Mnn1 lack-of-function mutants cannot be accounted for by mis-expression of Hsp70. Therefore, menin may promote genome stability by controlling the expression of other stress-responsive genes. In agreement with this notion, gene profiling reveals that Mnn1 is required for sustained expression of all heat shock protein genes but is dispensable for early induction of the heat shock response.

Conclusions/Significance

Mutants of the Mnn1 gene are hypersensitive to several stresses and display increased genome instability when subjected to conditions, such as heat shock, generally regarded as non-genotoxic. In this report, we describe a role for menin as a global regulator of heat shock gene expression and critical factor in the maintenance of genome integrity. Therefore, menin links the stress response to the control of genome stability in Drosophila melanogaster.     

Genetic history of hepatitis C virus in Venezuela: high diversity and long time of evolution of HCV genotype 2

Background

The subtype diversity of the hepatitis C virus (HCV) genotypes is unknown in Venezuela.

Methodology/Principal Findings

Partial sequencing of the NS5B region was performed in 310 isolates circulating in patients from 1995 to 2007. In the samples collected between 2005 and 2007, HCV genotype 1 (G1) was the most common genotype (63%), composed as expected of mainly G1a and G1b. G2 was the second most common genotype (33%), being G2a almost absent and G2j the most frequent subtype. Sequence analysis of the core region confirmed the subtype assignment performed within the NS5b region in 63 isolates. The complete genome sequence of G2j was obtained. G2j has been described in France, Canada and Burkina Fasso, but it was not found in Martinique, where several subtypes of G2 circulate in the general population. Bayesian coalescence analysis indicated a most recent common ancestor (MRCA) of G2j around 1785, before the introduction of G1b (1869) and G1a (1922). While HCV G1a and G1b experienced a growth reduction since 1990, coincident with the time when blood testing was implemented in Venezuela, HCV G2j did not seem to reach growth equilibrium during this period.

Conclusions/Significance

Assuming the introduction of G2j from Africa during the slave trade, the high frequency of G2j found in Venezuela could suggest: 1- the introduction of African ethnic groups different from the ones introduced to Martinique or 2- the occurrence of a founder effect. This study represents an in-depth analysis of the subtype diversity of HCV in Venezuela, which is still unexplored in the Americas and deserves further studies.     

Functional Analysis of the Group 4 Late Embryogenesis Abundant Proteins Reveals Their Relevance in the Adaptive Response during Water Deficit in Arabidopsis

Late-Embryogenesis Abundant (LEA) proteins accumulate to high levels during the last stages of seed development, when desiccation tolerance is acquired, and in vegetative and reproductive tissues under water deficit, leading to the hypothesis that these proteins play a role in the adaptation of plants to this stress condition. In this work, we obtained the accumulation patterns of the Arabidopsis (Arabidopsis thaliana) group 4 LEA proteins during different developmental stages and plant organs in response to water deficit. We demonstrate that overexpression of a representative member of this group of proteins confers tolerance to severe drought in Arabidopsis plants. Moreover, we show that deficiency of LEA proteins in this group leads to susceptible phenotypes upon water limitation, during germination, or in mature plants after recovery from severe dehydration. Upon recovery from this stress condition, mutant plants showed a reduced number of floral and axillary buds when compared with wild-type plants. The lack of these proteins also correlates with a reduced seed production under optimal irrigation, supporting a role in fruit and/or seed development. A bioinformatic analysis of group 4 LEA proteins from many plant genera showed that there are two subgroups, originated through ancient gene duplication and a subsequent functional specialization. This study represents, to our knowledge, the first genetic evidence showing that one of the LEA protein groups is directly involved in the adaptive response of higher plants to water deficit, and it provides data indicating that the function of these proteins is not redundant to that of the other LEA proteins.Water deficit is a common environmental condition that leads to various responses that help in the adaptation or adjustment of an organism to this stress, considered one of the most important environmental stresses influencing plant productivity (Bray, 1997; Morison et al., 2008). The adverse effects of this environmental stress need to be counteracted, mainly because of the increasing soil desertification in cultivated and uncultivated regions. This fact demands that plants tolerate drying periods and elevated salt concentrations in the soil, which may be accompanied by extreme temperatures. Also, an interest in understanding the mechanisms by which plants sense and respond to these environmental cues accounts for the most important reasons to study in detail the responses that have been selected in plants to cope with water deficit.The acquisition of desiccation tolerance during late stages of seed development is correlated with the induction of a set of small, highly hydrophilic proteins called Late-Embryogenesis Abundant (LEA) proteins (Dure et al., 1989). These proteins are ubiquitous in plants, and although there are several classifications, we will follow that of Battaglia et al. (2008), where they are classified into seven groups on the basis of sequence similarity. Analysis of the protein sequences in these groups from different plant species defined distinctive motifs within groups (Dure, 1993; Battaglia et al., 2008). The number of members is different for each LEA protein group and varies according to the plant species. Most LEA proteins are hydrophilins, a set of proteins characterized by their biased amino acid composition, richness in Gly and other small and/or charged residues, and high hydrophilicity index (Garay-Arroyo et al., 2000). This amino acid composition promotes their flexible structure in solution, existing mainly as random coils, with the exception of the hydrophobic or atypical LEA proteins (Singh et al., 2005). Moreover, hydrophilic LEA proteins from groups 2, 3, and 4 show a prevalence of typical spectroscopic patterns of intrinsically unstructured proteins, with the occurrence of transitions from intrinsically unstructured proteins to ordered conformations in the presence of helix-promoting solvents or air drying (McCubbin et al., 1985; Russouw et al., 1995; Eom et al., 1996; Lisse et al., 1996; Ismail et al., 1999; Wolkers et al., 2001; Soulages et al., 2002, 2003; Goyal et al., 2003; Shih et al., 2004; Tolleter et al., 2007). Their high content of water-interacting residues facilitates the scavenging of water molecules, which is of special importance during developmental stages where a programmed desiccation of tissues takes place, as in the dry seed (Dure et al., 1989), or when cells experience changes in their water status (Colmenero-Flores et al., 1999). Remarkably, there is also an elevated induction in the expression of these proteins in vegetative tissues after exposure to water deficit in basically all plants that have been analyzed. In recent years, proteins with similar characteristics and expression patterns have also been detected to be induced in response to osmotic stress in bacteria and yeast (Stacy and Aalen, 1998; Garay-Arroyo et al., 2000), algae (Honjoh et al., 1995, 2000; Tanaka et al., 2004), nematodes (Solomon et al., 2000; Browne et al., 2004), rotifers (Tunnacliffe et al., 2005), and arthropods (Menze et al., 2009).One of the hypotheses regarding their function is that these proteins may act as protectors of macromolecules and/or some cellular structures during water deficit, by preferentially interacting with the available water molecules and providing a hydration shell to protect “target” integrity and function (Bray, 1997; Garay-Arroyo et al., 2000; Hoekstra et al., 2001). The use of an in vitro dehydration assay, in which the activity of malate dehydrogenase and lactate dehydrogenase was measured in the presence or absence of a hydrophilic protein, showed that plant hydrophilins (LEA proteins from groups 2, 3, and 4) and hydrophilins from Saccharomyces cerevisiae and Escherichia coli were able to protect these enzymatic activities under low water availability conditions (Reyes et al., 2005). Similarly, in vitro assays using the same or other enzymes have been used to assess the protective capacities of LEA proteins under dehydration and cold (Honjoh et al., 2000; Hara et al., 2001; Bravo et al., 2003; Goyal et al., 2005; Grelet et al., 2005; Nakayama et al., 2007; Reyes et al., 2008). In some of these assays, the ratio of LEA protein to enzyme was 1:1, suggesting that the LEA protein protective activity is not only due to the formation of a preferential hydration shell but also to an additional effect probably related to a direct interaction with their targets (Reyes et al., 2005, 2008).There are many reports of LEA proteins expressed in transgenic plants under the control of regulated or constitutive promoters, showing tolerant phenotypes under drought, high salinity, or freezing stress (Xu et al., 1996; Sivamani et al., 2000; NDong et al., 2002; Chandra Babu et al., 2004; Puhakainen et al., 2004; Fu et al., 2007; Lal et al., 2007; Xiao et al., 2007; Dalal et al., 2009). Also, the heterologous expression in bacteria and yeast of some LEA proteins confers salt and freezing tolerance (Imai et al., 1996; Zhang et al., 2000; Liu and Zheng, 2005). However, this “gain-of-function” approach does not necessarily reflect their direct participation in the plant adjustment or adaptation to these stress conditions but rather their potential to confer tolerance when ectopically expressed. In contrast, the results of a “loss-of-function” approach will lead to a direct indication of the participation of a particular gene within this process. Even though there is a large extent of information regarding the different properties of LEA proteins, our knowledge concerning their role in plant adaptation to water-limiting conditions is insufficient.In this work, we focus on the study of the group 4 LEA proteins of Arabidopsis (Arabidopsis thaliana). With only three genes in the genome (AtLEA4-1, AtLEA4-2, and AtLEA4-5), the AtLEA4 group is one of the smallest groups in Arabidopsis (Battaglia et al., 2008; Hundertmark and Hincha, 2008), which makes it accessible for a loss-of-function analysis. The LEA4 proteins are characterized by a high content of A, T, and G amino acid residues, the latter highly represented in unstructured proteins. They have a conserved N-terminal domain of 70 to 80 residues, predicted to form amphipathic α-helices, and a less conserved C-terminal region with variable size and random coil structure (Dure, 1993). Like other LEA proteins, the LEA4 group is highly accumulated in all embryo tissues of dry seeds (Roberts et al., 1993). Recently, Wise (2002) performed a bioinformatics analysis and questioned the existence of a group 4 of LEA proteins as a distinct group of LEA proteins from group 3. The algorithm used the overrepresentation/underrepresentation of particular amino acids within small motifs in the protein, giving rise to a different classification for these proteins (Wise, 2003). In support of the original classification proposed by Dure et al. (1989) and because of the high sequence conservation within this group in plants, in this work, we present genetic and functional evidence that group 4 of LEA proteins is indeed a distinct group conserved in the plant kingdom. The results reported here show that overexpression of one of the AtLEA4 proteins in Arabidopsis leads to a tolerant phenotype compared with their wild-type counterparts in their capability to endure severe water deficit and that the reduction in the accumulation levels of these proteins leads to plants more sensitive to water-limiting conditions than their wild-type genotypes. Altogether, these data constitute, to our knowledge, the first direct evidence indicating that LEA4 proteins are involved in the adaptive response of vascular plants to withstand water deficit.     

Effects of stretching on morphological and biochemical aspects of the extracellular matrix of the rat calcaneal tendon

Several studies have demonstrated the relationship between exercise and the extracellular matrix of muscle tendons, and have described alterations in their structural and biochemical properties when subjected to strenuous exercise. However, little is known about what happens to tendons when they are subjected to stretching. We evaluated the changes in the composition and structure of rat calcaneal tendons subjected to a stretching program. The animals had their muscles stretched for 30 s with 30 s of rest, with 10 repetitions, three and five times a week for 21 days. For morphological analysis, the sections were stained with hematoxylin-eosin and toluidine blue. For biochemical analysis, the tendons were treated with 4 M guanidine hydrochloride and analyzed in SDS-PAGE. The contents of total proteins and glycosaminoglycans were also measured. In the sections stained with toluidine blue, we could observe an increase of rounded cells, especially in the enthesis region. In the region next to the enthesis was a metachromatic region, which was more intensely stained in the stretched groups. In the tension regions, the cells appeared more aligned. Cellularity increased in both regions. The SDS-PAGE analysis showed a larger amount of collagen in the stretched groups and a polydispersed component of 65 kDa in all the groups. The amounts of proteins and glycosaminoglycans were also larger in the stretched tendons. The agarose-gel electrophoresis confirmed the presence of dermatan sulfate in the tension and compression regions, and of chondroitin sulfate only in the latter. Our results showed that the stretching stimulus changed the cellularity and the amount of the extracellular matrix compounds, confirming that tendons are dynamic structures with a capacity to detect alterations in their load.     

Occurrence of Monosporascus cannonballus in Watermelon Fields in Tunisia and Factors Associated with Ascospore Density in Soil

Surveys of 11 watermelon fields throughout production areas of this crop in southern and central regions in Tunisia were conducted in 2007 to determine the aetiology and distribution of watermelon vine decline. Monosporascus cannonballus was isolated from diseased roots in all surveyed fields. All the isolates were identified according to morphological features and confirmed by amplification of a fragment of the ITS region with specific primers. Ascospores of M. cannonballus were recovered from soil in all watermelon fields surveyed and the average population densities ranged from 3.65 to 10.14 ascospores per g of soil. Multiple linear regression analysis revealed that only four of the crop and soil factors evaluated had a significant correlation with ascospore density at the end of the growing season: vertisol vs. other soils, disease incidence, percentage of clay and pH. The pH of the soil showed a strong significant negative linear relationship with ascospore density, while the other three factors correlated positively.     

Studies toward the structural optimization of novel thiazolylhydrazone-based potent antitrypanosomal agents

In previous studies, we identified promising anti-Trypanosoma cruzi cruzain inhibitors based on thiazolylhydrazones. To optimize this series, a number of medicinal chemistry directions were explored and new thiazolylhydrazones and thiosemicarbazones were thus synthesized. Potent cruzain inhibitors were identified, such as thiazolylhydrazones 3b and 3j, which exhibited IC(50) of 200-400nM. Furthermore, molecular docking studies showed concordance with experimentally derived structure-activity relationships (SAR) data. In the course of this work, lead compounds exhibiting in vitro activity against both the epimastigote and trypomastigote forms of T. cruzi were identified and in vivo general toxicity analysis was subsequently performed. Novel SAR were documented, including the importance of the thiocarbonyl carbon attached to the thiazolyl ring and the direct comparison between thiosemicarbazones and thiazolylhydrazones.     

Modeling relationships between calving traits: a comparison between standard and recursive mixed models

Background

The use of structural equation models for the analysis of recursive and simultaneous relationships between phenotypes has become more popular recently. The aim of this paper is to illustrate how these models can be applied in animal breeding to achieve parameterizations of different levels of complexity and, more specifically, to model phenotypic recursion between three calving traits: gestation length (GL), calving difficulty (CD) and stillbirth (SB). All recursive models considered here postulate heterogeneous recursive relationships between GL and liabilities to CD and SB, and between liability to CD and liability to SB, depending on categories of GL phenotype.

Methods

Four models were compared in terms of goodness of fit and predictive ability: 1) standard mixed model (SMM), a model with unstructured (co)variance matrices; 2) recursive mixed model 1 (RMM1), assuming that residual correlations are due to the recursive relationships between phenotypes; 3) RMM2, assuming that correlations between residuals and contemporary groups are due to recursive relationships between phenotypes; and 4) RMM3, postulating that the correlations between genetic effects, contemporary groups and residuals are due to recursive relationships between phenotypes.

Results

For all the RMM considered, the estimates of the structural coefficients were similar. Results revealed a nonlinear relationship between GL and the liabilities both to CD and to SB, and a linear relationship between the liabilities to CD and SB.Differences in terms of goodness of fit and predictive ability of the models considered were negligible, suggesting that RMM3 is plausible.

Conclusions

The applications examined in this study suggest the plausibility of a nonlinear recursive effect from GL onto CD and SB. Also, the fact that the most restrictive model RMM3, which assumes that the only cause of correlation is phenotypic recursion, performs as well as the others indicates that the phenotypic recursion may be an important cause of the observed patterns of genetic and environmental correlations.