Search results for: Mitochondrial DNA Isolation Kit50 assays
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Molecular identification and histological aspects of Renicola sloanei (Digenea: Renicolidae) in Puffinus puffinus (Procellariiformes): a first record.Renicolids are parasites that inhabit the renal tubules and ureters of molluscivorous and piscivorous birds. Puffinus puffinus is a migratory seabird that was identified as the definitive host of Renicola spp. Studies focusing on the renicolid species and the resulting renal lesions are valuable for their association with causes of stranding in seabirds. The aim of this study was to identify the renicolid trematodes and evaluate the histological findings in two P. puffinus stranded on the coast of Paraná state, Brazil. The parasites were evaluated by histologic, ultrastructural and molecular assays, while tissue changes were analyzed by histologic methods. The morphological and morphometrical characteristics of the parasites, along with polymerase chain reaction and sequencing assays (ribosomal and mitochondrial regions), identified the species as Renicola sloanei. The results also suggest that this helminth can be the adult form of Cercaria pythionike. The dilation of collecting ducts was the main histological finding in the kidneys. In conclusion, R. sloanei was identified, and for the first time, P. puffinus was described as a host of this digenean inducing mild renal changes.
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A qPCR Assay for the Detection of Including an mRNA Protocol Designed to Establish Propagule Viability in Environmental Samples.causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against and two closely related clade 7 species, and . From these tests, five primer pairs were selected based on specificity and, with a species-specific probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of DNA and showed no cross-reaction with other species, except for , a very closely related species to , which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with . Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of in recalcitrant environmental samples.
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A nanoscale, multi-parametric flow cytometry-based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics.Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi-parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell.
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Triptolide induces toxicity in inner ear stem cells via promoting DNA damage.Emerging evidence and clinical case reports have observed a risk of cytotoxic effects of triptolide in patients. We aimed to investigate the triptolide-induced toxicity in mouse inner ear stem cells. The utricular sensory epithelium from adult BALB/C6 mice was used for the isolation of inner ear stem cells. Sphere formation assay was applied to examine the stemness of the cells. Cell count kit-8 and Bromodeoxyuridine assays were employed to detect the cell proliferation ability. Cell apoptosis was measured with Annexin V-FITC & propidium iodide Apoptosis kit. The relative expression levels of gamma H2A histone family member X (γH2AX), tumor suppressor p53-binding protein 1 (53BP1) and optic atrophy 1 (OPA-1) were measured by Western Blot. Mitochondrial function was analyzed by the MitoGreen green-fluorescent mitochondrial dye kit. Triptolide significantly inhibited the cell viability and proliferation and suppressed the capability of sphere formation. Furthermore, triptolide induced apoptosis as indicated by increased expression of DNA damage repair markers γH2AX and 53BP1. Moreover, triptolide influenced the function of mitochondria by inducing the cleavage of OPA-1. Our work clarifies the toxicity of triptolide in mouse inner ear stem cells, which provides clues of the toxicology mechanism for future studies and basis for clinical use.
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Molecular analysis reveals expansion of Fasciola hepatica distribution from Afghanistan to China.Recently, phosphoenol pyruvate carboxykinase (pepck) and DNA polymerase delta (pold) were established as reliable nuclear markers for species identification of Fasciola spp. in multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism-based assays, respectively. Currently, little is known about Fasciola species distribution in Central Asia. Therefore, the objective of the present study was to perform precise molecular species identification of liver flukes from Afghanistan and to reveal their dispersal route(s) via phylogenetic analysis based on mitochondrial nad1 haplotypes. Ninety-two Fasciolaflukes collected from sheep in Kabul, Afghanistan, were identified as F. hepatica based on pepck and pold screening. Although the pepck fragment pattern obtained via multiplex PCR analysis could not distinguish the species of the seven Fasciola flukes, the pepck nucleotide sequence data confirmed that they were F. hepatica.The 20 nad1 haplotypes detected among the Afghani liver flukes were closely related to those from China and Egypt, with the Fvalue (-0.003, P = .41) between the F. hepatica populations from Afghanistan and China confirming a very close relationship. Nucleotide diversity was greater in the population from Afghanistan compared with that from China, indicating that the Afghani population was older, and that the dispersal direction of F. hepatica was from Afghanistan to China. The results of the present study contribute to our understanding of the dispersal of F. hepatica from its predicted origin, the Fertile Crescent.
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A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR.Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable faecal host DNA studies in animal disease models as well.
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Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load.The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 °C ± 1 °C for satellite-DNA and 78.1 °C ± 1 °C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 × 10 parasite or 240 target copies, and for kDNA, 2 × 10 parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (±0.002) and 0.991 (±0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines.
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Detection, genotyping, and phylogenetic analysis of Leishmania isolates collected from infected Jordanian residents and Syrian refugees who suffered from cutaneous leishmaniasis.Leishmania is a parasitic protozoan which is transmitted to humans through the bite of an infected female Phlebotomus and Lutzomyia sand flies. Cutaneous leishmaniasis (CL), caused by Leishmania major and L. tropica, is an endemic disease in many areas of Jordan and considered as a major public health problem. The political instability in the Syrian Arab Republic has resulted in the immigration of large number of refugees into Jordan where most of them resided in camps near the Syrian borders. Therefore, the main objective of the present study was to inspect Leishmania species/genotypes which are responsible for CL infections among Syrian refugees and compare them with the recovered species/genotypes isolated from Jordanian patients. Three molecular-based assays (ITS1-PCR-RFLP, Nested ITS1-5.8S rDNA PCR, and Kinetoplast DNA PCR) followed by sequencing and phylogenetic analysis were undertaken and compared for their efficiency to confirm CL diagnosis and genotype the infecting Leishmania species. Thereafter, the evolutionary relationships among various Leishmania isolates from Syrian and Jordanian CL patients were elucidated. Results from the present study indicated that 20 and 9 out of the inspected 66 patients (39 Jordanian and 27 Syrian) were infected with L. major and L. tropica respectively. ITS1-PCR RFLP typing proved to be more sensitive in the detection of Leishmania species (positive in 44% of the isolates) compared to both ITS1-5.8S rDNA gene and Kinetoplast DNA PCR which were successful in identifying Leishmania species only in 23% and 33% of the isolates respectively. Sequencing and phylogenetic analysis of ITS1 and ITS1-5.8S rDNA genes revealed high levels of heterogeneity among the sequenced isolates. One sample typed as L. tropica from Jordanian patient showed high similarity with L. tropica sample isolated from a Syrian patient in a Lebanon refugee camp; therefore, the need for comprehensive studies to confirm if any new L. tropica strains might be introduced to Jordan by Syrian refugees is urgently indicated. These observations highlighted the need for further studies to clarify the risk status of species and strains which might be introduced from Syria to Jordan.
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The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis.Mitochondrial topoisomerase IB (TOP1MT) is a nuclear-encoded topoisomerase, exclusively localized to mitochondria, which resolves topological stress generated during mtDNA replication and transcription. Here, we report that TOP1MT is overexpressed in cancer tissues and demonstrate that TOP1MT deficiency attenuates tumor growth in human and mouse models of colon and liver cancer. Due to their mitochondrial dysfunction, TOP1MT-KO cells become addicted to glycolysis, which limits synthetic building blocks and energy supply required for the proliferation of cancer cells in a nutrient-deprived tumor microenvironment. Mechanistically, we show that TOP1MT associates with mitoribosomal subunits, ensuring optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The TOP1MT genomic signature profile, based on Top1mt-KO liver cancers, is correlated with enhanced survival of hepatocellular carcinoma patients. Our results highlight the importance of TOP1MT for tumor development, providing a potential rationale to develop TOP1MT-targeted drugs as anticancer therapies.
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