Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG), depending on the tissue and experimental treatment, the gene has been us Results To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT) PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and N Conclusion As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

Why dig looted tombs? Two examples and some answers from Keushu (Ancash highlands, Peru)

Looted tombs at Andean archaeological sites are largely the result of a long tradition of trade in archaeological artefacts coupled with the 17th century policy of eradicating ancestor veneration and destroying mortuary evidence in a bid to “extirpate idolatry”. On the surface, looted funerary contexts often present abundant disarticulated and displaced human remains as well as an apparent absence of mortuary accoutrements. What kind of information can archaeologists and biological anthropologists hope to gather from such contexts? In order to gauge the methodological possibilities and interpretative limitations of targeting looted tombs, we fully excavated two collective funerary contexts at the archaeological site of Keushu (district and province of Yungay, Ancash, Peru; c. 2000 B.C.-A.D. 1600), which includes several dozen tombs, many built under large boulders or rock shelters, all of which appear disturbed by looting. The first is located in the ceremonial sector and excavation yielded information on four individuals; the second, in the funerary and residential sector, held the remains of seventy individuals - adults and juveniles. Here, we present and discuss the recovered data and suggest that careful, joint excavations by archaeologists and biological anthropologists can retrieve evidence of past mortuary practices, aid the biological characterisation of mortuary populations and help distinguish between a broad range of looting practices and post-depositional processes

Identification of stable reference genes for quantitative PCR in koalas

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations

Parasitic nematodes exert antimicrobial activity and benefit from microbiota-driven support for host immune regulation

Intestinal parasitic nematodes live in intimate contact with the host microbiota. Changes in the microbiome composition during nematode infection affect immune control of the parasites and shifts in the abundance of bacterial groups have been linked to the immunoregulatory potential of nematodes. Here we asked if the small intestinal parasite Heligmosomoides polygyrus produces factors with antimicrobial activity, senses its microbial environment and if the anti-nematode immune and regulatory responses are altered in mice devoid of gut microbes. We found that H. polygyrus excretory/secretory products exhibited antimicrobial activity against gram(+/-) bacteria. Parasites from germ-free mice displayed alterations in gene expression, comprising factors with putative antimicrobial functions such as chitinase and lysozyme. Infected germ-free mice developed increased small intestinal Th2 responses coinciding with a reduction in local Foxp3(+)RORγt(+) regulatory T cells and decreased parasite fecundity. Our data suggest that nematodes sense their microbial surrounding and have evolved factors that limit the outgrowth of certain microbes. Moreover, the parasites benefit from microbiota-driven immune regulatory circuits, as an increased ratio of intestinal Th2 effector to regulatory T cells coincides with reduced parasite fitness in germ-free mice

18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells

Background: One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. Results: The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. Conclusions: Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation

Parasitic Nematodes Exert Antimicrobial Activity and Benefit From Microbiota-Driven Support for Host Immune Regulation

Intestinal parasitic nematodes live in intimate contact with the host microbiota. Changes in the microbiome composition during nematode infection affect immune control of the parasites and shifts in the abundance of bacterial groups have been linked to the immunoregulatory potential of nematodes. Here we asked if the small intestinal parasite Heligmosomoides polygyrus produces factors with antimicrobial activity, senses its microbial environment and if the anti-nematode immune and regulatory responses are altered in mice devoid of gut microbes. We found that H. polygyrus excretory/secretory products exhibited antimicrobial activity against gram+/− bacteria. Parasites from germ-free mice displayed alterations in gene expression, comprising factors with putative antimicrobial functions such as chitinase and lysozyme. Infected germ-free mice developed increased small intestinal Th2 responses coinciding with a reduction in local Foxp3+RORγt+ regulatory T cells and decreased parasite fecundity. Our data suggest that nematodes sense their microbial surrounding and have evolved factors that limit the outgrowth of certain microbes. Moreover, the parasites benefit from microbiota-driven immune regulatory circuits, as an increased ratio of intestinal Th2 effector to regulatory T cells coincides with reduced parasite fitness in germ-free mice.Peer Reviewe

Novel Mycobacterium tuberculosis Complex Isolate from a Wild Chimpanzee

Tuberculosis (TB) is caused by gram-positive bacteria known as the Mycobacterium tuberculosis complex (MTBC). MTBC include several human-associated lineages and several variants adapted to domestic and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild chimpanzee in Côte d’Ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of MTBC, closer to the human-associated lineage 6 (also known as M. africanum West Africa 2) than to the other classical animal-associated MTBC strains. These results show that the general view of the genetic diversity of MTBC is limited and support the possibility that other MTBC variants exist, particularly in wild mammals in Africa. Exploring this diversity is crucial to the understanding of the biology and evolutionary history of this widespread infectious disease

Parasitic Nematodes Exert Antimicrobial Activity and Benefit From Microbiota-Driven Support for Host Immune Regulation

Intestinal parasitic nematodes live in intimate contact with the host microbiota. Changes in the microbiome composition during nematode infection affect immune control of the parasites and shifts in the abundance of bacterial groups have been linked to the immunoregulatory potential of nematodes. Here we asked if the small intestinal parasite Heligmosomoides polygyrus produces factors with antimicrobial activity, senses its microbial environment and if the anti-nematode immune and regulatory responses are altered in mice devoid of gut microbes. We found that H. polygyrus excretory/secretory products exhibited antimicrobial activity against gram+/− bacteria. Parasites from germ-free mice displayed alterations in gene expression, comprising factors with putative antimicrobial functions such as chitinase and lysozyme. Infected germ-free mice developed increased small intestinal Th2 responses coinciding with a reduction in local Foxp3+RORγt+ regulatory T cells and decreased parasite fecundity. Our data suggest that nematodes sense their microbial surrounding and have evolved factors that limit the outgrowth of certain microbes. Moreover, the parasites benefit from microbiota-driven immune regulatory circuits, as an increased ratio of intestinal Th2 effector to regulatory T cells coincides with reduced parasite fitness in germ-free mice

Genome-wide and comparative phylogenetic analysis of senescence-associated NAC transcription factors in sunflower (Helianthus annuus)

Background: Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. Results: To clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes (Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate. Conclusions: This finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower.Fil: Bengoa Luoni, Sofia Ailin. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Cenci, Alberto. Institut National de la Recherche Agronomique; FranciaFil: Moschen, Sebastián Nicolás. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Tucuman-Santiago del Estero. Estación Experimental Agropecuaria Famaillá; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Nicosia, Salvador. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Radonic, Laura Mabel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Sabio y Garcia, Julia Veronica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Langlade, Nicolas B.. Institut National de la Recherche Agronomique; FranciaFil: Vile, Denis. Université Montpellier II; FranciaFil: Vazquez Rovere, Cecilia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Fernández, Paula del Carmen. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

RNA isolation for transcriptomics of human and mouse small skin biopsies

<p>Abstract</p> <p>Background</p> <p>Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies.</p> <p>Findings</p> <p>We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms.</p> <p>Conclusions</p> <p>Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.</p