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 β-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

A review of plastic-associated pressures: Cetaceans of the Mediterranean Sea and Eastern Australian Shearwaters as case studies

Impacts of debris on marine fauna occur throughout the marine ecosystems, with adverse impacts documented on over 1,400 species; impacts can be divided into those arising from entanglement, and those from ingestion. Ingestion of, and entanglement in, debris has been documented in over 60% of all cetacean species. Seabirds are also impacted by debris predominately through entanglement and ingestion, with the number of species negatively impacted increasing from 138 to 174 over the past two decades. In the marine environment, cetaceans and seabirds are widely regarded as reliable sentinels due to their position near the top of the marine food web, conspicuous nature, and reliance on marine resources; for this reason, this paper is focused on seabirds and cetaceans as sentinels of ocean change. In particular, two case studies are considered in relation to different levels of environmental anthropogenic impact: the cetaceans of the Mediterranean Sea and seabirds of eastern Australia. Here we describe two recent studies used to diagnose the toxicological stress related to debris-associated pressures in cetaceans and seabirds. These studies highlight the diversity and scale of impacts being felt by marine species and the role these organisms can play in our society as charismatic sentinels of ocean health. Seabirds and marine mammals are exposed, in these key areas, to a variety of adversities that potentially decrease their survival or reproductive success. These include weather, food shortages, predators, competitors, parasites, disease, and human-induced effects and plastic pollution. Each factor affects seabirds and marine mammals in a different way, but more importantly, factors can also interact and create impacts far greater than any one factor alone. The Australian and Mediterranean case studies presented here emphasize the need to consider multiple sources of mortality when developing management plans for the conservation of vulnerable species

Editorial: Microplastics in the marine environment: Sources, distribution, biological effects and socio-economic impacts

inexistenteinfo:eu-repo/semantics/publishedVersio

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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), β-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.</p> <p>Conclusion</p> <p>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 (1.9× 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.</p

“Test Tube Cetaceans”: From the Evaluation of Susceptibility to the Study of Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures

Population estimates of various species of cetaceans indicate that certain species have declined dramatically this century. Most studies of contamination and biomarker responses in marine mammals have been conducted using animals killed by hunting, tacitly approving this activity. The development of a series of nondestructive techniques to evaluate biomarker responses and residue levels is strongly recommended for the hazard assessment, protection and conservation of endangered species of marine mammals. A non-invasive sampling method, represented by skin biopsy or integument biopsy (epidermis, dermis and blubber), have been developed and validated in cetaceans. In this paper we present the cetacean fibroblast cell cultures obtained from the skin biopsies as the “test tube cetacean” for evaluate both the susceptibility that the genotoxicity of different environmental contaminants. Fibroblast cell cultures were obtained from many species of cetaceans sampled in Mediterranean Sea (Italy) and in the Mar de Cortez (Mexico). Using test tube cetaceans we can study the relationships between contamination and biochemical responses. One of the principal applications of this developed in vitro system was the assessment of interspecies differences in the mixed function oxidase activity (Cyp1A1 and Cyp2B) induced by in vitro treatment of various contaminants, such as some Persistent Organic Pollutants (POPs) but also emerging contaminants (such as bisphenol A (BpA)) and nanoparticles, added at different concentrations. The induction of Cyp1A1 and Cyp2B was evaluated with the indirect immunofluorescence technique. Therefore, another purpose of this work was to evaluate the qualitative and quantitative MICA protein expression in fibroblast cell cultures with immunofluorescence technique as toxicological stress marker of the immune system of different species of cetaceans. Finally, In this paper we will present how to evaluate the presence of DNA damage by comet assay in test tube cetaceans, after treatment with different genotoxic compounds (for example PCBs, DDTs, PAHs, BPA)

Microplastic occurrence and phthalate ester levels in neuston samples and skin biopsies of filter-feeding megafauna from La Paz Bay (Mexico)

The impacts of microplastics on filter feeders megafauna have recently received increased attention. These or-ganisms are potentially exposed to plastic ingestion and the release of added/sorbed contaminants during feeding activities. An assessment of microplastic abundance and the chemical impact of Phthalates esters (PAEs) were performed in neustonic samples and skin biopsies of Balaenoptera physalus and Rhincodon typus inhabiting the Gulf of California (Mexico). Sixty-eight percent of the net tows contained plastics with a maximum of 0.24 items/ m3 mainly composed of polyethylene fragments. PAE levels were detected both in environmental and skin biopsy samples, with the highest values in the fin whale specimens (5291 ng/g d.w). Plasticizer fingerprint showed a similar distribution pattern between neustonic samples and filter-feeding species, with DEHP and MBP having the highest concentrations. The detection of PAE levels confirmed their potential role as plastic tracers and give preliminary information about the toxicological status of these species feeding in La Paz Bay

Female scientists of the Museo Argentino de Ciencias Naturales: the first arachnologists

"Pioneras: Las Científicas del Museo Argentino de Ciencias Naturales" es un ciclo que se inició en elmes de marzo del año 2019 en el marco del Día Internacional de la Mujer. Este ciclo surgió a partir de un grupo de mujeres autoconvocadas del Museo y tiene por objetivo reconocer y homenajear la labor de las primeras mujeres que desempeñaron actividades científicas dentro de nuestra Institución. En su tercera edición (2021), el ciclo rindió homenaje a tres brillantes profesionales y pioneras de la aracnología: Berta S. Gerschman de Pikelin, Rita D. E. Schiapelli y María Elena Galiano. Estas tres investigadoras han dedicado su carrera científica al estudio de arañas neotropicales. A lo largo de su trayectoria, no sólo han dado origen a la Colección Nacional de Aracnología del Museo, sino que también han dejado un legado de más de 150 trabajos científicos publicados y alrededor de 200 nuevas especies descriptas, construyendo una base sólida para investigaciones futuras.“Pioneers: Women Scientists of the Museo Argentino de Ciencias Naturales’’ is a cycle that began in March 2019 within the framework of the International Women’s Day. It began as an initiative of a self-convened group of working women of the Museum, with the aim of recognizing and honoring the trajectories of the first women who carried out scientific activities within our Institution. In the third edition (2021), the cycle paid tribute to three brilliant professionals and pioneers in arachnology: Berta S. Gerschman de Pikelin, Rita D. E. Schiapelli, and María Elena Galiano. These three researchers dedicated their scientific careers to studying Neotropical spiders. Along their careers, they built up the Arachnology National Collection. Furthermore, they left a legacy of more than 150 published papers and nearly 200 new described species, providing a solid basis for future scientific research.Fil: Ferraro, Daiana Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Panti, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Tancoff, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: de Cabo, Laura Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Chornogubsky Clerici, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: D'hiriart, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Grismado, Cristian José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Ramirez, Martin Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Scioscia, Cristina Luisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Cruz, Laura Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentin

Marine mammal ecotoxicology: Impacts of multiple stressors on population health

Marine Mammal Ecotoxicology: Impacts of Multiple Stressors on Population Health provides tactics on how to develop a comprehensive methodology for the study of existing threats to marine mammals. By presenting a conservation-biology approach and new and emerging technologies, this work helps provide crucial knowledge on the status of marine mammal populations that not only helps readers understand the ecosystem's health, but also instigate mitigation measures. This volume provides information that helps investigators unravel the relationships between exposure to environmental stressors (e.g., climate change, pollutants, marine litter, pathogens and biotoxins) and a range of endpoints in marine mammal species. The application of robust examination procedures and biochemical, immunological, and molecular techniques, combined with pathological examination and feeding ecology, has led to the development of health assessment methods at the individual and population levels in wild marine mammals

Mill dynamics: Exploring factors that influence closure patterns of primary forest product mills in the Southern U.S.

Timber is the most marketable product obtained from the majority of forests. Approximately 60 percent of U.S. timber products are produced by mills in the Southern U.S. (Prestemon and Abt 2002). The USDA Forest Service Forest Inventory and Analysis (FIA) southern region conducts biennial surveys of Timber Products Output (TPO) to assess uses of roundwood in 12 southern states. The TPO survey targets the entire population of primary wood-using mills, this study included a set of selected mill types for surveys conducted between 2005 and 2017. Primary wood-using mills included sawmills, plywood, veneer, poles, and post mills. This historical TPO mill information was analyzed using a time to event methodology also called a survival analysis approach, to evaluate internal and external factors affecting mill survival. Internal variables included plant size, type, structure and ownership, tree species group (hardwood and softwood), and equipment. External factors included clustering, plant competition at 10 and 30 miles, as well as the socio-economic variables - population density and unemployment. Mill numbers declined by approximately 39% between 2005 (start of study) and 2017 (end of study). Smaller numbers were observed for the years 2007 to 2009, coincident with the Great Recession in the U.S., but consumption volumes gradually increased after that period. The results showed most of the variables had a significant correlation with the probability of closure. The exception was the socioeconomic variables, which were not correlated with mill exit. The analysis showed significant changes in the density of mills with a downward trend in mill numbers regardless of socio-economic conditions