Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between ...different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR.
In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH.
Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Strong signatures of selection in the domestic pig genome Rubin, Carl-Johan; Megens, Hendrik-Jan; Barrio, Alvaro Martinez ...
Proceedings of the National Academy of Sciences - PNAS,
11/2012, Letnik:
109, Številka:
48
Journal Article
Recenzirano
Odprti dostop
Domestication of wild boar (Sus scrofa) and subsequent selection have resulted in dramatic phenotypic changes in domestic pigs for a number of traits, including behavior, body composition, ...reproduction, and coat color. Here we have used whole-genome resequencing to reveal some of the loci that underlie phenotypic evolution in European domestic pigs. Selective sweep analyses revealed strong signatures of selection at three loci harboring quantitative trait loci that explain a considerable part of one of the most characteristic morphological changes in the domestic pig—the elongation of the back and an increased number of vertebrae. The three loci were associated with the NR6A1, PLAG1 , and LCORL genes. The latter two have repeatedly been associated with loci controlling stature in other domestic animals and in humans. Most European domestic pigs are homozygous for the same haplotype at these three loci. We found an excess of derived nonsynonymous substitutions in domestic pigs, most likely reflecting both positive selection and relaxed purifying selection after domestication. Our analysis of structural variation revealed four duplications at the KIT locus that were exclusively present in white or white-spotted pigs, carrying the Dominant white , Patch , or Belt alleles. This discovery illustrates how structural changes have contributed to rapid phenotypic evolution in domestic animals and how alleles in domestic animals may evolve by the accumulation of multiple causative mutations as a response to strong directional selection.
Obesity is a complex condition that increases the risk of life threatening diseases such as cardiovascular disease and diabetes. Studying the gene regulation of obesity is important for understanding ...the molecular mechanisms behind the obesity derived diseases and may lead to better intervention and treatment plans. MicroRNAs (miRNAs) are short non-coding RNAs regulating target mRNA by binding to their 3'UTR. They are involved in numerous biological processes and diseases, including obesity. In this study we use a mixed breed pig model designed for obesity studies to investigate differentially expressed miRNAs in subcutaneous adipose tissue by RNA sequencing (RNAseq). Both male and female pigs are included to explore gender differences. The RNAseq study shows that the most highly expressed miRNAs are in accordance with comparable studies in pigs and humans. A total of six miRNAs are differentially expressed in subcutaneous adipose tissue between the lean and obese group of pigs, and in addition gender specific significant differential expression is observed for a number of miRNAs. The differentially expressed miRNAs have been verified using qPCR. The results of these studies in general confirm the trends found by RNAseq. Mir-9 and mir-124a are significantly differentially expressed with large fold changes in subcutaneous adipose tissue between lean and obese pigs. Mir-9 is more highly expressed in the obese pigs with a fold change of 10 and a p-value < 0.001. Mir-124a is more highly expressed in the obese pigs with a fold change of 114 and a p-value < 0.001. In addition, mir-124a is significantly higher expressed in abdominal adipose tissue in male pigs with a fold change of 119 and a p-value < 0.05. Both miRNAs are also significantly higher expressed in the liver of obese male pigs where mir-124a has a fold change of 12 and mir-9 has a fold change of 1.6, both with p-values < 0.05.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with a complex microenvironment. Dichotomous tumour-promoting and -restrictive roles have been ascribed to the tumour microenvironment, ...however the effects of individual stromal subsets remain incompletely characterised. Here, we describe how heterocellular Oncostatin M (OSM) - Oncostatin M Receptor (OSMR) signalling reprograms fibroblasts, regulates tumour growth and metastasis. Macrophage-secreted OSM stimulates inflammatory gene expression in cancer-associated fibroblasts (CAFs), which in turn induce a pro-tumourigenic environment and engage tumour cell survival and migratory signalling pathways. Tumour cells implanted in Osm-deficient (Osm
) mice display an epithelial-dominated morphology, reduced tumour growth and do not metastasise. Moreover, the tumour microenvironment of Osm
animals exhibit increased abundance of α smooth muscle actin positive myofibroblasts and a shift in myeloid and T cell phenotypes, consistent with a more immunogenic environment. Taken together, these data demonstrate how OSM-OSMR signalling coordinates heterocellular interactions to drive a pro-tumourigenic environment in PDA.
Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel ...extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α
/α
signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.
Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not ...known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.
We investigate the "law of small numbers" using a data set on lotto gambling that allows us to measure players' reactions to draws. While most players pick the same set of numbers week after week, we ...find that those who do change, react on average as predicted by the law of small numbers as formalized in recent behavioral theory. In particular, players tend to bet less on numbers that have been drawn in the preceding week, as suggested by the "gambler's fallacy", and bet more on a number if it was frequently drawn in the recent past, consistent with the "hot-hand fallacy".
Abstract
Urban cloudburst management may include the intentional temporary storage of flood water in green recreational areas. In cities with combined sewers, this will expose the population visiting ...the area to sewage and increase the risk of diarrhoeal disease. We present a unique approach to estimate the risk of diarrhoeal disease after urban flooding. The exposure scenario was: rainwater mixed with sewage flows into a park; sewage with pathogens deposit on the grass; after discharge, a baby plays on the grass and is exposed to the pathogens in the deposited sewage by hand-to-mouth transfer. The work included modelling the transport of sewage into four parks intended to be flooded during future cloudbursts. A flood simulation experiment was conducted to estimate the deposition of pathogens from sewage to grass and transfer from grass to hand. Hand-to-mouth transfer, based on literature values, was used to estimate the ingested dose of pathogens. The probability of illness was estimated by QMRA. The estimated average probability of illness varied between 0.03 and 17%. If the probability of illness is considered unacceptable, the cloudburst plans should be changed, or interventions, e.g. informing the public about the risk or restricting access to the flooded area, should be implemented.
A better understanding of factors that influence functioning may improve the identification of patients with distal radius fractures (DRFs) who need hand therapy. The purpose of this scoping review ...was to provide a comprehensive overview of factors that have been evaluated for their influence on hand functioning following volar plate fixation of DRFs.
6 databases were searched from 2005 to 2021 for publications regarding surgical treatment for a DRF with a volar locking plate. Included studies evaluated demographic, perioperative, and postoperative factors within the 6 weeks post-surgery for their influence on functioning at least 3 months post-surgery. Functioning was assessed with patient-reported outcome measures. The factors were categorized into themes and mapped to the International Classification of Functioning, Disability and Health (ICF).
148 studies were included. 708 factors were categorized into 39 themes (e.g. pain) and mapped to the ICF components. The themes were primarily mapped to "body functions and structures" (n = 26) and rarely to "activities and participation" (n = 5). Fracture type (n = 40), age (n = 38), and sex (n = 22) were the most frequently evaluated factors.
This scoping review identified an extensive number of factors evaluated within 6 weeks after surgery for their influence on functioning at least 3 months after volar plate fixation of a DRF and the existing research has primarily evaluated factors related to "body functions and structures," with limited focus on factors related to "activities and participation."
Celotno besedilo
Dostopno za:
DOBA, FSPLJ, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Protein kinases enable cellular information processing. Although numerous human phosphorylation sites and their dynamics have been characterized, the evolutionary history and physiological importance ...of many signaling events remain unknown. Using target phosphoproteomes determined with a similar experimental and computational pipeline, we investigated the conservation of human phosphorylation events in distantly related model organisms (fly, worm, and yeast). With a sequence-alignment approach, we identified 479 phosphorylation events in 344 human proteins that appear to be positionally conserved over approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly related organisms. Thus, we devised a network-alignment approach to reconstruct conserved kinase-substrate networks, which identified 778 phosphorylation events in 698 human proteins. Both methods identified proteins tightly regulated by phosphorylation as well as signal integration hubs, and both types of phosphoproteins were enriched in proteins encoded by disease-associated genes. We analyzed the cellular functions and structural relationships for these conserved signaling events, noting the incomplete nature of current phosphoproteomes. Assessing phosphorylation conservation at both site and network levels proved useful for exploring both fast-evolving and ancient signaling events. We reveal that multiple complex diseases seem to converge within the conserved networks, suggesting that disease development might rely on common molecular networks.
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