scholarly journals Specific and Sensitive Diagnosis of BCOR-ITD in Various Cancers by Digital PCR

2021 ◽  
Vol 11 ◽  
Author(s):  
Doriane Barets ◽  
Romain Appay ◽  
Marie Heinisch ◽  
Maxime Battistella ◽  
Corinne Bouvier ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Dna Sequences ◽  
Endometrial Stromal Sarcoma ◽  
Digital Pcr ◽  
Genetic Alteration ◽  
Human Tumors ◽  
Central Nervous System Tumor ◽  
Cell Sarcoma ◽  
Normal Sequence ◽  
Wide Range

BCOR is an epigenetic regulator altered by various mechanisms including BCOR-internal tandem duplication (BCOR-ITD) in a wide range of cancers. Six different BCOR-ITD in the 3’-part of the coding sequence of exon 15 have been reported ranging from 89 to 114 bp in length. BCOR-ITD is a common genetic alteration found in clear cell sarcoma of the kidney and primitive myxoid mesenchymal tumor of infancy (PMMTI) and it characterizes a new type of central nervous system tumor: “CNS tumor with BCOR-ITD”. It can also be detected in undifferentiated round cell sarcoma (URCS) and in high-grade endometrial stromal sarcoma (HGESS). Therefore, it is of utmost importance to search for this genetic alteration in these cancers with the most frequent technique being RNA-sequencing. Here, we developed a new droplet PCR assay (dPCR) to detect the six sequences characterizing BCOR-ITD. To achieve this goal, we used a single colored probe to detect both the duplicated region and the normal sequence that acts as a reference. We first generated seven synthetic DNA sequences: ITD0 (the normal sequence) and ITD1 to ITD6 (the duplicated sequences described in the literature) and then we set up the optima dPCR conditions. We validated our assay on 19 samples from a representative panel of human tumors (9 HGNET-BCOR, 5 URCS, 3 HGESS, and 2 PMMTI) in which BCOR-ITD status was known using at least one other method including RNA sequencing, RT-PCR or DNA-methylation profiling for CNS tumors. Our results showed that our technique was 100% sensitive and specific. DPCR detected BCOR-ITD in 13/19 of the cases; in the remaining 6 cases additional RNA-sequencing revealed BCOR gene fusions. To conclude, in the era of histomolecular classification of human tumors, our modified dPCR assay is of particular interest to detect BCOR-ITD since it is a robust and less expensive test that can be applied to a broad spectrum of cancers that share this alteration.

scholarly journals A New Statistic for Detecting Genetic Differentiation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 2011-2014 ◽  
Author(s):  
Richard R Hudson
Keyword(s):  
Genetic Differentiation ◽  
Dna Sequences ◽  
Genetic Data ◽  
Island Model ◽  
Wide Range ◽  
Infinite Sites Model ◽  
Parameter Values

Abstract A new statistic for detecting genetic differentiation of subpopulations is described. The statistic can be calculated when genetic data are collected on individuals sampled from two or more localities. It is assumed that haplotypic data are obtained, either in the form of DNA sequences or data on many tightly linked markers. Using a symmetric island model, and assuming an infinite-sites model of mutation, it is found that the new statistic is as powerful or more powerful than previously proposed statistics for a wide range of parameter values.

Parasites in Antarctic krill guts inferred from DNA sequences

2019 ◽  
Vol 31 (1) ◽  
pp. 16-22 ◽  
Author(s):  
Alison C. Cleary ◽  
Maria C. Casas ◽  
Edward G. Durbin ◽  
Jaime Gómez-Gutiérrez
Keyword(s):  
Dna Sequences ◽  
High Frequency ◽  
Dna Analysis ◽  
Antarctic Krill ◽  
Euphausia Superba ◽  
Gut Contents ◽  
The West ◽  
West Antarctic ◽  
Wide Range

AbstractThe keystone role of Antarctic krill,Euphausia superbaDana, in Southern Ocean ecosystems, means it is essential to understand the factors controlling their abundance and secondary production. One such factor that remains poorly known is the role of parasites. A recent study of krill diet using DNA analysis of gut contents provided a snapshot of the parasites present within 170E. superbaguts in a small area along the West Antarctic Peninsula. These parasites includedMetschnikowiaspp. fungi,Haptoglossasp. peronosporomycetes,LankesteriaandParalecudinaspp. apicomplexa,Stegophorussp. nematodes, andPseudocolliniaspp. ciliates. Of these parasites,Metschnikowiaspp. fungi andPseudocolliniaspp. ciliates had previously been observed inE. superba, as had other genera of apicomplexans, though notLankesteriaandParalecudina.In contrast, nematodes had previously only been observed in eggs ofE. superba, and there are no literature reports of peronosporomycetes in euphausiids.Pseudocolliniaspp., parasitoids which obligately kill their host, were the most frequently observed infection, with a prevalence of 12%. The wide range of observed parasites and the relatively high frequency of infections suggest parasites may play a more important role than previously acknowledged inE. superbaecology and population dynamics.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes

1983 ◽  
Vol 3 (4) ◽  
pp. 596-604
Author(s):  
C A Whitlock ◽  
S F Ziegler ◽  
O N Witte
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Lymphoid Cells ◽  
Growth Potential ◽  
Malignant Growth ◽  
Wide Range ◽  
Feeder Layers ◽  
Growth Phenotype ◽  
Abelson Virus

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.

scholarly journals High viral load of Merkel cell polyomavirus DNA sequences in Langerhans cell sarcoma tissues

2014 ◽  
Vol 9 (1) ◽  
Author(s):  
Ichiro Murakami ◽  
Michiko Matsushita ◽  
Takeshi Iwasaki ◽  
Satoshi Kuwamoto ◽  
Masako Kato ◽  
...  
Keyword(s):  
Viral Load ◽  
Dna Sequences ◽  
High Viral Load ◽  
Merkel Cell Polyomavirus ◽  
Langerhans Cell ◽  
Merkel Cell ◽  
Cell Sarcoma ◽  
Langerhans Cell Sarcoma

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals Cross-border Investigations on the Prevalence and Transmission Dynamics of Cryptosporidium Species in Dairy Cattle Farms in Western Mainland Europe

2021 ◽  
Author(s):  
Pedro Pinto ◽  
Claudia A Ribeiro ◽  
Sumaiya Hoque ◽  
Ourida Hammouma ◽  
Hélène Leruste ◽  
...  
Keyword(s):  
The Netherlands ◽  
Human Health ◽  
Dna Sequences ◽  
Significant Risk ◽  
Dairy Farms ◽  
Economic Loss ◽  
Cryptosporidium Spp ◽  
Wide Range ◽  
Parasitic Protist ◽  
Cattle Farms

Cryptosporidium is comprised an apicomplexan parasitic protist, which infects a wide range of hosts, causing cryptosporidiosis. In cattle farms, the incidence of cryptosporidiosis results in high mortality in calves leading to considerable economic loss in the livestock industry. Infected animals may also act as a major reservoir of Cryptosporidium spp., in particular C. parvum, the most common cause of cryptosporidiosis in calves. This poses a significant risk to other farms via breeding centres, to trading of livestock and to human health. This study, funded by the Interreg-2-seas programme, is a part of a global project aimed at strategies to tackle cryptosporidiosis. To reach this target, it was essential to determine whether prevalence was dependent on the studied countries or if the issue was borderless. Indeed, C. parvum occurrence was assessed across dairy farms in certain regions of Belgium, France and the Netherlands. At the same time, the animal-to-animal transmission of the circulating C. parvum subtypes was studied. To accomplish this, 1084 faecal samples, corresponding to 57 dairy-farms from all three countries, were analysed. Well-established protocols amplifying the 18S rDNA and gp60 genes fragments, followed by DNA sequencing, were used for the detection and subtyping C. parvum; the DNA sequences obtained were further characterised using a combination of bioinformatics and phylogenetics methods. Our results show 25.7%, 24.9% and 20.8% prevalence of Cryptosporidium spp. in Belgium, France and the Netherlands respectively. Overall, 93% of the farms were Cryptosporidium positive. The gp60 subtyping demonstrated a significant number of the C. parvum positives belonged to the IIa allelic family, which has been also detected in humans. Consequently, this study highlights how widespread is C. parvum in dairy farms and endorses cattle as a major carrier of zoonotic C. parvum subtypes, which subsequently pose a significant threat to human health.

scholarly journals TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Acta Naturae ◽  
2014 ◽  
Vol 6 (3) ◽  
pp. 19-40 ◽  
Author(s):  
A. A. Nemudryi ◽  
K. R. Valetdinova ◽  
S. P. Medvedev ◽  
S. M. Zakian
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genome Engineering ◽  
Disease Modeling ◽  
Regulation Of Gene Expression ◽  
Hereditary Disease ◽  
Transcription Activator ◽  
Wide Range ◽  
High Standards ◽  
Phenotype Formation

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isnt enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

scholarly journals Diverse tumour susceptibility in Collaborative Cross mice: identification of a new mouse model for human gastric tumourigenesis

Gut ◽  
2019 ◽  
Vol 68 (11) ◽  
pp. 1942-1952 ◽  
Author(s):  
Pin Wang ◽  
Yunshan Wang ◽  
Sasha A Langley ◽  
Yan-Xia Zhou ◽  
Kuang-Yu Jen ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Mouse Model ◽  
Rna Sequencing ◽  
Genetic Association ◽  
Association Analysis ◽  
Collaborative Cross ◽  
Sequencing Analysis ◽  
Genetic Association Analysis ◽  
Mouse Population ◽  
Wide Range

ObjectiveThe Collaborative Cross (CC) is a mouse population model with diverse and reproducible genetic backgrounds used to identify novel disease models and genes that contribute to human disease. Since spontaneous tumour susceptibility in CC mice remains unexplored, we assessed tumour incidence and spectrum.DesignWe monitored 293 mice from 18 CC strains for tumour development. Genetic association analysis and RNA sequencing were used to identify susceptibility loci and candidate genes. We analysed genomes of patients with gastric cancer to evaluate the relevance of genes identified in the CC mouse model and measured the expression levels of ISG15 by immunohistochemical staining using a gastric adenocarcinoma tissue microarray. Association of gene expression with overall survival (OS) was assessed by Kaplan-Meier analysis.ResultsCC mice displayed a wide range in the incidence and types of spontaneous tumours. More than 40% of CC036 mice developed gastric tumours within 1 year. Genetic association analysis identified Nfκb1 as a candidate susceptibility gene, while RNA sequencing analysis of non-tumour gastric tissues from CC036 mice showed significantly higher expression of inflammatory response genes. In human gastric cancers, the majority of human orthologues of the 166 mouse genes were preferentially altered by amplification or deletion and were significantly associated with OS. Higher expression of the CC036 inflammatory response gene signature is associated with poor OS. Finally, ISG15 protein is elevated in gastric adenocarcinomas and correlated with shortened patient OS.ConclusionsCC strains exhibit tremendous variation in tumour susceptibility, and we present CC036 as a spontaneous laboratory mouse model for studying human gastric tumourigenesis.

scholarly journals Molecular Diversity of Nematode Parasites in Afrotropical Reed Frogs (Hyperolius spp.)

Diversity ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 265
Author(s):  
Ulrich Sinsch ◽  
J. Maximilian Dehling ◽  
Patrick Scheid ◽  
Carsten Balczun
Keyword(s):  
Dna Sequences ◽  
Nematode Species ◽  
Sub Saharan Africa ◽  
Parasitic Nematodes ◽  
Marker Genes ◽  
Nematode Parasites ◽  
Diversity Assessment ◽  
Wide Range ◽  
Nematode Diversity ◽  
Sub Saharan

The diversity of nematodes infecting amphibians is understudied in tropical Africa and unknown in Rwanda. Diversity assessment is hampered by the fact that species descriptions refer mostly to morphological features that are unlinked to DNA sequences of marker genes available in public databases. In this paper, we explore the abundance and diversity of parasitic nematodes in reed frogs Hyperolius kivuensis (n = 115), H. parallelus (n = 45) and H. viridiflavus (n = 100) collected in Rwanda. Five nematode species were identified morphologically as Orneoascaris chrysanthemoides, O. schoutedeni, Gendria leberrei, Aplectana chamaeleonis and Rhabdias collaris. Corresponding DNA sequences of 18S and COI genes were determined and subsequently deposited in GenBank. Aplectana chamaeleonis showed the highest prevalence (8.7%), but O. chrysanthemoides the highest mean intensity of infection (6.0) and largest number (24) of individuals in H. kivuensis. To the best of our knowledge, all amphibian hosts are new records for these nematode species, which are known to infect a wide range of amphibian and reptile species. Our findings suggest that nematode diversity is probably lower than previously assumed due to low host specificity. As morphological species identification is often challenging, our data facilitate molecular identification of adult and specifically larval nematodes found in amphibians of Sub-Saharan Africa.

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