Validation of a simplified small-scale DNA extraction protocol from wine by quantitative real-time PCR.

In the present study, we compared a simplified small-scale purification protocol to obtain DNA admixtures out of wine, with our large-scale published method. The extraction methods must provide DNA free of PCR inhibitors, that can interfere with DNA amplification. To evaluate the efficiency of grapevine's nuclear DNA extraction from wine, the new protocol was also compared in terms of purity and yield to the DNA obtained out of grapevine's (Vitis vinifera) leaf tissue, using a commercial kit. Two single-copy nuclear genes, nine-cis-epoxy carotenoid dioxygenase 2 (NCED2), and prefoldin subunit 5-like (PS5) were amplified in DNA extracted from wine and grapevine by real-time TaqMan PCR to determine the presence of inhibitors in relation to the diversity of starting biological matrix. This study showed that the small-scale, simpler method for extracting DNA from wine produced effective results in terms of inhibitor presence and purity. Furthermore, even though the initial biological matrix was more complicated, the grapevine nuclear DNA that was removed from wine was qualitatively equivalent to the DNA that was isolated from the leaves. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03992-x.

aTBP: A versatile tool for fish genotyping.

Animal Tubulin-Based-Polymorphism (aTBP), an intron length polymorphism method recently developed for vertebrate genotyping, has been successfully applied to the identification of several fish species. Here, we report data that demonstrate the ability of the aTBP method to assign a specific profile to fish species, each characterized by the presence of commonly shared amplicons together with additional intraspecific polymorphisms. Within each aTBP profile, some fragments are also recognized that can be attributed to taxonomic ranks higher than species, e.g. genus and family. Versatility of application across different taxonomic ranks combined with the presence of a significant number of DNA polymorphisms, makes the aTBP method an additional and useful tool for fish genotyping, suitable for different purposes such as species authentication, parental recognition and detection of allele variations in response to environmental changes.

An "ex vivo" model to evaluate toxicological responses to mixtures of contaminants in cetaceans: integumentum biopsy slices.

The need for powerful new tools to detect the effects of chemical pollution, in particular of persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs) on Mediterranean cetaceans led us to develop and apply a suite of sensitive biomarkers for integument biopsies of stranded and free-ranging animals. This multi-response ex vivo method has the aim to detect toxicological effects of contaminant mixtures. In the present study, we applied an ex vivo assay using skin biopsy and liver slices, combining molecular biomarkers [Western blot of Cytochrome P450 1A1 (CYP1A1) and Cytochrome P450 2B (CYP2B)] and gene expression biomarkers (Quantitative real-time PCR of CYP1A1, heat shock protein 70, estrogen receptor alpha and E2F transcription factor) in response to chemical exposure [organochlorines compounds (OCs), polybrominated diphenyl ethers (PBDEs), and PAHs] for stranded Mediterranean Stenella coeruleoalba. The main goal of this experiment was to identify the biomarker and/or a suite of biomarkers that could best detect the presence of a specific class of pollutants (OCs, PBDEs, and PAHs) or a mixture of them. This multi-response biomarker methodology revealed an high sensitivity and selectivity of responses (such as CYP1A and ER α mRNA variations after OCs and PAHs exposure) and could represent a valid future approach for the study of inter- and intra-species sensitivities to various classes of environmental contaminants.

The Pelagos Sanctuary for Mediterranean marine mammals: Marine Protected Area (MPA) or marine polluted area? The case study of the striped dolphin (Stenella coeruleoalba).

The concurrence of man-made pressures on cetaceans in the Mediterranean Sea is potentially affecting population stability and marine biodiversity. This needs to be proven for the only pelagic marine protected area in the Mediterranean Sea: the Pelagos Sanctuary for Mediterranean Marine Mammals. Here we applied a multidisciplinary tool, using diagnostic markers elaborated in a statistical model to rank toxicological stress in Mediterranean cetaceans. As a case study we analyzed persistent, bioaccumulative and toxic chemicals combined with a wide range of diagnostic markers of exposure to anthropogenic contaminants and genetic variation as marker of genetic erosion in striped dolphin (Stenella coeruleoalba) skin biopsies. Finally, a statistical model was applied to obtain a complete toxicological profile of the striped dolphin in the Pelagos Sanctuary and other Mediterranean areas (Ionian Sea and Strait of Gibraltar). Here we provide the first complete evidence of the toxicological stress in cetaceans living in Pelagos Sanctuary.

Ecotoxicological diagnosis of striped dolphin (Stenella coeruleoalba) from the Mediterranean basin by skin biopsy and gene expression approach.

Mediterranean cetacean odontocetes are exposed to environmental stress, in particular to persistent organic pollutants, polycyclic aromatic hydrocarbons and trace elements. In the present study, the response of "gene-expression biomarkers" was evaluated in Mediterranean striped dolphin (Stenella coeruleoalba) skin biopsies collected in three sampling areas: Pelagos sanctuary (Ligurian sea), Ionian sea, and Strait of Gibraltar. The mRNA levels of five putative biomarker genes (aryl hydrocarbon receptor, E2F-1 transcription factor, cytochrome P450 1A, estrogen receptor 1, and heat shock protein 70) were measured for the first time by quantitative real-time PCR in cetacean skin biopsies. The different responses of most of the genes reflected contamination levels in the three sampling areas. Pelagos sanctuary dolphins appeared to be the most exposed to toxicological stress, having the highest up-regulation of CYP1A and AHR. Moreover, a cluster analysis distinguished the populations on the basis of the gene expression biomarker used in our study, showing different pattern between Mediterranean sea and Strait of Gibraltar. Our results suggest that this molecular approach applied to non-destructive biopsy material is a powerful diagnostic tool for evaluating ecotoxicological impact on cetacean populations.

Lymphocyte TRPV 1-4 gene expression and MIF blood levels in a young girl clinically diagnosed with HSAN IV.

OBJECTIVE: Patients with congenital insensitivity to pain are unable to sense pain and temperature. They undergo many injuries, inflammatory state, and infections. Various mutations in the neurotrophic tyrosine kinase receptor gene have been implicated in this disorder. We measured the leukocyte expression of transient receptor potential vanilloid (TRPV) 1-4 genes and the blood macrophage migration inhibitory factor (MIF) concentration in a young girl clinically diagnosed with congenital insensitivity to pain. The investigation may help to define the interplay between nerve growth factor and TRPV 1-4 channels and between these sensors and MIF in this disease, and in broader terms in nociception. METHODS: TRPV 1-4 gene expression (real-time polymerase chain reaction) and MIF concentration (enzyme-linked immunosorbent assay) were determined in the blood of the girl, her family, and control participants. Statistical analysis of gene expression was carried out between samples and controls with a mathematical model based on the correction for exact polymerase chain reaction efficiencies, and the mean crossing point deviation between samples and controls. RESULTS: The TRPV 1--4 gene expression rates did not significantly differ from the values found in the control group. TRPV1 was almost doubly upregulated. MIF levels were much higher than the reference value. DISCUSSION: The high increase in the MIF concentration (likely due to the chronic or recurrent inflammatory state) may have contributed to the normal expression of TRPV 1-4 and to the relative upregulation of TRPV1. The role of this cytokine on the expression of these genes deserves further investigation.

Athletic humans and horses: comparative analysis of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) expression in peripheral blood mononuclear cells in trained and untrained subjects at rest.

BACKGROUND: Horses and humans share a natural proclivity for athletic performance. In this respect, horses can be considered a reference species in studies designed to optimize physical training and disease prevention. In both species, interleukin-6 (IL-6) plays a major role in regulating the inflammatory process induced during exercise as part of an integrated metabolic regulatory network. The aim of this study was to compare IL-6 and IL-6 receptor (IL-6R) mRNA expression in peripheral blood mononuclear cells (PBMCs) in trained and untrained humans and horses. RESULTS: Nine highly trained male swimmers (training volume: 21.6 ± 1.7 h/wk in 10-12 sessions) were compared with two age-matched control groups represented by eight lightly trained runners (training volume: 6.4 ± 2.6 h/wk in 3-5 sessions) and nine untrained subjects. In addition, eight trained horses (training volume: 8.0 ± 2.1 h/wk in 3-4 sessions) were compared with eight age-matched sedentary mares. In humans, IL-6 mRNA levels in PBMCs determined by quantitative reverse transcription-polymerase chain reaction were significantly higher in highly trained subjects, whereas IL-6R expression did not differ among groups. In horses, transcripts of both IL-6 and IL-6R were significantly up-regulated in the trained group. CONCLUSIONS: Up-regulation of IL-6R expression in PBMCs in horses could reflect a mechanism that maintains an adequate anti-inflammatory environment at rest through ubiquitous production of anti-inflammatory cytokines throughout the body. These findings suggest that the system that controls the inflammatory response in horses is better adapted to respond to exercise than that in humans.

The putative-farnesoic acid O-methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre-imaginal life expression.

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative-FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real-Time PCR in medfly pre-imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre-imaginal putative-FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well-known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control.

A multi-trial diagnostic tool in fin whale (Balaenoptera physalus) skin biopsies of the Pelagos Sanctuary (Mediterranean Sea) and the Gulf of California (Mexico).

The main objective of this study was to apply a set of sensitive non-lethal biomarkers in skin biopsies of fin whales (Balaenoptera physalus) to evaluate the toxicological status of this mysticete in the Pelagos Sanctuary (Mediterranean Sea) and in the Gulf of California (Sea of Cortez-Mexico). We developed a "multi-trial diagnostic tool" (based on field and in vitro studies), combining molecular biomarkers (western blot of CYP1A1, CYP2B) and gene expression (qRT-PCR of HSP70, ERα, AHR, E2F-1) with the analysis of OCs, PAHs and PBDEs. The study revealed a higher level of toxicological stress in the Mediterranean fin whales.

Quantitative Real-Time PCR detection of TRPV1-4 gene expression in human leukocytes from healthy and hyposensitive subjects.

BACKGROUND: Besides functioning as chemosensors for a broad range of endogenous and synthetic ligands, transient receptor potential vanilloid (TRPV) 1-4 channels have also been related to capsaicin (TRPV1), pain, and thermal stimuli perception, and itching sensation (TRPV1-4). While the expression of the TRPV1-4 genes has been adequately proved in skin, sensory fibres and keratinocytes, less is known about TRPV3 and TRPV4 expression in human blood cells. RESULTS: To study the gene expression of TRPV1-4 genes in human leukocytes, a quantitative Real-Time PCR (qRT-PCR) method, based on the calculation of their relative expression, has been developed and validated. The four commonly used house-keeping genes (HKGs), beta-Actin (Act-B), glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT1), and cyclophilin B (hCyPB), were tested for the stability of their expression in several human leukocyte samples, and used in the normalization procedure to determine the mRNA levels of the TRPV 1-4 genes in 30 healthy subjects. cDNAs belonging to all the TRPV1-4 genes were detected in leukocytes but the genes appear to be expressed at different levels. Our analysis did not show significant sex differences in TRPV1-4 cDNA levels in the 30 healthy subjects. The same qRT-PCR assay was used to compare TRPV1-4 expression between healthy controls and patients hyposensitive to capsaicin, pain and thermal stimuli: an almost doubled up-regulation of the TRPV1 gene was found in the pathological subjects. CONCLUSION: The qRT-PCR assay developed and tested in this study allowed us to determine the relative expression of TRPV1-4 genes in human leukocytes: TRPV3 is the least expressed gene of this pool, followed by TRPV4, TRPV1 and TRPV2. The comparison of TRPV1-4 gene expression between two groups of healthy and hyposensitive subjects highlighted the evident up-regulation of TRPV1, which was almost doubly expressed (1.9x normalized fold induction) in the latter group. All the four house-keeping genes tested in this work (Act-B, GAPDH, hCyPB, HPRT1) were classified as optimal controls and showed a constant expression in human leukocytes samples. We recommend the use of these genes in similar qRT-PCR studies on human blood cells.

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

BACKGROUND: 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. 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. 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. RESULTS: 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 geNorm, NormFinder and BestKeeper). Succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyltransferase (HPRT) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transferrin receptor (TFRC) and ribosomal protein L32 (RPL32) were constantly classified as the less reliable controls. CONCLUSION: 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 SDHA and HPRT as the most stable reference genes of our pool.

Selection of reliable reference genes for qRT-PCR studies on cetacean fibroblast cultures exposed to OCs, PBDEs, and 17beta-estradiol.

Quantitative real-time PCR (qRT-PCR) represents an effective molecular technique for the detection of mRNA expression in biological samples. Its sensitivity allows the quantification of slight changes in the regulation of gene transcription but is strictly dependent upon the method followed during the normalization procedure. Relative quantification determines changes in the steady-state mRNA levels of genes across multiple samples and it is assessed by comparison with the levels of one or more internal control RNA. In this context, the choice of constitutively expressed control genes, whose transcription is not affected by the contaminants, appears to be fundamental for the reliability of this technique. During this study, fibroblast cell cultures originated from integumentum biopsies, sampled in the cetacean species Stenella coeruleoalba, have been exposed for 6h to increasing concentrations of different mixtures of compounds with endocrine disruptor capacities (EDCs): organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), and 17beta-estradiol. Ten common housekeeping genes have been tested for the expression of their transcripts in exposed cell cultures using qRT-PCR assays and raw data were analyzed with the two Excel applets geNorm and NormFinder. The genes encoding for SDHA, GAPDH and YWHAZ appear to be the most reliable controls, respectively, for the OC, PBDE and 17beta-estradiol treatments. These results clearly show that the transcription of even widely diffused control genes can be regulated by different treatments and underlie the importance of a careful selection of the optimal housekeeping genes in toxicological studies.

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies.

BACKGROUND: Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. RESULTS: Ten commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and beta-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes for data normalization. CONCLUSION: In this work, we have provided essential background information for the selection of control genes in qRT-PCR studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals. Of 10 HKGs tested, those encoding for YWHAZ and GAPDH appear as the most reliable control genes for the normalization of qRT-PCR data in the analysis of striped dolphin skin biopsies. Potentially useful reference genes are also those encoding for ribosomal proteins L4 and S18.

Hexapod origins: monophyletic or paraphyletic?

Recent morphological and molecular evidence has changed interpretations of arthropod phylogeny and evolution. Here we compare complete mitochondrial genomes to show that Collembola, a wingless group traditionally considered as basal to all insects, appears instead to constitute a separate evolutionary lineage that branched much earlier than the separation of many crustaceans and insects and independently adapted to life on land. Therefore, the taxon Hexapoda, as commonly defined to include all six-legged arthropods, is not monophyletic.