Telomere Length Measurement in Human Tissue Sections by Quantitative Fluorescence In Situ Hybridization (Q-FISH).

Telomeres in most somatic cells shorten with each cell division, and critically short telomeres lead to cellular dysfunction, cell cycle arrest, and senescence. Thus, telomere shortening is an important hallmark of human cellular senescence. Quantitative fluorescence in situ hybridization (Q-FISH) using formalin-fixed paraffin-embedded (FFPE) tissue sections allows the estimation of telomere lengths in individual cells in histological sections. In our Q-FISH method, fluorescently labelled peptide nucleic acid (PNA) probes are hybridized to telomeric and centromeric sequences in FFPE human tissue sections, and relative telomere lengths (telomere signal intensities relative to centromere signal intensities) are measured. This chapter describes our Q-FISH protocols for assessing relative telomere lengths in FFPE human tissue sections.

How does adulteration of wax foundation affect phenoloxidase and lysozyme activities as selected parameters of immunity in Apis mellifera?

Introduction: The adulteration of wax foundation is, for many reasons, a growing problem of modern beekeeping not only in Europe but also around the world. Wax foundation contaminated with stearin addition leads to a brood die-off, while paraffin addition negatively affects the strength of combs. It is tenable that such adulterated wax foundation reduces bees' immunity. The aim of the study was to determine the activities of two bee immune enzymes, lysozyme and phenoloxidase, in the haemolymph of worker bees which had emerged from combs with wax foundations contaminated with stearin or paraffin. Material and Methods: Combs built with stearin- or paraffin-adulterated wax (both adulterants at concentrations of 10%, 30% or 50%) or pure wax (0% adulterated) foundations were placed in the colonies, one for each adulterant and percentage. The workers were marked upon emergence from these combs and those bees were introduced into one strong colony per adulterant and percentage. Phenoloxidase and lysozyme activities were determined in the haemolymph of 1-, 7- and 14-day-old workers. Results: The higher the concentrations of stearin and paraffin in the wax foundation, the lower the phenoloxidase activities were. These activities increased with the bee age. In contrast, the trends in lysozymes were opposite. Paraffin seems to be less toxic than stearin. Conclusion: Adulteration of wax foundation with even a small amount of stearin or paraffin has negative effects on the functioning of the bee.

Application of pattern deconvolution strategies for the estimation of bromochloro alkane concentrations in indoor dust samples.

Bromochloro alkanes (BCAs) are a class of flame retardants similar in structure to polychlorinated alkanes (PCAs), which are the major component of short-chain chlorinated paraffins (SCCPs) listed as Persistent Organic Pollutants under the Stockholm Convention. BCAs have recently been detected for the first time in environmental samples. Due to the complete lack of commercially available analytical standards, no method for quantifying BCAs has been reported to date. In this study, 16 custom-synthesised standards with mixed bromine and chlorine halogenation and carbon chain lengths ranging from C10 to C17 were characterized by liquid chromatography and Orbitrap high-resolution mass spectrometry and used to assess the applicability of pattern deconvolution quantification strategies for BCAs in indoor dust. Br1-9 and Cl1-8 BCAs were detected as [M + Cl]- adduct ions among the C10 to C17 standards, as well as numerous PCA homologues. After applying correction factors to account for the presence of PCAs in the standards, triplicate fortification experiments using varied halogenation composition and concentration determined an average measurement accuracy of 81% over the carbon chain lengths studied and coefficient of variance ≤20% between replicates. Overall, approximately 89% of the ΣBCA concentrations quantified in the fortification trials met the European Union Reference Laboratory's accuracy acceptability criteria recommended for PCAs, between 50 and 150%. Application of the BCA pattern deconvolution quantification procedure to seven representative indoor dust samples from the United States of America revealed a low correlation between the homologue distribution in the samples and the prototype standards (R2 ≤ 0.40), which precluded reliable quantification. This study indicates that pattern deconvolution is an appropriate strategy for quantifying BCAs in environmental samples, but that a large set of appropriate mixture standards will be required before more reliable estimates of BCA concentrations can be achieved in indoor dust.

Exchange of CO2 with CO as Reactant Switches Selectivity in Photoreduction on Co-ZrO2 from C1-3 Paraffin to Small Olefins.

Photocatalytic reduction of CO2 into C2,3 hydrocarbons completes a C-neutral cycle. The reaction pathways of photocatalytic generation of C2,3 paraffin and C2H4 from CO2 are mostly unclear. Herein, a Co0-ZrO2 photocatalyst converted CO2 into C1-3 paraffin, while selectively converting CO into C2H4 and C3H6 (6.0 ± 0.6 µmol h-1 gcat-1, 70 mol%) only under UV-visible light. The photocatalytic cycle was conducted under 13CO and H2, with subsequent evacuation and flushing with CO. This iterative process led to an increase in the population of C2H4 and C3H6 increased up to 61-87 mol%, attributed to the accumulation of CH2 species at the interface between Co0 nanoparticles and the ZrO2 surface. CO2 adsorbed onto the O vacancies of the ZrO2 surface, with resulting COH species undergoing hydrogenation on the Co0 surface to yield C1-3 paraffin using either H2 or H2O (g, l) as the reductant. In contrast, CO adsorbed on the Co0 surface, converted to HCOH species, and then split into CH and OH species at the Co and O vacancy sites on ZrO2, respectively. This comprehensive study elucidates intricate photocatalytic pathways governing the transformation of CO2 into paraffin and CO to olefins.

Utilizing decellularized bio-membranes to optimize histopathological embedding of small tissues.

In recent years, minimally invasive biopsy techniques have been widely used to generate small tissue samples that require processing in clinical pathology. However, small paraffin-embedded tissues are prone to loss due to their small size. To prevent the loss of small tissues, researchers have employed nonbiological embedding materials for preembedding, but this approach can lead to cumbersome experimental procedures and increase the chances of tissue loss. This study aimed to develop a convenient decellularized embedding material derived from biological membrane tissues to effectively protect small tissues from loss during paraffin embedding. This study decellularized three types of fresh animal-derived membrane tissues and selected the small intestine as the most suitable decellularized raw material through attempts at softening, comparing physical properties, and using tissue as the starting material. Subsequently, small tissues from various tissue sources were embedded, followed by H&E staining, Masson staining, immunofluorescence staining, and immunohistochemical staining. The decellularized material derived from biomembrane tissues (DMBT) developed in this study can reduce the loss of small tissues without the need for preembedding, thereby shortening the embedding process. This provides a new pathological embedding tool for future laboratory and clinical research and work.•The fat layer of the pig's small intestine is scraped off, and chemical reagents are used to defat and decellularize it.•Chemical reagents are used to soften and make the pig's small intestine transparent, and the decellularized pig's small intestine is dried.•DMBT is used for embedding and staining the biological tissue.

Retrospective clinical and microbiologic analysis of metagenomic next-generation sequencing in the microbiological diagnosis of cutaneous infectious granulomas.

BACKGROUND: Cutaneous infectious granulomas (CIG) are localized and chronic skin infection caused by a variety of pathogens such as protozoans, bacteria, worms, viruses and fungi. The diagnosis of CIG is difficult because microbiological examination shows low sensitivity and the histomorphological findings of CIG caused by different pathogens are commonly difficult to be distinguished. OBJECTIVE: The objective of this study is to explore the application of mNGS in tissue sample testing for CIG cases, and to compare mNGS with traditional microbiological methods by evaluating sensitivity and specificity. METHODS: We conducted a retrospective study at the Department of Dermatology of Sun Yat-sen Memorial Hospital, Sun Yat-sen University from January 1st, 2020, to May 31st, 2024. Specimens from CIG patients with a clinical presentation of cutaneous infection that was supported by histological examination were retrospectively enrolled. Specimens were delivered to be tested for microbiological examinations and mNGS. RESULTS: Our data show that mNGS detected Non-tuberculosis mycobacteria, Mycobacterium tuberculosis, fungi and bacteria in CIG. Compared to culture, mNGS showed a higher positive rate (80.77% vs. 57.7%) with high sensitivity rate (100%) and negative predictive value (100%). In addition, mNGS can detect more pathogens in one sample and can be used to detect variable samples including the samples of paraffin-embedded tissue with shorter detective time. Of the 21 patients who showed clinical improvement within a 30-day follow-up, eighteen had their treatments adjusted, including fifteen who continued treatment based on the results of mNGS. CONCLUSIONS: mNGS could provide a potentially rapid and effective alternative detection method for diagnosis of cutaneous infectious granulomas and mNGS results may affect the clinical prognosis resulting from enabling the patients to initiate timely treatment.

Microbiological investigation of pregnancies following vaginal radical trachelectomy using 16S rRNA sequencing of FFPE placental specimens.

This study examined the risk of intrauterine infection associated with radical trachelectomy (RT) in early-stage cervical cancer patients. This procedure preserves fertility but is linked to increased risk of intrauterine infection due to cervical defects during pregnancy. DNA was extracted from the formalin-fixed paraffin-embedded (FFPE) placental specimens of 23 pregnant post-RT patients and 16S rRNA gene sequencing was used for bacterial identification. The prevalence of Lactobacillus crispatus and Burkholderia stabilis was significantly higher in the non-chorioamnionitis group. In contrast, alpha diversity analysis using the PD index showed significantly higher diversity in the chorioamnionitis group (P = 0.04). The demonstrated relationship between chorioamnionitis and microbial diversity affirms the importance of controlling the genital bacterial flora in pregnancies following RT.

A set of pretreatment reagents including improved formula fixation and decalcification facilitating immunohistochemistry and DNA analyses of formalin-fixed paraffin-embedded bone marrow trephine biopsy.

Bone marrow biopsy depends on tissue morphology, immunohistochemical staining, and moleculardetection. Tissue pretreatment is required for bone marrow samples, from clinical specimen acquisition to pathological reporting, but during the process, proteins and nucleic acids are often altered because of the acid in fixation and decalcification solutions. In our study, we present an easy and effective pretreatment protocol and compared this novel pretreatment protocol (Set 2) with an existing traditional pretreatment process (Set 1) using tissue morphology, IHC staining, and molecular pathological analyses. Granulocytic IHC markers showed more intensive staining in samples of Set 2 than in those of Set 1. The Set 2 protocol provided a higher DNA yield and less fragmentation; moreover, samples processed with the Set 2 protocol could be subsequently used in FISH and DNA sequencing assays. Our optimized novel pretreatment protocol could better protect proteins and DNA molecules while maintaining good cell morphology compared to traditional pretreatment The novel pretreatment reagents could role as a reference by more laboratories for pretreating bone marrow biopsy samples and scientific research.

Aqueous extraction of formalin-fixed paraffin-embedded tissue and detection of prion disease using real-time quaking-induced conversion.

OBJECTIVE: The goal of the research presented here is to determine if methods previously developed for the aqueous extraction of PrPSc from formalin-fixed paraffin-embedded tissue (FFPET) are applicable to the detection PrPSc by real-time quaking induced conversion (RT-QuIC). Previous work has utilized aqueous extraction of FFPET for detection of transmissible spongiform encephalopathies (TSEs) utilizing western blot and ELISA. This research extends the range of suitable methods for detection of TSEs in FFPET to RT-QuIC, which is arguably the most sensitive method to detect TSEs. RESULTS: We found complete agreement between the TSE status and the results from RT-QuIC seeded with the aqueous extract of FFPET samples. The method affords the diagnostic assessment TSE status by RT-QuIC of FFPET without the use of organic solvents that would otherwise create a mixed chemical-biological waste for disposal.

Challenges and solutions in FISH for formalin-fixed paraffin-embedded tissue: A scoping review.

Fluorescence in situ hybridization (FISH) has revolutionized molecular cytogenetic analysis since the 1980s, enabling precise localization of DNA sequences in cells and tissues. Despite its relevance, applying FISH to formalin-fixed paraffin-embedded (FFPE) tissue samples encounters significant technical challenges. This review addresses the main issues encountered in this context, such as inadequate fixation, contamination, block and slide age, inadequate pretreatment, and FISH technique. Proposed solutions include optimized pretreatment protocols, monitoring of blockage, careful selection of probes, and thorough analysis of results. Implementing good laboratory practices and quality control strategies are essential to ensure reliable results. Additionally, the use of emerging technologies such as artificial intelligence and digital pathology offers new perspectives for improving the efficiency and accuracy of FISH in FFPE samples. This review highlights the importance of a careful and personalized approach to overcome the technical challenges associated with FISH in FFPE samples, strengthening its role in research and clinical diagnosis. RESEARCH HIGHLIGHTS: Few FISH studies on FFPE: The scarcity of studies specifically addressing FISH applications in FFPE tissues highlights a critical gap in the literature. Troubleshooting FISH in FFPE tissues: Identifying and addressing common challenges in FISH techniques when applied to FFPE samples, such as signal quality and hybridization efficiency. Critical aspects of FISH technique: Discuss the main technical considerations crucial for successful FISH in FFPE tissues, including sample preparation, probe selection, and protocol optimization.