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Molecular characterization of human isolates of Strongyloides stercoralis and Rhabditis spp. based on mitochondrial cytochrome c oxidase subunit 1 (cox1).

Due to the similarity of Strongyloides stercoralis with free-living nematodes of Rhabditis species they might be miss-diagnosed with each other in microscopical examination of stool samples. The aim of this study was molecular characterization and differentiation of human derived isolates of S. stercoralis and Rhabditis species based on the mitochondrial gene of cytochrome c oxidase subunit 1 (cox1) amplification.

1209 related Products with: Molecular characterization of human isolates of Strongyloides stercoralis and Rhabditis spp. based on mitochondrial cytochrome c oxidase subunit 1 (cox1).

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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.

1821 related Products with: Molecular identification and histological aspects of Renicola sloanei (Digenea: Renicolidae) in Puffinus puffinus (Procellariiformes): a first record.

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PCR monitoring of parasitemia during drug treatment for canine Chagas disease.

To date, there is no clear standard to monitor drug treatment for canine Chagas disease. We used 2 real-time PCR (rtPCR) assays targeting kinetoplast DNA (kDNA) and nuclear satellite DNA (nDNA) to detect in canine whole blood. Samples were collected randomly from 131 untreated dogs with unknown infection status in Texas. The kDNA-based rtPCR was slightly more sensitive (diagnostic sensitivity of kDNA = 49% vs. nDNA = 44%; = 0.5732) but slightly less specific (diagnostic specificity of kDNA = 96% vs. nDNA = 97%; > 0.9999) than the nDNA-based rtPCR. However, the differences in sensitivity and specificity between the nDNA- and kDNA-based rtPCR assays were not statistically significant. Using the nDNA- and kDNA-based qualitative rtPCR assays to monitor parasitemia from 137 itraconazole- and amiodarone-treated cases with nDNA- and kDNA-based PCR-positive baselines showed that the PCR positive rate decreased to 0% in 30 d. Using kDNA-based quantitative rtPCR to monitor normalized DNA copies in 4 representative dogs demonstrated that drug treatment could reduce parasite loads within 7-30 d. The kDNA-based qualitative rtPCR may be used for routine parasitemia screening of drug-treated Chagas-positive dogs, whereas nDNA-based qualitative rtPCR may be used for confirmation.

2863 related Products with: PCR monitoring of parasitemia during drug treatment for canine Chagas disease.

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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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Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening.

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

2478 related Products with: Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening.

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