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Glomerular Diseases 2024The first spatially resolved transcriptomics platforms, GeoMx (Nanostring) and Visium (10x Genomics) were launched in 2019 and were recognized as the method of the year... (Review)
Review
BACKGROUND
The first spatially resolved transcriptomics platforms, GeoMx (Nanostring) and Visium (10x Genomics) were launched in 2019 and were recognized as the method of the year by in 2020. The subsequent refinement and expansion of these and other technologies to increase -plex, work with formalin-fixed paraffin-embedded tissue, and analyze protein in addition to gene expression have only added to their significance and impact on the biomedical sciences. In this perspective, we focus on two platforms for spatial transcriptomics, GeoMx and Visium, and how these platforms have been used to provide novel insight into kidney disease. The choice of platform will depend largely on experimental questions and design. The application of these technologies to clinically sourced biopsies presents the opportunity to identify specific tissue biomarkers that help define disease etiology and more precisely target therapeutic interventions in the future.
SUMMARY
In this review, we provide a description of the existing and emerging technologies that can be used to capture spatially resolved gene and protein expression data from tissue. These technologies have provided new insight into the spatial heterogeneity of diseases, how reactions to disease are distributed within a tissue, which cells are affected, and molecular pathways that predict disease and response to therapy.
KEY MESSAGE
The upcoming years will see intense use of spatial transcriptomics technologies to better define the pathophysiology of kidney diseases and develop novel diagnostic tests to guide personalized treatments for patients.
PubMed: 38600956
DOI: 10.1159/000538344 -
Cancers Dec 2023Gastric cancer (GC) is one of the most commonly diagnosed cancers and the fourth cause of cancer death worldwide. Personalised treatment improves GC outcomes. A...
Gastric cancer (GC) is one of the most commonly diagnosed cancers and the fourth cause of cancer death worldwide. Personalised treatment improves GC outcomes. A molecular classification is needed to choose the appropriate therapy. A classification that uses on-slide biomarkers and formalin-fixed and paraffin-embedded (FFPE) tissue is preferable to comprehensive genomic analysis. In 2016, Setia and colleagues proposed an on-slide classification; however, this is not in widespread use. We propose a modification of this classification that has six subgroups: GC associated with Epstein-Barr virus (GC EBV+), GC with mismatch-repair deficiency (GC dMMR), GC with epithelial-mesenchymal transformation (GC EMT), GC with chromosomal instability (GC CIN), CG that is genomically stable (GC GS) and GC not otherwise specified (GC NOS). This classification also has a provision for biomarkers for current or emerging targeted therapies (Her2, PD-L1 and Claudin18.2). Here, we assess the implementation and feasibility of this inclusive working classification. : We constructed a tissue microarray library from a cohort of 79 resection cases from FFPE tissue archives. We used a restricted panel of on-slide markers (EBER, MMR, E-cadherin, beta-catenin and p53), defined their interpretation algorithms and assigned each case to a specific molecular subtype. : GC EBV(+) cases were 6%, GC dMMR cases were 20%, GC EMT cases were 14%, GC CIN cases were 23%, GC GS cases were 29%, and GC NOS cases were 8%. : This working classification uses markers that are widely available in histopathology and are easy to interpret. A diagnostic subgroup is obtained for 92% of the cases. The proportion of cases in each subgroup is in keeping with other published series. Widescale implementation appears feasible. A study using endoscopic biopsies is warranted.
PubMed: 38201483
DOI: 10.3390/cancers16010055 -
International Journal of Reproductive... Aug 2023Polycystic ovary syndrome (PCOS) is a heterogeneous medical condition with a cluster of metabolic and endocrine disorders including dyslipidemia, insulin resistance, and...
BACKGROUND
Polycystic ovary syndrome (PCOS) is a heterogeneous medical condition with a cluster of metabolic and endocrine disorders including dyslipidemia, insulin resistance, and hyperandrogenism.
OBJECTIVE
The present study aimed to determine the effects of single-dose and co-supplementation of vitamin D (vit D) and omega-3 (O3) on anthropometric and several biochemical factors in women with PCOS.
MATERIALS AND METHODS
In this double-blind, randomized clinical trial, 80 PCOS women referred to Shahid Motahhari Clinic, Shiraz, Iran, from April to October 2017 were studied in 4 groups (n = 20/each) for 8 wk. The placebo group received the placebo capsule (paraffin oil); 1 weekly and 2/daily; the vit D group received vit D (50,000 IU/weekly) + 2 placebo capsules daily, O3 group, 2, O3 capsules daily + 1 placebo capsule weekly, and vit D + O3 (50000 IU/weekly vit D + 2, O3 capsules daily). Before and after 8 wk of intervention, height, weight, body mass index, waist circumference, triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, fasting blood sugar, homeostasis model of insulin resistance index, and sex hormone binding globulin were compared between groups.
RESULTS
The significant reduction was detected in serum triglyceride (p = 0.002), TC (p = 0.04), fasting blood sugar (p = 0.02), insulin (p = 0.001), and homeostasis model of insulin resistance index (p = 0.001) concentrations in all vit D, O3, and vit D + O3 supplemented groups compared to the placebo group. Furthermore, in comparison with the placebo group, a significant increase was observed in serum sex hormone binding globulin levels after vit D, O3, and vit D + O3 treatments. Nevertheless, no significant changes were observed in serum high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and anthropometric indices in all treated participants.
CONCLUSION
The current study indicated that single dose and co-supplementation of vit D and O3 for 8 wk was associated with beneficial effects on serum triglyceride, TC, insulin, and sex hormone binding globulin concentrations among women suffering from PCOS.
PubMed: 37727395
DOI: 10.18502/ijrm.v21i7.13889 -
STAR Protocols Jun 2024Here, we present a protocol for using spatial transcriptomics in bone and multi-tissue musculoskeletal formalin-fixed paraffin-embedded (FFPE) samples from mice. We...
Here, we present a protocol for using spatial transcriptomics in bone and multi-tissue musculoskeletal formalin-fixed paraffin-embedded (FFPE) samples from mice. We describe steps for tissue harvesting, sample preparation, paraffin embedding, and FFPE sample selection. We detail procedures for sectioning and placement on spatial slides prior to imaging, decrosslinking, library preparation, and final analyses of the sequencing data. The complete protocol takes ca. 18 days for mouse femora with adjacent muscle; of this time, >50% is required for mineralized tissue decalcification. For complete details on the use and execution of this protocol, please refer to Wehrle et al. and Mathavan et al..
Topics: Animals; Mice; Paraffin Embedding; Formaldehyde; Tissue Fixation; Transcriptome; Gene Expression Profiling; Bone and Bones; Musculoskeletal System
PubMed: 38555590
DOI: 10.1016/j.xpro.2024.102986 -
Cureus Jan 2024The proper regulations for storage, retention, and use of archived specimens in pathology laboratories and academic institutions are yet to be established. These... (Review)
Review
The proper regulations for storage, retention, and use of archived specimens in pathology laboratories and academic institutions are yet to be established. These specimens could be used appropriately for research purposes. Ideal storage and retention in a controlled environment is necessary, and there is a lack of established rules regarding the ownership of the tissue specimens, paraffin blocks, and slides. Though there are numerous uses of formalin-fixed tissue specimens, blocks, and slides, there are also problems in archiving them that hinder their use. This review article addresses the above issues and proposes simple guidelines for the effective use of archived specimens.
PubMed: 38410328
DOI: 10.7759/cureus.53025 -
BMC Genomics Dec 2023RNA-Seq analysis of Formalin-Fixed and Paraffin-Embedded (FFPE) samples has emerged as a highly effective approach and is increasingly being used in clinical research... (Review)
Review
RNA-Seq analysis of Formalin-Fixed and Paraffin-Embedded (FFPE) samples has emerged as a highly effective approach and is increasingly being used in clinical research and drug development. However, the processing and storage of FFPE samples are known to cause extensive degradation of RNAs, which limits the discovery of gene expression or gene fusion-based biomarkers using RNA sequencing, particularly methods reliant on Poly(A) enrichment. Recently, researchers have developed an exome targeted RNA-Seq methodology that utilizes biotinylated oligonucleotide probes to enrich RNA transcripts of interest, which could overcome these limitations. Nevertheless, the standardization of this experimental framework, including probe designs, sample multiplexing, sequencing read length, and bioinformatic pipelines, remains an essential requirement. In this study, we conducted a comprehensive comparison of three main commercially available exome capture kits and evaluated key experimental parameters, to provide the overview of the advantages and limitations associated with the selection of library preparation protocols and sequencing platforms. The results provide valuable insights into the best practices for obtaining high-quality data from FFPE samples.
Topics: Exome; Formaldehyde; Gene Expression Profiling; Paraffin; Paraffin Embedding; RNA; Sequence Analysis, RNA; Tissue Fixation
PubMed: 38102591
DOI: 10.1186/s12864-023-09886-1 -
Journal of Pathology and Translational... Nov 2023The Wnt signaling pathway regulates crucial cellular processes, including stem cell development and tissue repair. Dysregulation of this pathway, particularly β-catenin...
BACKGROUND
The Wnt signaling pathway regulates crucial cellular processes, including stem cell development and tissue repair. Dysregulation of this pathway, particularly β-catenin stabilization, is linked to colorectal carcinoma and other tumors. Axin2, a critical component in the pathway, plays a role in β-catenin regulation. This study examines Axin2 expression in normal gastric mucosa and various gastric pathologies.
METHODS
Formalin-fixed and paraffin-embedded tissue samples from normal stomach, gastritis, intestinal metaplasia (IM), and gastric carcinoma were collected. Axin2 and β-catenin expression were evaluated using RNA in situ hybridization and immunohistochemistry, respectively. Histo-scores (H-scores) were calculated to quantify expression levels of Axin2. Associations between Axin2 expression and clinicopathological variables were examined.
RESULTS
Axin2 expression was examined in normal stomach, gastritis, and IM tissues. Axin2 expression was mainly observed in the surface and isthmus areas in the normal stomach and gastritis, whereas Axin2 expression was markedly higher at the bases of IM. Axin2 H-scores were significantly elevated in IM (mean ± standard deviation [SD], 87.0 ± 38.9) compared to normal (mean ± SD, 18.0 ± 4.5) and gastritis tissues (mean ± SD, 33.0 ± 18.6). In total, 30% of gastric carcinomas showed higher Axin2 expression. Axin2 expression did not have significant associations with age, sex, Lauren classification, histological differentiation, invasion depth, and lymph node metastasis. However, a strong positive correlation was observed between Axin2 and nuclear β-catenin in gastric carcinomas (p < .001).
CONCLUSIONS
Axin2 expression was significantly increased in IM compared to normal and gastritis cases. In addition, Axin2 showed a strong positive association with nuclear β-catenin expression in gastric carcinomas, demonstrating a close relationship with abnormal Wnt/β-catenin signaling pathway.
PubMed: 37926983
DOI: 10.4132/jptm.2023.10.12 -
Indian Journal of Pathology &... 2023Cyclic adenosine monophosphate (cAMP) is an intracellular signal transmitter involved in the regulation of melanocyte growth, proliferation, and melanogenesis. R21 is a...
BACKGROUND
Cyclic adenosine monophosphate (cAMP) is an intracellular signal transmitter involved in the regulation of melanocyte growth, proliferation, and melanogenesis. R21 is a monoclonal antibody against the soluble adenylyl cyclase (sAC) protein. Various nuclear and cytoplasmic R21 expression patterns in melanocytic lesions have been previously reported. Pan-nuclear staining was defined as specific for melanoma and was found supportive in the assessment of surgical margins.
AIMS
The aim of this study is to evaluate the different expression patterns of R21 immunostain and investigate its effectiveness in the differential diagnosis of cutaneous malignant and benign melanocytic lesions.
SETTINGS AND DESIGN
Fifty invasive cutaneous melanoma and 50 benign melanocytic proliferation were included in the study.
MATERIALS AND METHODS
Paraffin blocks that best reflected tumor morphology were studied via immunohistochemical staining for R21. For all patterns, the cases showing staining in 25% or more tumor cells were considered as positive.
STATISTICAL ANALYSIS USED
Yates' Chi-square, Pearson Chi-square exact test, Spearman correlation were used.
RESULTS AND CONCLUSIONS
Dot-like Golgi staining was characteristic for nevi (12/50) and seen only in one melanoma. Pan-nuclear staining was striking for melanoma (36/50). This pattern was observed in 2 dysplastic and 3 common melanocytic nevi too. None of the Spitz nevi expressed R21 in pan-nuclear pattern. For the diagnosis of melanoma, sensitivity and specificity of the pan-nuclear expression were 72% and 90%, respectively. Positive and negative predictive values were found as 87% and 76%. R21, a second-generation immunohistochemical marker, can be used in the differential diagnosis of benign and malignant melanocytic lesions.
Topics: Humans; Melanoma; Skin Neoplasms; Diagnosis, Differential; Biomarkers, Tumor; Melanocytes; Nevus
PubMed: 38084536
DOI: 10.4103/ijpm.ijpm_1146_21 -
ACS Applied Engineering Materials Dec 2023The design of shielding materials against ionizing radiation while simultaneously displaying enhanced multifunctional characteristics remains challenging. Here, for the...
The design of shielding materials against ionizing radiation while simultaneously displaying enhanced multifunctional characteristics remains challenging. Here, for the first time, we present moldable paraffin-based iron nano- and microcomposites attenuating γ- and X-radiation. The moldability was gained by the warmth-of-hands-driven plasticity, which allowed for obtaining a specific shape of the composites at room temperature. The manufactured composites contained iron particles of various sizes, ranging from 22 nm to 63 μm. The target materials were widely characterized using XRD, NMR, Raman, TGA, SEM, and EDX. In the case of microcomposites, the shielding properties were developed at two concentrations: 10 and 50 wt %. The statistically significant results indicate that the iron particle size has a negligible effect on the shielding properties of the nano- and microcomposites. On the other hand, the higher iron particle contents significantly affected the attenuating ability, which emerged even as superior to the elemental aluminum in the X-ray range: at a 70 kV anode voltage, the half value layer was 6.689, 1.882, and 0.462 cm for aluminum, paraffin + 10 wt % Fe 3.5-6.5 μm, and paraffin + 50 wt % Fe 3.5-6.5 μm microcomposites, respectively. Importantly, the elaborated methodology-in situ cross-verified in the hospital studies recording real-life sampling-opens the pathway to high-performance, eco-friendly, lightweight, and recyclable shields manufactured via fully reproducible and scalable protocols.
PubMed: 38148950
DOI: 10.1021/acsaenm.3c00557 -
Food Science and Biotechnology Nov 2023A time-temperature indicator (TTI) based on acid-base reaction was developed by applying a new pH dye composed of cysteine-loaded chitosan (Cys-CS) microspheres and...
A time-temperature indicator (TTI) based on acid-base reaction was developed by applying a new pH dye composed of cysteine-loaded chitosan (Cys-CS) microspheres and silver nanoparticles (AgNPs). It was hypothesized that cysteine released by the disintegration of Cys-CS microspheres at a critical pH would cause AgNPs to aggregate, leading to color change. Cys-CS microspheres were produced as water-in-oil (paraffin oil, MCT oil, soybean oil) emulsions according to the KOH addition method. An enzymatic TTI was made using glucose oxidase, glucose, and catalase. Only paraffin oil produced Cys-CS microspheres (average diameter, 335 ± 100 µm), whereas the others did not, probably due to saponification with KOH. FTIR analysis confirmed that cysteine was encapsulated in the microspheres. The microspheres disintegrated at pH 6.18 in a titration test. The TTI pH gradually decreased and showed a sudden color change at pH 6.10, which was similar to the critical pH of microsphere disintegration.
PubMed: 37781051
DOI: 10.1007/s10068-023-01369-z