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Molecular & Cellular Proteomics : MCP Dec 2022Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from...
Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles specific to features within the islet. These features include the islet-acinar interface vascular tissue, inner islet vasculature, isolated endocrine cells, whole islet with vasculature, and acinar tissue from around the islet. Compared to interface vasculature, unique protein signatures observed in the inner vasculature indicate increased innervation and intra-islet neuron-like crosstalk. We also demonstrate the utility of these data for identifying localized structure-specific drug-target interactions using existing protein/drug binding databases.
Topics: Islets of Langerhans; Proteomics; Proteins; Laser Capture Microdissection
PubMed: 36244662
DOI: 10.1016/j.mcpro.2022.100426 -
Expert Review of Proteomics Oct 2021Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic... (Review)
Review
INTRODUCTION
Laser Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic visualization. Recently, there has been a surge of publications using LCM for tissue spatial molecular profiling relevant to a wide range of research topics.
AREAS COVERED
We summarize the many advances in tissue Laser Capture Proteomics (LCP) using mass spectrometry for discovery, and protein arrays for signal pathway network mapping. This review emphasizes: a) transition of LCM phosphoproteomics from the lab to the clinic for individualized cancer therapy, and b) the emerging frontier of LCM single cell molecular analysis combining proteomics with genomic, and transcriptomic analysis. The search strategy was based on the combination of MeSH terms with expert refinement.
EXPERT OPINION
LCM is complemented by a rich set of instruments, methodology protocols, and analytical A.I. (artificial intelligence) software for basic and translational research. Resolution is advancing to the tissue single cell level. A vision for the future evolution of LCM is presented. Emerging LCM technology is combining digital and AI guided remote imaging with automation, and telepathology, to a achieve multi-omic profiling that was not previously possible.
Topics: Artificial Intelligence; Laser Capture Microdissection; Lasers; Precision Medicine; Proteomics
PubMed: 34607525
DOI: 10.1080/14789450.2021.1984886 -
Expert Reviews in Molecular Medicine Jun 2022Non-obstructive azoospermia (NOA), the most severe type of male infertility, affects approximately 1% of men worldwide. However, the aetiology of most NOA cases is not... (Review)
Review
Non-obstructive azoospermia (NOA), the most severe type of male infertility, affects approximately 1% of men worldwide. However, the aetiology of most NOA cases is not definite, that is defined as idiopathic NOA (INOA), posing a clinical conundrum worldwide. Most of these patients must receive donor sperm treatment until the emergence of microdissection testicular sperm extraction (micro-TESE). Although this procedure has recently become a promising treatment for INOA, the low sperm retrieval rate and testicular trauma have prompted us to explore appropriate non-invasive molecular biomarkers to predict the outcomes of sperm recovery preoperatively. Previous studies have identified a spectrum of biomarkers to address this challenging issue at various levels in different tissues, such as DNAs, RNAs, protein and steroid levels in the blood and seminal fluid. To better understand and assess the predictive values of diverse molecular biomarkers from different tissues on the outcome of sperm retrieval by micro-TESE in patients with INOA, we summarised recent findings and discussed the potential applications of these methods. The ultimate goal of this study was to provide references for further studies and clinical management.
Topics: Azoospermia; Biomarkers; Humans; Male; Microdissection; Retrospective Studies; Semen; Spermatozoa; Testis
PubMed: 35659383
DOI: 10.1017/erm.2022.17 -
Hypertension (Dallas, Tex. : 1979) Oct 2020
Topics: Denervation; Humans; Hypertension; Kidney; Microdissection; Nervous System
PubMed: 32903105
DOI: 10.1161/HYPERTENSIONAHA.120.15834 -
Laboratory Investigation; a Journal of... Jul 2023Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained...
Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.
Topics: MicroRNAs; Microdissection; Epithelial Cells; Formaldehyde; Gene Expression Profiling
PubMed: 36990152
DOI: 10.1016/j.labinv.2023.100133 -
Frontiers in Endocrinology 2022Most of data available in the literature reported the sperm retrieval rate and limited intracytoplasmic sperm injection (ICSI) results of microdissection testicular...
Clinical Outcomes and Live Birth Rate Resulted From Microdissection Testicular Sperm Extraction With ICSI-IVF in Non-Obstructive Azoospermia: A Single-Center Cohort Study.
BACKGROUND
Most of data available in the literature reported the sperm retrieval rate and limited intracytoplasmic sperm injection (ICSI) results of microdissection testicular sperm extraction (micro-TESE) in non-obstructive azoospermia (NOA) patients with different etiologies. Unfortunately, there is currently a lack of comprehensive data to guide clinicians in conducting comprehensive consultations with NOA patients.
OBJECTIVES
To obtain more comprehensive evidence-based data and clinical outcomes for better consultation of NOA patients who opted to undergo micro-TESE combined with ICSI-IVF.
METHODS
It was a retrospective study involved 968 NOA patients underwent micro-TESE during January 2015 to December 2019. Embryological, clinical, and live birth outcomes were demonstrated comprehensively and three kinds of stratification analyses were performed based on ICSI-IVF cycles using frozen and fresh sperm, different etiologies of NOA and various amounts of sperm retrieved.
RESULTS
The sperm retrieval rate was 44.6%, and ICSI was performed in 299 couples leading to 150 clinical pregnancies and 140 live-birth deliveries. The clinical pregnancy rate (CPR) was 50.17%, and the cumulative live birth rate (LBR) was 46.82%, and the low birth defects rate was 1.43%. No significant difference was observed about cumulative LBR in frozen sperm group and fresh sperm group (47.5% vs 42.9%, 0.05). NOA patients with AZFc microdeletions had the lowest rate of a high-score embryo on day 3 (4.4%, <0.05) and the lowest cumulative LBR (19.4%, <0.05). NOA patients with lower sperm count (having fewer than 20 sperms retrieved) had significantly lower cumulative LBR than those with higher sperm count (having more than 20 sperms retrieved) (28.1% vs 51.9%, <0.05).
CONCLUSIONS
For those NOA patients who stepped in ICSI-IVF cycles, the cumulative LBR was 46.82%. No significant difference was indicated in the LBR between ICSI-IVF cycles using frozen or fresh testicular sperm. Compared to other etiologies, NOA caused by AZFc microdeletions have the poorest embryological and clinical outcomes. Patients with less testicular sperm retrieved have poorer embryological and clinical outcomes.
Topics: Azoospermia; Birth Rate; Cohort Studies; Female; Humans; Male; Microdissection; Pregnancy; Retrospective Studies; Semen; Sperm Injections, Intracytoplasmic; Spermatozoa
PubMed: 35813616
DOI: 10.3389/fendo.2022.893679 -
Bio-protocol Dec 2020Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain...
Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain areas to perform molecular or anatomical analyses. The parafilm-assisted microdissection (PAM) was previously described and involves the microdissection of tissue sections mounted on parafilm-covered glass slides. In this work, we describe the use of the PAM method to microdissect rodent nucleus accumbens (NAc). (1) We first describe the best way to perform the mouse euthanasia and how to remove the brain. (2) Next, we describe how to prepare the slides with parafilm that will be used to receive the brain slices. (3) Following, we describe how to handle the brain in the cryostat, how to align the hemispheres and how to identify the NAc antero-posterior limits. (4) We also describe how to perform the staining and dehydration of the slices, a critical step to facilitate the microdissection and preserve macromolecules. (5) In the final step, we describe how to identify the dorso-ventral and latero-medial limits of the NAc and, finally, how to perform the manual microdissection of the area. This is a low-cost technique that allows the researcher to specifically microdissect any brain region, from which intact RNA and proteins can be extracted to perform several molecular analyses (., real-time PCR, Western blot, and RNA-seq).
PubMed: 33659485
DOI: 10.21769/BioProtoc.3836 -
Journal of Visualized Experiments : JoVE Nov 2019Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such...
Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.
Topics: Animals; Cochlea; Epithelium; Hair Cells, Auditory; Hair Cells, Auditory, Inner; Mice; Microdissection; Organ of Corti
PubMed: 31762458
DOI: 10.3791/60299 -
Frontiers in Neural Circuits 2022Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly , in particular, we...
Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly , in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the brain. These requirements include: good preservation of ultrastructural detail, high level of staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly's delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging.
Topics: Animals; Drosophila; Microscopy, Electron, Scanning; Volume Electron Microscopy; Connectome; Synapses; Brain
PubMed: 36589862
DOI: 10.3389/fncir.2022.917251 -
The Urologic Clinics of North America May 2020Guiding a couple with nonobstructive azoospermia requires an integrated approach to care by the urologist and the reproductive endocrinologist. After informing the... (Review)
Review
Guiding a couple with nonobstructive azoospermia requires an integrated approach to care by the urologist and the reproductive endocrinologist. After informing the couple of the implications of the diagnosis, care must be taken to outline the options of parenthood. Most experts agree that sperm retrieval in men can be challenging. This article describes various options of sperm retrieval, historic and contemporary, and highlights the advantages and disadvantages of each. The authors find that using a testicular map can invariably help guide sperm retrieval and overall fertility care. The right approach is one that involves a shared decision with the couple.
Topics: Azoospermia; Biopsy, Fine-Needle; Humans; Male; Microdissection; Semen Analysis; Sperm Retrieval; Spermatozoa; Testis
PubMed: 32272987
DOI: 10.1016/j.ucl.2019.12.013