Exercise-induced up-regulation of MMP-1 and IL-8 genes in endurance horses

<p>Abstract</p> <p>Background</p> <p>The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.</p> <p>In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs).</p> <p>Results</p> <p>Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (<it>MMP-1</it>) and interleukin 8 (<it>IL-8</it>), which were derived from previous genome-wide expression analysis. Significant up-regulation of these two genes was found in 10 horses that had completed a race of 90–120 km in a time-course experimental design.</p> <p>Conclusion</p> <p>These results suggest that <it>MMP-1 </it>and <it>IL-8 </it>are both involved in the exercise-induced stress response, and this represents a starting point from which to understand the adaptive responses to this phenomenon.</p

Exercise induced stress in horses: Selection of the most stable reference genes for quantitative RT-PCR normalization

<p>Abstract</p> <p>Background</p> <p>Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare.</p> <p>In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response.</p> <p>Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability.</p> <p>Results</p> <p>The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in <it>geNorm</it>, <it>NormFinder </it>and <it>BestKeeper</it>). Succinate dehydrogenase complex subunit A (<it>SDHA</it>) and hypoxanthine phosphoribosyltransferase (<it>HPRT</it>) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), transferrin receptor (<it>TFRC</it>) and ribosomal protein L32 (<it>RPL32</it>) were constantly classified as the less reliable controls.</p> <p>Conclusion</p> <p>This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate <it>SDHA </it>and <it>HPRT </it>as the most stable reference genes of our pool.</p

Involvement of XRCC1 and DNA Ligase III Gene Products in DNA Base Excision Repair

DNA ligase III and the essential protein XRCC1 are present at greatly reduced levels in the xrcc1 mutant CHO cell line EM-C11. Cell-free extracts prepared from these cells were used to examine the role of the XRCC1 gene product in DNA base excision repair in vitro. EM-C11 cell extract was partially defective in ligation of base excision repair patches, in comparison to wild type CHO-9 extracts. Of the two branches of the base excision repair pathway, only the single nucleotide insertion pathway was affected; no ligation defect was observed in the proliferating cell nuclear antigen-dependent pathway. Full complementation of the ligation defect in EM-C11 extracts was achieved by addition to the repair reaction of recombinant human DNA ligase III but not by XRCC1. This is consistent with the notion that XRCC1 acts as an important stabilizing factor of DNA ligase III. These data demonstrate for the first time that xrcc1 mutant cells are partially defective in ligation of base excision repair patches and that the defect is specific to the polymerase beta-dependent single nucleotide insertion pathway

identifying the most promising agronomic adaptation strategies for the tomato growing systems in southern italy via simulation modeling

Abstract The main cultivation area of the Italian processing tomato is the Southern Capitanata plain. Here, the hardest agronomic challenge is the optimization of the irrigation water use, which is often inefficiently performed by farmers, who tend to over-irrigate. This could become unsustainable in the next years, given the negative impacts of climatic changes on groundwater availability and heat stress intensification. The aim of the study was to identify the most promising agronomic strategies to optimize tomato yield and water use in Capitanata, through a modeling study relying on an extensive dataset for model calibration and evaluation (22 data sets in 2005–2018). The TOMGRO simulation model was adapted to open-field growing conditions and was coupled with a soil model to reproduce the impact of water stress on yield and fruit quality. The new model, TomGro_field, was applied on the tomato cultivation area in Capitanata at 5 × 5 km spatial resolution using an ensemble of future climatic scenarios, resulting from the combination of four General Circulation Models, two extreme Representative Concentration Pathways and five 10-years time frames (2030–2070). Our results showed an overall negative impact of climate change on tomato yields (average decrease = 5–10%), which could be reversed by i) the implementation of deficit irrigation strategies based on the restitution of 60–70% of the crop evapotranspiration, ii) the adoption of varieties with longer cycle and iii) the anticipation of 1–2 weeks in transplanting dates. The corresponding irrigation amounts applied are around 360 mm, thus reinforcing that a rational water management could be realized. Our study provides agronomic indications to tomato growers and lays the basis for a bio-economic analysis to support policy makers in charge of promoting the sustainability of the tomato growing systems

Chapter Facing Malaria Parasite with Mosquito Symbionts

Optical physic

Facing Malaria Parasite with Mosquito Symbionts

Optical physic

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials

A self-repair history: compensatory effect of a de novo variant on the FANCA c.2778+83C>G splicing mutation

Introduction: Fanconi anemia (FA) is a genome instability condition that drives somatic mosaicism in up to 25% of all patients, a phenomenon now acknowledged as a good prognostic factor. Herein, we describe the case of P1, a FA proband carrying a splicing variant, molecularly compensated by a de novo insertion. Methods and Results: Targeted next-generation sequencing on P1's peripheral blood DNA detected the known FANCA c.2778 + 83C &gt; G intronic mutation and suggested the presence of a large deletion on the other allele, which was then assessed by MLPA and RT-PCR. To determine the c.2778 + 83C &gt; G splicing effect, we performed a RT-PCR on P1's lymphoblastoid cell line (LCL) and on the LCL of another patient (P2) carrying the same variant. Although we confirmed the expected alternative spliced form with a partial intronic retention in P2, we detected no aberrant products in P1's sample. Sequencing of P1's LCL DNA allowed identification of the de novo c.2778 + 86insT variant, predicted to compensate 2778 + 83C &gt; G impact. Albeit not found in P1's bone marrow (BM) DNA, c.2778 + 86insT was detected in a second P1's LCL established afterward, suggesting its occurrence at a low level in vivo. Minigene assay recapitulated the c.2778 + 83C &gt; G effect on splicing and the compensatory role of c.2778 + 86insT in re-establishing the physiological mechanism. Accordingly, P1's LCL under mitomycin C selection preserved the FA pathway activity in terms of FANCD2 monoubiquitination and cell survival. Discussion: Our findings prove the role of c.2778 + 86insT as a second-site variant capable of rescuing c.2778 + 83C &gt; G pathogenicity in vitro, which might contribute to a slow hematopoietic deterioration and a mild hematologic evolution

Hypomorphic FANCA mutations correlate with mild mitochondrial and clinical phenotype in Fanconi anemia

Fanconi anemia is a rare disease characterized by congenital malformations, aplastic anemia, and predisposition to cancer. Despite the consolidated role of the Fanconi anemia proteins in DNA repair, their involvement in mitochondrial function is emerging. The purpose of this work was to assess whether the mitochondrial phenotype, independent of genomic integrity, could correlate with patient phenotype. We evaluated mitochondrial and clinical features of 11 affected individuals homozygous or compound heterozygous for p.His913Pro and p.Arg951Gln/Trp, the two residues of FANCA that are more frequently affected in our cohort of patients. Although p.His913Pro and p.Arg951Gln proteins are stably expressed in cytoplasm, they are unable to migrate in the nucleus, preventing cells from repairing DNA. In these cells, the electron transfer between respiring complex I-III is reduced and the ATP/AMP ratio is impaired with defective ATP production and AMP accumulation. These activities are intermediate between those observed in wild-type and FANCA-/- cells, suggesting that the variants at residues His913 and Arg951 are hypomorphic mutations. Consistent with these findings, the clinical phenotype of most of the patients carrying these mutations is mild. These data further support the recent finding that the Fanconi anemia proteins play a role in mitochondria, and open up possibilities for genotype/phenotype studies based on novel mitochondrial criteria

Exploring the Italian equine gene pool via high-throughput genotyping

Introduction: The Italian peninsula is in the center of the Mediterranean area, and historically it has been a hub for numerous human populations, cultures, and also animal species that enriched the hosted biodiversity. Horses are no exception to this phenomenon, with the peculiarity that the gene pool has been impacted by warfare and subsequent “colonization”. In this study, using a comprehensive dataset for almost the entire Italian equine population, in addition to the most influential cosmopolitan breeds, we describe the current status of the modern Italian gene pool.Materials and Methods: The Italian dataset comprised 1,308 individuals and 22 breeds genotyped at a 70 k density that was merged with publicly available data to facilitate comparison with the global equine diversity. After quality control and supervised subsampling to ensure consistency among breeds, the merged dataset with the global equine diversity contained data for 1,333 individuals from 54 populations. Multidimensional scaling, admixture, gene flow, and effective population size were analyzed.Results and Discussion: The results show that some of the native Italian breeds preserve distinct gene pools, potentially because of adaptation to the different geographical contexts of the peninsula. Nevertheless, the comparison with international breeds highlights the presence of strong gene flow from renowned breeds into several Italian breeds, probably due to historical introgression. Coldblood breeds with stronger genetic identity were indeed well differentiated from warmblood breeds, which are highly admixed. Other breeds showed further peculiarities due to their breeding history. Finally, we observed some breeds that exist more on cultural, traditional, and geographical point of view than due to actual genetic distinctiveness