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Frontiers in Cellular and Infection... 2022Humans are exposed to infection as pet cats gradually become family members and represent an increasing public health risk worldwide. Toxoplasmosis diagnosis...
Humans are exposed to infection as pet cats gradually become family members and represent an increasing public health risk worldwide. Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this study, real-time fluorescence quantitative loop-mediated isothermal amplification (qLAMP) and visual LAMP detection technologies were established to conduct tests of based on the membrane DNA extraction method, and the optimal detection mix was determined by adding the protective reagent trehalose and screening the concentrations of Mg and dNTPs. Paraffin and lyophilization were used to reduce and even remove aerosol pollution, constructing a detailed anti-contamination protocol. Based on the positive standard plasmid DNA, the LODs of qLAMP and visual LAMP were 92 copies/μL and 92 copies/μL, and the standard curve of qLAMP was Y=2.9503X+20.8992 with R 0.99. The applicability of the qLAMP and visual LAMP assays in disease diagnosis was assessed by evaluating 200 clinical cat faeces samples. The assays showed good diagnostic consistency, with kappa values of 1.0 and 0.99 compared with Man qPCR, respectively. Compared with Man qPCR, the diagnostic specificity/sensitivity of qLAMP and visual LAMP were 100%/100% and 100%/80%, respectively. The qLAMP and visual LAMP assays reported here are rapid and simple tests without extensive sample preparation and have a short turnaround time within 60 min, making them suitable for point-of-care testing.
Topics: Animals; Cats; DNA, Protozoan; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Paraffin; Sensitivity and Specificity; Toxoplasma; Toxoplasmosis; Trehalose
PubMed: 36225232
DOI: 10.3389/fcimb.2022.1024690 -
Respiratory Research May 2023The role of epigenetic modifications in tumorigenesis has been widely reported. However, the role and mechanism of H3K4me3 modification in lung adenocarcinoma (LUAD) are...
BACKGROUD
The role of epigenetic modifications in tumorigenesis has been widely reported. However, the role and mechanism of H3K4me3 modification in lung adenocarcinoma (LUAD) are rarely reported systematically. We, therefore, sought to analyze the characteristics of LUAD associated with H3K4me3 modification, build an H3K4me3-lncRNAs score model to predict the prognosis of patients with LUAD and clarify the potential value of H3K4me3 in immunotherapy of LUAD.
METHODS
We evaluated H3K4me3-lncRNA patterns and H3K4me3-lncRNA scores of 477 LUAD samples based on 53 lncRNAs closely correlated to H3K4me3 regulators and comprehensive analyzed the role of these patterns in tumorigenesis and tumor immunity. Using Gene set variation analysis (GSVA), we systematically evaluated the H3K4me3 level of every sample and deeply analyzed the effect of H3K4me3 on the prognosis of LUAD. In addition, we included two independent immunotherapy cohorts to study the impact of high H3K4me3 score on the prognosis of patients. We also used an independent cohort with 52 matched paraffin specimens of LUAD to verify the impact of high H3K3me3 expression on the prognosis of patients.
RESULTS
We identified three H3K4me3-lncRNA patterns that exhibited specific immune characteristics. Characterized by immunosuppressive and increased TGFβ-mediated epithelial-mesenchymal transition (EMT), patients with high H3K4me3-lncRNA score had a poor overall survival and decreased H3K4me3 score. H3K4me3 score was significantly positively correlated with CD4T-cell and CD8T-cell activation, programmed cell death and immune checkpoints (ICs) expression, and was negatively correlated with MYC pathway, TP53 pathway, and cell proliferation. Patients with high H3K4me3 score showed elevated expression of ICs, potentiated CD4 T-cell and CD8 T-cell activation, increased programmed cell death, and suppressed cell proliferation and TGFβ-mediated EMT. Patients with high H3K4me3 score and high expression of CTLA4, ICOS, TIGIT, PDCD1LG2, IDO1, CD274, PDCD1, LAG3, or HAVCR2 had the best survival advantage. Two independent immunotherapy cohorts verified that patients with high H3K4me3 score showed an increased inflamed tumor microenvironment (TME) phenotype and enhanced anti-PD-1/L1 immunotherapy response. Immunohistochemistry (IHC) data from 52 matched paraffin specimens of LUAD confirmed that the protein level of H3K4me3 in tumor was significantly lower than that of paracancerous tissues and H3K4me3 brought significant survival benefits to patients with LUAD.
CONCLUSIONS
We build an H3K4me3-lncRNAs score model to predict the prognosis of patients with LUAD. More importantly, this study revealed characteristics of H3K4me3 modification in LUAD and clarified the important potential role of H3K4me3 on tumor immunotherapy and patients' survival.
Topics: Humans; RNA, Long Noncoding; Paraffin; Carcinogenesis; Adenocarcinoma; Lung; Lung Neoplasms; Tumor Microenvironment
PubMed: 37131252
DOI: 10.1186/s12931-023-02418-1 -
Current Protocols in Cytometry Apr 2010This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in... (Review)
Review
This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and "mass action law" characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units.
Topics: Antigens; Cell Membrane; Cell Nucleus; Chromatin; DNA; Flow Cytometry; Fluorescent Dyes; Humans; Immunophenotyping; Neoplasms; Paraffin; Ploidies; Reproducibility of Results
PubMed: 20373495
DOI: 10.1002/0471142956.cy0702s52 -
International Journal of Molecular... Nov 2023The aim of this study was to identify metabolomic signatures associated with the gliomagenesis pathway (-mutant or -wt) and tumor grade of diffuse gliomas (DGs)...
The aim of this study was to identify metabolomic signatures associated with the gliomagenesis pathway (-mutant or -wt) and tumor grade of diffuse gliomas (DGs) according to the 2021 WHO classification on frozen samples and to evaluate the diagnostic performances of these signatures in tumor samples that are formalin-fixed and paraffin-embedded (FFPE). An untargeted metabolomic study was performed using liquid chromatography/mass spectrometry on a cohort of 213 DG samples. Logistic regression with LASSO penalization was used on the frozen samples to build classification models in order to identify -mutant vs. -wildtype DG and high-grade vs low-grade DG samples. 2-Hydroxyglutarate (2HG) was a metabolite of interest to predict mutational status and aminoadipic acid (AAA) and guanidinoacetic acid (GAA) were significantly associated with grade. The diagnostic performances of the models were 82.6% AUC, 70.6% sensitivity and 80.4% specificity for 2HG to predict status and 84.7% AUC, 78.1% sensitivity and 73.4% specificity for AAA and GAA to predict grade from FFPE samples. Thus, this study showed that AAA and GAA are two novel metabolites of interest in DG and that metabolomic data can be useful in the classification of DG, both in frozen and FFPE samples.
Topics: Humans; Adult; Brain Neoplasms; Formaldehyde; Paraffin; Paraffin Embedding; Isocitrate Dehydrogenase; Glioma; Mutation
PubMed: 38069019
DOI: 10.3390/ijms242316697 -
STAR Protocols Sep 2023Organoids are unique tools to mimic how tumors evolve in a 3D environment. Here, we present a protocol to embed spheroids invading a 3D matrix into a paraffin mold. We...
Organoids are unique tools to mimic how tumors evolve in a 3D environment. Here, we present a protocol to embed spheroids invading a 3D matrix into a paraffin mold. We describe steps for preparing spheroids, collagen and agarose inclusion, and paraffinization. We then detail procedures for sectioning, staining, and visualization. This protocol allows histological identification of markers expressed in cells escaping the tumor. For complete details on the use and execution of this protocol, please refer to Guyon et al. (2022)..
Topics: Humans; Glioblastoma; Organoids; Collagen; Histological Techniques; Paraffin
PubMed: 37597188
DOI: 10.1016/j.xpro.2023.102521 -
Journal of Visualized Experiments : JoVE Jun 2018Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell...
Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 µm sections can be cut from the same islet block. Using this method, histological and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.
Topics: Animals; Histological Techniques; Humans; Islets of Langerhans; Mice; Paraffin; Rats
PubMed: 30010652
DOI: 10.3791/57931 -
Environmental Monitoring and Assessment Apr 2021Chlorinated paraffins (CPs) are anthropogenic pollutants of growing environmental concern. These highly complex mixtures of thousands of homologs and congeners are...
Chlorinated paraffins (CPs) are anthropogenic pollutants of growing environmental concern. These highly complex mixtures of thousands of homologs and congeners are usually applied as additives in lubricants or as flame retardants and plasticizers in polymers and paints. Recent studies indicated the presence of high amounts of CPs in the kitchen environment whose sources could not be unequivocally identified. One option was the use of CPs as or in lubricants of hinges. To test this hypothesis, we performed wipe tests on lubricants on 29 hinges of different types of kitchen appliances (refrigerators, baking ovens, dishwashers, freezers, microwave oven, pasta machine, food processor, steam cooker) and analyzed them for short-chain CPs (SCCPs) and medium-chain CPs (MCCPs). CPs were detected in 21 samples (72%). Per wipe, SCCP concentrations ranged between 0.02 and 10 µg (median 0.23 µg), while MCCPs ranged from 0.09 to 750 µg (median 1.0 µg). Highest MCCP amounts (380 and 750 µg per wipe, respectively) were determined in new and unused appliances. A medium correlation between SCCP content and appliance age was observed, but no additional statistic correlation between SCCP/MCCP amount and appliance type or manufacturer could be observed. CPs released from hinges by volatilization, abrasion, and cleaning processes could enter the environment and come in contact with persons living in the corresponding households.
Topics: China; Environmental Monitoring; Environmental Pollutants; Flame Retardants; Hydrocarbons, Chlorinated; Paraffin
PubMed: 33829339
DOI: 10.1007/s10661-021-09023-z -
Biomolecules Jun 2023Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using...
Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-µm-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific αSyn seeding behaviour in FFPE human brain. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.
Topics: Humans; alpha-Synuclein; Lewy Body Disease; Multiple System Atrophy; Synucleinopathies; Paraffin; Paraffin Embedding; Formaldehyde; Brain
PubMed: 37371515
DOI: 10.3390/biom13060936 -
Lab on a Chip Feb 2009A disposable, self-contained polymerase chain reaction (PCR) chip with on-board stored, just-on-time releasable, paraffin-passivated, dry reagents is described. During...
A disposable, self-contained polymerase chain reaction (PCR) chip with on-board stored, just-on-time releasable, paraffin-passivated, dry reagents is described. During both storage and sample preparation, the paraffin immobilizes and protects the stored reagents. Fluid flow through the reactor leaves the reagents undisturbed. Prior to the amplification step, the chamber is filled with target analyte suspended in water. Upon heating the PCR chamber to the DNA's denaturation temperature, the paraffin melts and moves out of the way, and the reagents are released and hydrated. To better understand the reagent release process, a scaled up model of the reactor was constructed and the paraffin migration was visualized. Experiments were carried out with a 30 microl reactor demonstrating detectable amplification (with agarose gel electrophoresis) of 10 fg ( approximately 200 copies) of lambda DNA template. The in-reactor storage and on-time release of the PCR reagents reduce the number of needed operations and significantly simplifies the flow control that would, otherwise, be needed in lab-on-chip devices.
Topics: Desiccation; Equipment Design; Heating; Indicators and Reagents; Lab-On-A-Chip Devices; Microchip Analytical Procedures; Paraffin; Polymerase Chain Reaction
PubMed: 19190797
DOI: 10.1039/b807915c -
Environmental Pollution (Barking, Essex... Jan 2022There are large knowledge gaps concerning environmental levels and fate of many organic pollutants, particularly for chemicals of emerging concern in tropical regions of...
There are large knowledge gaps concerning environmental levels and fate of many organic pollutants, particularly for chemicals of emerging concern in tropical regions of the Global South. In this study, we investigated the levels of chlorinated paraffins (CPs) and dechloranes in air and soil in rural, suburban, and urban regions in and around Dar es Salaam, Tanzania. Samples were also collected near the city's main municipal waste dumpsite and an electronic waste (e-waste) handling facility. In passive air samples, short chain CPs (SCCPs) dominated, with an average estimated concentration of 22 ng/m, while medium chain CPs (MCCPs) had an average estimated concentration of 9 ng/m. The average estimated air concentration of ∑dechloranes (Dechlorane Plus (DP) + Dechlorane 602 + Dechlorane 603) was three to four orders of magnitudes lower, 2 pg/m. In soil samples, MCCPs dominated with an average concentration of 640 ng/g dw, followed by SCCPs with an average concentration of 330 ng/g dw, and ∑dechloranes with an average concentration of 0.9 ng/g dw. In both air and soil, DP was the dominating dechlorane compound. Urban pulses were observed for CPs and dechloranes in air and soil. CPs were in addition found in elevated levels at the municipal waste dumpsite and the e-waste handling facility, while DPs were found in elevated levels at the e-waste handling facility. This suggests that waste handling sites represent important emission sources for these pollutants. Investigations into seasonal trends and environmental fate of CPs and dechloranes showed that monsoonal rain patterns play a major role in governing air concentrations and mobility, particularly for the less volatile MCCPs and dechloranes. This study is the first to report levels of CPs in air from sub-Saharan Africa, and DP, Dechlorane 602, and Dechlorane 603 in soil from sub-Saharan Africa.
Topics: China; Environmental Monitoring; Hydrocarbons, Chlorinated; Paraffin; Soil; Tanzania
PubMed: 34626702
DOI: 10.1016/j.envpol.2021.118298