Proteomic approaches to identifying carbonylated proteins in brain tissue

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.     

Effects of potyvirus effective population size in inoculated leaves on viral accumulation and the onset of symptoms

Effective population size (N(e)) is a key parameter for understanding evolutionary processes, but it is generally not considered in epidemiological studies or in studying infections of individual hosts. Whether N(e) has an effect on the onset of symptoms and viral accumulation in Tobacco etch virus (TEV) infection of Nicotiana tabacum plants is considered here. Using mixtures of TEV variants carrying fluorescent markers, the dose dependence of N(e) was confirmed, and the inoculation procedure was found to be the main source of variation in these experiments. Whereas the onset of symptoms was independent of N(e), there was less and more variable accumulation at 6 days postinoculation for small N(e) values (N(e) < 5). The observed variation in accumulation was not heritable, however, suggesting that this variation was not due to the fixation of deleterious mutations in the small founder populations. On the other hand, virus-induced fluorescence and accumulation in the inoculated leaf were strongly N(e) dependent. Systemic accumulation was independent of N(e), although removal of the inoculated leaf led to a small reduction in systemic accumulation for small N(e) values. For whole plants, N(e)-dependent effects on accumulation were no longer observed at 9 days postinoculation. Therefore, the effects of N(e) on accumulation are due mainly to limited expansion in the inoculated leaf and are transient. In this system, N(e)-dependent effects will be strongest at low doses and early in infection. We conclude that N(e) can have implications for epidemiology and infection at the individual host level, beyond determining the rate of mixed-genotype infection.     

High genetic divergence of hormogastrid earthworms (Annelida, Oligochaeta) in the central Iberian Peninsula: evolutionary and demographic implications

Hormogastridae earthworms are highly important for the functioning of the Mediterranean soil system. However, little is known about the species distribution and genetic diversity of these soil invertebrates. In the present study, the genetic differentiation and gene flow were studied among populations of hormogastrids from the central Iberian Peninsula. A 648-bp portion of the mitochondrial cytochrome c oxidase I gene was sequenced for 82 individuals from 7 localities, resulting in the identification of 38 haplotypes exclusive to localities. All of the individuals were morphologically identified as Hormogaster elisae , but the high genetic divergence found among populations (up to 20.20%) suggests the occurrence of more than one cryptic species within this region. Further analysis of the phylogenetic relationships revealed six different evolutionary lineages coincident with geographical location, including the two nearest populations Molar and Redueña as one evolutionary unit. From these results, at least three new species could be inferred, in addition to the morphospecies H. elisae s.s . Partitioning of genetic variance among populations indicated that isolation by distance was the primary agent for differentiation of the investigated hormogastrid populations. Our data suggest that the evolutionary lineages for H. elisae s.l. originated between the late Miocene and the early Pleistocene, but that mtDNA genealogies coalesce on a more recent scale of a few thousand years.     

Tire-breeding mosquitoes of public health importance along an urbanisation gradient in Buenos Aires, Argentina

Used vehicle tires are a source of mosquito vectors and a means of their introduction and expansion. With the aim of assessing the effects of urbanisation on the main mosquito vectors in temperate Argentina, the infestation levels of Aedes aegypti (L.) and Culex pipiens L. were studied in used tires from highly urbanised cities to low-urbanised small towns in Buenos Aires. Immatures of both species accounted for 96% of the 9,722 individuals collected; the total individuals collected represented seven species. The percentage of water-filled tires containing mosquitoes [container index (CI)] was 33% and the percentage of infested sites [site index (SI)] was 65.2%. These indexes decreased significantly from low to high urbanisation levels for both mosquito species. The relative abundance (RA) of Ae. aegypti immatures was slightly higher toward large cities, but showed no difference for Cx. pipiens. The CI of shaded tires was significantly higher than the CI of exposed tires for both mosquito species. There was no difference in RA values between shaded and sunlit tires. The CI and the SI were highest during the summer across the urbanisation levels, except for Cx. pipiens, which continued to increase during the autumn in small towns. Results related to urbanisation gradient, sunlit exposure and seasonality are discussed.     

Analysis of diversity and activity of sulfate-reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and dsrB genes as molecular markers

Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.     

Studies on the Pathogenic Potential and Life Cycle of Reniform Nematode on Broccoli

The significant reduction in the plant growth was observed at an initial inoculum level of 2 immature females of Rotylenchulus reniformis/g soil and hence this inoculum level was considered as the minimum damaging threshold level for broccoli. The population of reniform nematode increased with the increasing levels of inoculum. Though, the maximum nematode population was recorded at the highest level of inoculum, the rate of reproduction of nematode was found to be highest at the lowest level of initial inoculum. Symptoms like chlorosis, stunted growth, shedding of leaves, early sprouting of inflorescence and sparsely developed roots were found during the experimental studies. Rotylenchulus reniformis required 30 days to complete the life cycle on broccoli.     

Promising applications of cold plasma for microbial safety,chemical decontamination and quality enhancement in fruits

Consumers’ demand is increasing for safe foods without impairing the phytochemical and sensory quality. In turn, it has increased research interest in the exploration of innovative food processing technologies. Cold plasma technology is getting popularity now days owing to its high efficacy in decontamination of microbes in fruit and fruit-based products. As a on-thermal approach, plasma processing maintains the quality of fruits and minimizes the thermal effects on nutritional properties. Cold plasma is also exploited for inactivating enzymes and degrading pesticides as both are directly related with quality loss and presently are most important concerns in fresh produce industry. The present review covers the influence of cold plasma technology on reducing microbial risks and enhancing the quality attributes in fruits.     

Phenotypic,Genetic, and Cytogenetic Evidence of Hybridization Between Species of Trans-Andean Tamarins (Genus <Emphasis Type="Italic">Saguinus</Emphasis>)

Incomplete reproductive isolation and hybridization is relatively frequent in primates. However, no cases of hybridization between formally recognized species have been reported in tamarins (genus Saguinus), a highly specious group of Neotropical primates. Here, we provide evidence from different sources to demonstrate three cases of hybridization in captivity between species of Saguinus distributed west of the Andes (trans-Andean). To do this, we described fur color patterns, genotyped 12 microsatellite loci, sequenced the mitochondrial hypervariable region I, and generated chromosomal R bands for the three formally recognized species and the new hybrids of trans-Andean tamarins. We identified one case of interbreeding between the white-footed tamarin (Saguinus leucopus) and the cotton-top tamarin (S. ?dipus) and two independent reciprocal crosses of S. leucopus and the Geoffroy’s tamarin (S. geoffroyi). All these hybrids exhibit intermediate phenotypes between parental species, and genetic data are consistent with first-generation hybridization. Cytogenetic data suggest that the S. leucopus × S. ?dipus hybrid is sterile, as it is a female with XY karyotype apparently affected by a condition known as gonadal dysgenesis. Trans-Andean tamarin species occur in northwest Colombia with parapatric distributions bounded by major rivers. Potential contact zones, either natural or anthropogenic, might facilitate hybridization in the wild, but this scenario remains to be assessed. Our findings warrant future studies focused on the evolutionary mechanisms of reproductive isolation in tamarins. Given the risk of hybridization, caution should be taken in management and conservation of tamarins.     

The multiple‐specificity landscape of modular peptide recognition domains

Modular protein interaction domains form the building blocks of eukaryotic signaling pathways. Many of them, known as peptide recognition domains, mediate protein interactions by recognizing short, linear amino acid stretches on the surface of their cognate partners with high specificity. Residues in these stretches are usually assumed to contribute independently to binding, which has led to a simplified understanding of protein interactions. Conversely, we observe in large binding peptide data sets that different residue positions display highly significant correlations for many domains in three distinct families (PDZ, SH3 and WW). These correlation patterns reveal a widespread occurrence of multiple binding specificities and give novel structural insights into protein interactions. For example, we predict a new binding mode of PDZ domains and structurally rationalize it for DLG1 PDZ1. We show that multiple specificity more accurately predicts protein interactions and experimentally validate some of the predictions for the human proteins DLG1 and SCRIB. Overall, our results reveal a rich specificity landscape in peptide recognition domains, suggesting new ways of encoding specificity in protein interaction networks.