PhD

dissertationThe in vivo effects of Ftorafur (FT) and 5-fluorouracil (FU) were evaluated at several different organizational levels within the same animal model. On a molar basis, FU was found to be two to three times more potent than FT with respect to growth inhibition of murine mammary adenocarcinomas. However, administration of FT produced less host toxicity at a dose level which resulted in similar antitumor activity. Drug-induced perturbations in cell cycle phase distributions were analyzed by the flow cytometer (FCM). Although FCM analysis is limited by lack of information on the extent of dead and dying cells, data obtained with both drugs were consistent with the possibility of S phase cytotoxicity and drug-induce G1/S block or delay. Changes in the age distribution of perturbed tumor cells, is detected by FCM analysis, correlated with 3H-TdR autoradiography and with 32P incorporation into DNA but not with 3H-deoxyuridine incorporation into DNA. Both drugs depressed 3H-deosyuridine incorporation into DNA; however, incorporation in both the tumor and small intestine returned to control levels earlier with FT than with an equimolar dose of FU. However, both drugs produced similar patters of 3H-deoxyuridine incorporation when FT was administered at three times the molar equivalent dose of FU. The results of these studies also suggest that deoxyuridine incorporation primarily reflects drug effects on thymidylate synthetase and not necessarily the overall rate of DNA synthesis. This study also involved the development of techniques for the dispersal of solid tumors and intestinal lining epithelium into single cell suspensions for FCM analysis. Tetraphenylboron (TPB) was utilized as an aid to solid tumor dispersal. TPB-dispersed tumors yield DNA distributions with minimal cell clumping and low levels of cellular debris. Fluorescent enzyme histochemistry with subsequent FCM analysis was utilized to separate dispersed epithelial cells of the small intestine into crypt and villus subpopulations. When fully optimized, the epithelial cell separation should offer a rapid method by which perturbations in the age distribution of crypt cells can easily be evaluated by FCM

Comparative analysis of novel and common reference genes in adult tissues of the mussel Mytilus galloprovincialis

Background Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates. Results In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1 alpha, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues. Conclusion Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species

Serum uric acid, creatinine, and the assessment of antioxidant capacity in critical illness

No Abstract - The letter addresses the issue of low plasma urate as a marker of impaired antioxidant capacity in critical illness

The small non-coding RNA processing machinery of two living fossil species, lungfish and coelacanth, gives new insights into the evolution of the Argonaute protein family

Argonaute (AGO) family proteins play many roles in epigenetic programming, genome rearrangement, mRNA breakdown, inhibition of translation, and transposon silencing. Despite being a hotspot in current scientific research, their evolutionary history is still poorly understood and consequently the identification of evolutionary conserved structural features should also generate useful information for better understanding their functions. We report here for the first time the transcript sets of the two subfamilies, Ago and Piwi, in the West African lungfish Protopterus annectens and in the Indonesian coelacanth Latimeria menadoensis, two key species in the evolutionary lineage leading to tetrapods. The phylogenetic analysis of 142 inferred protein sequences in 22 fully sequenced species and the analysis of microsynteny performed in the major vertebrate lineages indicate an intricate pattern for the evolution of both subfamilies that has been shaped by whole genome duplications and lineage specific gains and losses. The argonaute subfamily was additionally expanded by local gene duplications at the base of the jawed vertebrate lineage. The subfamily of Piwi proteins is involved in several processes such as spermatogenesis, piRNA biogenesis, and transposon repression. Expression assessment of AGO genes and genes coding for proteins involved in small RNA biogenesis suggests a limited activity of the Piwi pathway in lungfish in agreement with the lungfish genome containing mainly old and inactive transposons

A comparative view on sex differentiation and gametogenesis genes in lungfish and coelacanths

none8siGonadal sex differentiation andreproductionare the keys totheperpetuationof favorable gene combinations andpositively selected traits. In vertebrates, several gonad development features that differentiate tetrapods and fishes are likely to be, at least in part, related to the water-to-land transition. The collection of information from basal sarcopterygians, coelacanths, and lungfishes, is crucial to improve our understanding of the molecular evolution of pathways involved in reproductive functions, since these organisms are generally regarded as “living fossils” and as the direct ancestors of tetrapods. Here, we report for the first time the characterization of >50 genes related to sex differentiation and gametogenesis in Latimeria menadoensis and Protopterus annectens. Although the expression profiles of most genes is consistent with the intermediate position of basal sarcopterygians between actinopterygian fish and tetrapods, their phylogenetic placement and presence/absence patterns often reveal a closer affinity to the tetrapod orthologs. On the other hand, particular genes, for example, the male gonad factor gsdf (Gonadal Soma-Derived Factor), provide examples of ancestral traits sharedwith actinopterygians,which disappeared in the tetrapod lineage.openMaria Assunta Biscotti, Mateus Contar Adolfi, Marco Barucca, Mariko Forconi, Alberto Pallavicini, Marco Gerdol, Adriana Canapa, Manfred SchartlBiscotti, Maria Assunta; Contar Adolfi, Mateus; Barucca, Marco; Forconi, Mariko'; Pallavicini, Alberto; Gerdol, Marco; Canapa, Adriana; Schartl, Manfre

Co-occurrence and diversity patterns of benthonic and planktonic communities in a shallow marine ecosystem

Marine microorganisms are involved in a variety of biogeochemical cycles and live in diverse ecological communities where they interact with each other and with other organisms to guarantee ecosystem functions. The present study focused on a shallow marine environment located in Ría de Vigo (NW, Spain), where sediment and size-fractionated plankton samples were collected from 2016 to 2018. DNA metabarcoding was used to describe the eukaryote and prokaryote composition and diversity in sediments and plankton and to depict possible associations among the most frequent and abundant organisms by co-occurrence network analysis. High eukaryote and prokaryote diversity indices were obtained in all compartments. Significant differences among eukaryote and prokaryote communities were found between sediment and plankton samples, with a high percentage of exclusive operational taxonomic units (OTUs) associated with each compartment, especially from sediment. Despite these differences, shared taxa between water and sediment were also obtained, suggesting a relatively meaningful exchange of organisms between both environmental compartments. Significant co-occurrences were mainly obtained between prokaryotes (41%), followed by eukaryotes–prokaryotes (32%) and between eukaryotes (27%). The abundant and strong positive correlations between organisms, including representatives from the sediment and the water column, suggested an essential role of biotic interactions as community-structuring factors in shallow waters where beneficial associations likely prevail. This study provides a novel approach for the detailed description of the eukaryote and prokaryote diversity and co-occurrence patterns in a shallow marine area, including both the sediment and different water-size fractions. The high diversity obtained and the detection of predominantly coexisting interactions among organisms from sediment and the overlying water column suggest a movement of species between both habitats and therefore confirm the importance of integratively studying shallow marine ecosystems.Xunta de Galicia | Ref. IN606A-2018/020Xunta de Galicia | Ref. IN607B 2019/01Agencia Estatal de Investigación | Ref. CTM2017-83362-RInterreg España-Portugal | Ref. 20200474_BLUEBIOLA

Light Emitting Molecular Devices Based on Transition Metals

Multicomponent systems have been designed, which are able to perform defined functions related to light emission and quenching. The desired function can be switched ON/OFF by the operator through a chemical input, either a change of pH or a variation of the redox potential. Transition metals (e.g. Ni(II), Cu(II)) are key constituents within the considered systems, playing a distinctive architectural role and favouring electron transfer processes

Citron Kinase Deficiency Leads to Chromosomal Instability and TP53-Sensitive Microcephaly

Mutations in citron (CIT), leading to loss or inactivation of the citron kinase protein (CITK), cause primary microcephaly in humans and rodents, associated with cytokinesis failure and apoptosis in neural progenitors. We show that CITK loss induces DNA damage accumulation and chromosomal instability in both mammals and Drosophila. CITK-deficient cells display "spontaneous" DNA damage, increased sensitivity to ionizing radiation, and defective recovery from radiation-induced DNA lesions. In CITK-deficient cells, DNA double-strand breaks increase independently of cytokinesis failure. Recruitment of RAD51 to DNA damage foci is compromised by CITK loss, and CITK physically interacts with RAD51, suggesting an involvement of CITK in homologous recombination. Consistent with this scenario, in doubly CitK and Trp53 mutant mice, neural progenitor cell death is dramatically reduced; moreover, clinical and neuroanatomical phenotypes are remarkably improved. Our results underscore a crucial role of CIT in the maintenance of genomic integrity during brain development

Grain quality as Influenced by the structural properties of weed communities in Mediterranean wheat crops

Weed community structure, including composition, taxonomic and functional diversity, may explain variability in crop quality, adding to the variability accounted by management, climatic and genetic factors. Focusing on Mediterranean rainfed wheat crops, we sampled weed communities from 26 fields in Spain that were either organically or conventionally managed. Weed communities were characterized by their abundance and taxonomic, compositional and trait-based measures. Grain protein concentration and the glutenin to gliadin ratio were used as indicators of wheat grain quality. Linear mixed effects models were used to analyze the relationship between crop quality and weed community variables, while accounting for environmental factors. Nitrogen fertilization, previous crop and precipitation explained a large portion of the variation in wheat grain protein concentration (R2marginal = 0.39) and composition (R2marginal = 0.26). Weed community measures had limited effects on grain quality (increasing R2marginal of models by 1% on average). The weed effects were related to the composition and the functional structure of their communities, but not to their abundance. Environmental conditions promoting higher protein concentration were also selecting for weed species with competitive attributes, whereas the role of weed functional diversity depended on the functional trait and on the resource limiting crop grain quality. Understanding the mechanisms of weed effects on crop quality could aid on designing sustainable weed management practices.This research was supported by grants AGL2012-33736 and AGL2015-64130-R funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. D.J.G. was partially supported by the United States National Science Foundation (DUE 1758497 and DUE 1949969)Postprint (published version

Lice, rodents, and many hopes: a rare disease in a young refugee

Borrelia recurrentis infection is a louse-borne disease and Leptospirosis is a rat-borne zoonosis, both endemic in areas characterized by a low hygiene condition. This is the first case of life-threatening Borrelia recurrentis and Leptospira species co-infectio