The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I-IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF.

In the past 20 years, laser microdissection (LMD) technology has been widely applied to plant tissues, allowing to obtain new information on the role of different cell-type populations during plant development and interactions, including plant-pathogen interactions. The application of a LMD approach allowed verifying the response of plant and pathogen during the progression of the infection in different cell types, focusing both on gene expression in host plants and pathogens. Here, a protocol to apply the LMD approach to study plant and fungal transcript profiles in different cell-type populations is described in detail, from the biological material preparation to RNA extraction and gene expression analyses.

Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new proteoform biomarkers of diseases. There are few published spatially resolved TDP studies. One of the challenges relates to the limited performance of TDP for the analysis of spatially isolated samples using, for example, laser capture microdissection (LCM) because those samples are usually mass-limited. We present the first pilot study of LCM-capillary zone electrophoresis (CZE)-MS/MS for spatially resolved TDP and used zebrafish brain as the sample. The LCM-CZE-MS/MS platform employed a non-ionic detergent and a freeze-thaw method for efficient proteoform extraction from LCM isolated brain sections followed by CZE-MS/MS without any sample cleanup step, ensuring high sensitivity. Over 400 proteoforms were identified in a CZE-MS/MS analysis of one LCM brain section via consuming the protein content of roughly 250 cells. We observed drastic differences in proteoform profiles between two LCM brain sections isolated from the optic tectum (Teo) and telencephalon (Tel) regions. Proteoforms of three proteins (npy, penkb, and pyya) having neuropeptide hormone activity were exclusively identified in the isolated Tel section. Proteoforms of reticulon, myosin, and troponin were almost exclusively identified in the isolated Teo section, and those proteins play essential roles in visual and motor activities. The proteoform profiles accurately reflected the main biological functions of the Teo and Tel regions of the brain. Additionally, hundreds of post-translationally modified proteoforms were identified.

Dopaminergic neurons (DA) of the substantia nigra pars compacta (SNpc) selectively and progressively degenerate in Parkinson\'s disease (PD). Until now, molecular analyses of DA in PD have been limited to genomic or transcriptomic approaches, whereas, to the best of our knowledge, no proteomic or combined multiomic study examining the protein profile of these neurons is currently available. In this exploratory study, we used laser capture microdissection to extract regions from DA in 10 human SNpc obtained at autopsy in PD patients and control subjects. Extracted RNA and proteins were identified by RNA sequencing and nanoliquid chromatography-mass spectrometry, respectively, and the differential expression between PD and control group was assessed. Qualitative analyses confirmed that the microdissection protocol preserves the integrity of our samples and offers access to specific molecular pathways. This multiomic analysis highlighted differential expression of 52 genes and 33 proteins, including molecules of interest already known to be dysregulated in PD, such as LRP2, PNMT, CXCR4, MAOA and CBLN1 genes, or the Aldehyde dehydrogenase 1 protein. On the other hand, despite the same samples were used for both analyses, correlation between RNA and protein expression was low, as exemplified by the CST3 gene encoding for the cystatin C protein. This is the first exploratory study analyzing both gene and protein expression of laser-dissected neuronal parts from SNpc in PD. Data are available via ProteomeXchange with identifier PXD024748 and via GEO with identifier GSE 169755.

The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.

Background and Objectives: Breast cancer (BC) remains one of the major causes of cancer death in women worldwide. The difficulties in assessing the deep molecular mechanisms involved in this pathology arise from its high complexity and diverse tissue subtypes. Long non-coding RNAs (lncRNAs) were shown to have great tissue specificity, being differentially expressed within the BC tissue subtypes. Materials and Methods: Herein, we performed lncRNA profiling by PCR array in triple negative breast cancer (TNBC) and luminal A tissue samples from 18 BC patients (nine TNBC and nine luminal A), followed by individual validation in BC tissue and cell lines. Tissue samples were previously archived in formalin-fixed paraffin-embedded (FFPE) samples, and the areas of interest were dissected using laser capture microdissection (LCM) technology. Results: Two lncRNAs (OTX2-AS1 and SOX2OT) were differentially expressed in the profiling analysis (fold change of 205.22 and 0.02, respectively, p < 0.05 in both cases); however, they did not reach statistical significance in the individual validation measurement (p > 0.05) when analyzed with specific individual assays. In addition, GAS5 and NEAT1 lncRNAs were individually assessed as they were previously described in the literature as being associated with BC. GAS5 was significantly downregulated in both TNBC tissues and cell lines compared to luminal A samples, while NEAT1 was significantly downregulated only in TNBC cells vs. luminal A. Conclusions: Therefore, we identified GAS5 lncRNA as having a differential expression in TNBC tissues and cells compared to luminal A, with possible implications in the molecular mechanisms of the TNBC subtype. This proof of principle study also suggests that LCM could be a useful technique for limiting the sample heterogeneity for lncRNA gene expression analysis in BC FFPE tissues. Future studies of larger cohort sizes are needed in order to assess the biomarker potential of lncRNA GAS5 in BC.

MicroRNAs are involved in various pathologies including cancer. The aim of the study was to assess the level of expression of miR-96-5p, -134-5p, -181b-5p, -200b-3p in FFPE samples of prostate cancer, adjacent cancer-free tissue, and benign prostatic hyperplasia. Samples of 23 FFPE prostate cancer and 22 benign prostatic hyperplasias were dissected and HE stained. Compartments of tumor tissue and adjacent healthy glandular tissue were isolated from each sample using Laser Capture Microdissection. Total RNA was isolated from dissected tissues. Expression of miR-96-5p, miR-134-5p, 181b-5p, and miR-200b-3p was determined by real-time RT-qPCR method. The expression of miR-200b-3p was significantly higher in cancerous prostate: both in adenocarcinomatous glands and in the adjacent, apparently unaffected glands compared to BPH samples. The expression of miR-181b-5p was lower in in both prostate cancer tissues and adjacent tissue compared to BPH samples. Expression of miR-96-5p and miR-134-5p was lower in prostate cancer tissues compared to BPH. Levels of miR-96-5p, miR-134-5p, and 181b-5p negatively correlated with the Gleason score. Given further studies, miR-96-5p, miR-134-5p and especially miR-200b-3p and miR-181b-5p may differentiate BPH and PC.

Laser microdissection (LMD) technology has been widely applied to plant tissues, offering novel information on the role of different cell-type populations during plant-microbe interactions. In this chapter, protocols to apply the LMD approach to study plant and fungal transcript profiles in different cell-type populations from arbuscular mycorrhizal (AM) roots are described in detail, starting from the biological material preparation to gene expression analyses by RT-PCR and RT-qPCR.

Penile intraepithelial neoplasia (PeIN) is currently classified in human papillomavirus (HPV)- and non-HPV-related subtypes with variable HPV genotypes. PeINs are frequently associated with other intraepithelial lesions in the same specimen. The aim of this study was to detect and compare HPV genotypes in PeINs and associated lesions using high-precision laser capture microdissection-polymerase chain reaction and p16INK4a immunostaining. We evaluated resected penile specimens from 8 patients and identified 33 PeINs and 54 associated lesions. The most common subtype was warty PeIN, followed by warty-basaloid and basaloid PeIN. Associated lesions were classical condylomas (17 cases), atypical classical condylomas (2 cases), flat condylomas (9 cases), atypical flat condylomas (6 cases), flat lesions with mild atypia (12 cases), and squamous hyperplasia (8 cases). After a comparison, identical HPV genotypes were found in PeIN and associated lesions in the majority of the patients (7 of 8 patients). HPV16 was the most common genotype present in both PeIN and corresponding associated lesion (50% of the patients). Nonspecific flat lesions with mild atypia, classical condylomas, and atypical condylomas were the type of associated lesions most commonly related to HPV16. Other high-risk HPV genotypes present in PeIN and associated nonspecific flat lesion with mild atypia were HPV35 and HPV39. In this study of HPV in the microenvironment of penile precancerous lesions, we identified identical high-risk HPV genotypes in PeIN and classical, flat, or atypical condylomas and, specially, in nonspecific flat lesions with mild atypia. It is possible that some of these lesions represent hitherto unrecognized precancerous lesions.

The availability of human pancreatic islets with characteristics closely resembling those present in vivo is instrumental for ex vivo studies in diabetes research.
In this review we propose metabolically phenotyped surgical patients as a novel source of pancreatic tissue for islet research. Laser Capture Microdissection from snap frozen surgical specimens is a relatively simple, reproducible and scalable method to isolate islets of highest purity for many types of \"omics\" analyses. Fresh pancreatic tissue slices enable the functional characterization of living islet cells in situ through dynamic experiments. Access to complete medical history and laboratory values for each donor offers the opportunity of direct correlations with different \"omics\" data and detailed metabolic profiling prior to pancreas surgery. Peripheral blood samples complete the picture of each patient and represent a platform for pursuit of biomarkers with uniquely comprehensive background information in regard to the donor\'s islet cells.
Living donors provide the scientific community with a steady and abundant supply of excellent material to study islets closest to their in situ environment, thus advancing our understanding of their physiology in health and diseases.