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Seminars in Reproductive Medicine Nov 2023Nonobstructive azoospermia (NOA) is among the most common causes of male infertility. For men with NOA seeking fertility treatment, microdissection testicular sperm...
Nonobstructive azoospermia (NOA) is among the most common causes of male infertility. For men with NOA seeking fertility treatment, microdissection testicular sperm extraction (microTESE) is the best option for retrieving sperm, which can be used with in vitro fertilization-intracytoplasmic sperm injection to achieve pregnancy in their partner. With the aid of the operating microscope, microTESE allows for thorough evaluation of the testis tissue and selection of seminiferous tubules that appear most capable of sperm production. Rates of success with microTESE vary depending on the underlying cause of NOA and the center at which the procedure is performed. Not all patients are candidates for microTESE, and those who are candidates should be counseled on the likelihood of sperm retrieval and the potential for changes in postoperative testis function.
Topics: Pregnancy; Female; Humans; Male; Testis; Microdissection; Retrospective Studies; Semen; Azoospermia; Spermatozoa
PubMed: 38262439
DOI: 10.1055/s-0043-1777833 -
Journal of Visualized Experiments : JoVE Apr 2023The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the...
The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye's spatial topography. In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade. The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.
Topics: Animals; Microdissection; Eye; Retina; Retinal Pigment Epithelium; Lens, Crystalline; Cornea
PubMed: 37154568
DOI: 10.3791/64414 -
Journal of the American Society of... Jan 2024Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by... (Review)
Review
Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by digestion of the dissected glomerular proteins by trypsin, and subsequently mass spectrometry to identify and semiquantitate the glomerular proteins. LCM/MS has played a crucial role in the identification of novel types of amyloidosis, biomarker discovery in fibrillary GN, and more recently discovery of novel target antigens in membranous nephropathy (MN). In addition, LCM/MS has also confirmed the role for complement proteins in glomerular diseases, including C3 glomerulopathy. LCM/MS is now widely used as a clinical test and considered the gold standard for diagnosis and typing amyloidosis. For the remaining glomerular diseases, LCM/MS has remained a research tool. In this review, we discuss the usefulness of LCM/MS in other glomerular diseases, particularly MN, deposition diseases, and diseases of complement pathways, and advocate more routine use of LCM/MS at the present time in at least certain diseases, such as MN, for target antigen detection. We also discuss the limitations of LCM/MS, particularly the difficulties faced from moving from a research-based technique to a clinical test. Nonetheless, the role of LCM/MS in glomerular diseases is expanding. Currently, LCM/MS may be used to identify the etiology in certain glomerular diseases, but in the future, LCM/MS can play a valuable role in determining pathways of complement activation, inflammation, and fibrosis.
Topics: Humans; Kidney Diseases; Kidney Glomerulus; Mass Spectrometry; Laser Capture Microdissection; Glomerulonephritis, Membranous; Amyloidosis
PubMed: 37749770
DOI: 10.1681/ASN.0000000000000221 -
Journal of the American Society For... Jul 2023Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed...
Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.
Topics: Humans; Workflow; Proteomics; High-Throughput Screening Assays; Peptides
PubMed: 37267530
DOI: 10.1021/jasms.3c00099 -
Computers in Biology and Medicine Nov 2023In the era of immunotherapy, the suboptimal response rate and the development of acquired resistance among the initial beneficiaries continue to present significant...
Microdissection of cancer-associated fibroblast infiltration subtypes unveils the secreted SERPINE2 contributing to immunosuppressive microenvironment and immuotherapeutic resistance in gastric cancer: A large-scale study integrating bulk and single-cell transcriptome profiling.
In the era of immunotherapy, the suboptimal response rate and the development of acquired resistance among the initial beneficiaries continue to present significant challenges across multiple malignancies, including gastric cancer (GC). Considering that the interactions of tumor stroma, especially the cancer-associated fibroblasts (CAFs), with immune and tumor cells, play indispensable roles in tumor progression, tumor microenvironment remodeling and therapeutic responsiveness, in-depth exploration on the roles of CAFs and pivotal mediators of their functions may provide novel clues to increase the effectiveness of current immunotherapeutic drugs and further achieve synergistic antitumor response. Herein, through the consensus clustering of canonical biomarkers, three GC subclasses with different abundance of CAFs were virtually microdissected in four integrated bulk cohorts encompassing 2148 GC patients from 11 independent datasets. An extensive immunogenomic analysis revealed that tumors with high CAFs infiltration were characterized with unfavorable outcomes, aggressive phenotypes, decreased tumor immunogenicity, high risk of immune evasion and thus immunotherapeutic resistance. By leveraging large-scale single-cell transcriptomic profiling, a series of CAF-secreted proteins were identified, among which the SERPINE2 was confirmed to be restrictively enriched in stromal fibroblasts of GC tissues and contribute to promoting a protumor milieu and fostering an immunosuppressive microenvironment via bioinformatics computations and tissue microarray analysis. Moreover, pan-cancer investigations generalized the immunological roles of SERPINE2, especially in pan-gastrointestinal malignancies, with multiple real-world immunotherapy cohorts further confirming its implications on predicting immunotherapeutic efficacy. In conclusion, these findings suggest that the CAF-derived SERPINE2 is a promising immune-oncology target with therapeutic implications to further synergize the immunotherapeutic combinations.
Topics: Humans; Stomach Neoplasms; Cancer-Associated Fibroblasts; Tumor Microenvironment; Single-Cell Analysis; Drug Resistance, Neoplasm; Gene Expression Profiling; Transcriptome; Immunotherapy; Gene Expression Regulation, Neoplastic
PubMed: 37729702
DOI: 10.1016/j.compbiomed.2023.107406 -
Methods in Molecular Biology (Clifton,... 2024Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic...
Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.
Topics: Cryptococcus neoformans; Microdissection; Cellular Senescence; Lab-On-A-Chip Devices; Single-Cell Analysis; Cryptococcosis
PubMed: 38758331
DOI: 10.1007/978-1-0716-3722-7_25 -
Molecular Biotechnology Mar 2023An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory...
An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory mechanism of woody plants. Laser microdissection is one technique that allows isolation of specific, target cells or tissue from a heterogeneous cell population. This technique entails microscopic visualization of the selected tissue and use a laser beam to separate the desired cells from surrounding tissue. Initial identification of these cells is made based on morphology and/or histological staining. Some works have been made in several tissues and plant models. However, there are few studies of laser microdissection application in woody species, particularly, lignified and suberized cells. Moreover, the presence of high level of suberin in cell walls can be a big challenge for the application of this approach. In our study it was developed a technique for tissue isolation, using laser microdissection of four different plant cell types (phellogen, lenticels, cortex and xylem) from woody tissues of cork oak (Quercus suber), followed by RNA extraction and RNA-Seq. We tested several methodologies regarding laser microdissection, cryostat equipments, fixation treatments, duration of single-cells collection and number of isolated cells by laser microdissection and RNA extraction procedures. A simple and efficient protocol for tissue isolation by laser microdissection and RNA purification was obtained, with a final method validation of RNA-Seq analysis. The optimized methodology combining RNA-Seq for expression analysis will contribute to elucidate the molecular pathways associated with different development processes of the xylem and phellem in oaks, including the lenticular channels formation.
Topics: Microdissection; RNA-Seq; Plants; Lasers; Quercus; RNA, Plant
PubMed: 35976558
DOI: 10.1007/s12033-022-00542-9 -
Cell Reports Jun 2023Gastric mixed adenoneuroendocrine carcinoma (MANEC) is a clinically aggressive and heterogeneous tumor composed of adenocarcinoma (ACA) and neuroendocrine carcinoma...
Gastric mixed adenoneuroendocrine carcinoma (MANEC) is a clinically aggressive and heterogeneous tumor composed of adenocarcinoma (ACA) and neuroendocrine carcinoma (NEC). The genomic properties and evolutionary clonal origins of MANEC remain unclear. We conduct whole-exome and multiregional sequencing on 101 samples from 33 patients to elucidate their evolutionary paths. We identify four significantly mutated genes, TP53, RB1, APC, and CTNNB1. MANEC resembles chromosomal instability stomach adenocarcinoma in that whole-genome doubling in MANEC is predominant and occurs earlier than most copy-number losses. All tumors are of monoclonal origin, and NEC components show more aggressive genomic properties than their ACA counterparts. The phylogenetic trees show two tumor divergence patterns, including sequential and parallel divergence. Furthermore, ACA-to-NEC rather than NEC-to-ACA transition is confirmed by immunohistochemistry on 6 biomarkers in ACA- and NEC-dominant regions. These results provide insights into the clonal origin and tumor differentiation of MANEC.
Topics: Humans; Phylogeny; Microdissection; Carcinoma, Neuroendocrine; Adenocarcinoma; Stomach Neoplasms; Genomics
PubMed: 37285266
DOI: 10.1016/j.celrep.2023.112576 -
Journal of Visualized Experiments : JoVE Mar 2021The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human...
The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.
Topics: Animals; Disease Models, Animal; Endolymphatic Sac; Humans; Mice
PubMed: 33843930
DOI: 10.3791/62375 -
Cytometry. Part a : the Journal of the... Dec 2019Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable...
Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific. However, a critical component to this process is the need for timely chromosome preparation via the air-drying method not to exceed a ~2-3 h before midi is performed. Failure of this time-sensitive step often results in the chromosomes drying out after dropping, and upon initiation of the midi technique, the dissected material can jump away while touching by the needle, and collection of a suitable sample is inhibited. Herein, we demonstrate with a simple adaptation of the standard procedure, midi can be performed on semi-archived material stored for longer periods at -20°C. Thus, the critical step to obtain well-spread chromosome preparations can be completed under established conditions, for example, in the primary laboratory, stored at -20°C, and sent directly to specialized reference laboratories offering midi. In our study, we were able to obtain high-quality DNA libraries, as verified by gel electrophoreses and reverse fluorescence in situ hybridization, via midi extracted chromosome spreads derived from human, fish, snake, lampbrush, and insect stored for up to 6 months. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Topics: Animals; Biological Specimen Banks; Chromosomes; DNA Probes; Humans; In Situ Hybridization, Fluorescence; Microdissection
PubMed: 31532073
DOI: 10.1002/cyto.a.23896