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Biomedical Optics Express Jun 2024Fast and efficient separation of target samples is crucial for the application of laser-assisted microdissection in the molecular biology research field. Herein, we...
Fast and efficient separation of target samples is crucial for the application of laser-assisted microdissection in the molecular biology research field. Herein, we developed a laser axial scanning microdissection (LASM) system with an 8.6 times extended depth of focus by using an electrically tunable lens. We showed that the ablation quality of silicon wafers at different depths became homogenous after using our system. More importantly, for those uneven biological tissue sections within a height difference of no more than 19.2 µm, we have demonstrated that the targets with a size of microns at arbitrary positions can be dissected efficiently without additional focusing and dissection operations. Besides, dissection experiments on various biological samples with different embedding methods, which were widely adopted in biological experiments, also have shown the feasibility of our system.
PubMed: 38867797
DOI: 10.1364/BOE.523954 -
Biomedical Optics Express Jun 2024The spatial omics information analysis of heterogeneous cells or cell populations is of great importance for biomedical research. Herein, we proposed a picosecond laser...
The spatial omics information analysis of heterogeneous cells or cell populations is of great importance for biomedical research. Herein, we proposed a picosecond laser capture microdissection boosted by edge catapulting combined with dielectrophoretic force (ps-LMED) that enables fast and non-invasive acquisition of uncontaminated cells and cell populations for downstream molecular assays. The target cells were positioned under a microscope and separated by a focused picosecond pulsed laser. The system employed the plasma expansion force during cutting to lift the target and captured it under dielectrophoretic force from the charged collection cap eventually. The principle of our system has been validated by both theoretical analysis and practical experiments. The results indicated that our system can collect samples ranging from a single cell with a diameter of a few microns to large tissues with a volume of 532,500 µm at the moment finishing the cutting, without further operations. The cutting experiments of living cells and ribonucleic acid (RNA) and protein omics analysis results of collected targets demonstrated the advantage of non-destructiveness to the samples and feasibility in omics applications.
PubMed: 38867793
DOI: 10.1364/BOE.525630 -
European Urology Open Science Jul 2024No clear-cut markers for predicting positive sperm retrieval (+SR) at microdissection testicular sperm extraction (mTESE) have been identified thus far. Our aim was to... (Review)
Review
Role of Follicle-stimulating Hormone, Inhibin B, and Anti-Müllerian Hormone in Predicting Sperm Retrieval from Men with Nonobstructive Azoospermia Undergoing Microdissection Testicular Sperm Extraction: A Systematic Review and Meta-analysis.
BACKGROUND AND OBJECTIVE
No clear-cut markers for predicting positive sperm retrieval (+SR) at microdissection testicular sperm extraction (mTESE) have been identified thus far. Our aim was to conduct a systematic review and meta-analysis to evaluate the ability of follicle-stimulating hormone (FSH), inhibin B (InhB), and anti-Müllerian hormone (AMH) to predict +SR in men with nonobstructive azoospermia (NOA) undergoing mTESE.
METHODS
We performed a search in the PubMed, EMBASE, Web of Science, and Scopus databases according to the Preferred Reporting Items for Systematic Review and Meta-analysis statement. Thirty-four publications were selected for inclusion in the analysis.
KEY FINDINGS AND LIMITATIONS
Overall, the mean +SR rate was 45%. Pooled standardized mean difference (SMD) values revealed significant hormonal differences between the +SR and -SR groups, with lower FSH (SMD -0.30), higher InhB (SMD 0.54), and lower AMH (SMD -0.56) levels in the +SR group. Pooled odds ratios (Ors) revealed no significant prediction of +SR by either FSH (OR 1.03, 95% confidence interval [CI] 1.00-1.06) or InhB (OR 1.01, 95% CI 1.00-1.02), despite variations in baseline levels and study heterogeneity. Conversely, AMH had significant predictive value (OR 0.82, 95% CI 0.73-0.92), with lower baseline levels in the +SR group. InhB and FSH levels were higher in the +SR group, while InhB exhibited the opposite trend.
CONCLUSIONS AND CLINICAL IMPLICATIONS
Despite study heterogeneity, our meta-analysis findings support the ability of AMH to predict +SR for men with NOA undergoing mTESE.
PATIENT SUMMARY
We conducted a review and analysis of results from previous studies. Our findings show that for men with an infertility condition called nonobstructive azoospermia, blood levels of anti-Müllerian hormone can predict successful extraction of sperm using a microsurgical technique. Levels of two other hormones did not predict successful sperm extraction.
PubMed: 38854995
DOI: 10.1016/j.euros.2024.05.001 -
The Journal of Heart and Lung... Jun 2024T cells drive acute cellular rejection (ACR) and its progression to chronic lung allograft dysfunction (CLAD) following lung transplantation. ISHLT grade A1 ACR without...
The CD8 T cell content of transbronchial biopsies from patients with a first episode of clinically stable grade A1 cellular rejection is associated with future chronic lung allograft dysfunction.
BACKGROUND
T cells drive acute cellular rejection (ACR) and its progression to chronic lung allograft dysfunction (CLAD) following lung transplantation. ISHLT grade A1 ACR without associated allograft dysfunction is often untreated, yet some patients develop progressive graft dysfunction. T cell composition of A1 ACR lesions may have prognostic value, therefore protein-level and epigenetic techniques were applied to transbronchial biopsy (TBB) tissue to determine whether differential infiltration of T cells in recipients experiencing a first episode of stable grade A1 ACR (StA1R) is associated with early CLAD.
METHODS
Sixty-two patients experiencing a first episode of StA1R were divided into those experiencing CLAD within 2 years (n=13) and those remaining CLAD-free for 5≤ years (n=49). Imaging mass cytometry (IMC) was used to profile the spectrum and distribution of intragraft T cell phenotypes on a subcohort (n=16; 8 early-CLAD and 8 no-early-CLAD). Immunofluorescence was used to quantify CD4, CD8 and FOXP3 cells. Separately, CD3 cells were fluorescently labelled, micro-dissected and, using bisulfite conversion and pyrosequencing, the degree of Treg-specific demethylated region methylation was determined.
RESULTS
PhenoGraph unsupervised clustering on IMC revealed 50 unique immune cell subpopulations. Methylation and immunofluorescence analyses demonstrated no significant differences in Tregs between early-CLAD and no-early-CLAD groups. Immunofluorescence revealed that patients who developed CLAD within 2 years of LTx showed greater CD8 T cell infiltration compared to those who remained CLAD-free for 5 or more years.
CONCLUSIONS
In asymptomatic patients with a first episode of A1 rejection, greater CD8 T cell content may be indicative of worse long-term outlook.
PubMed: 38852935
DOI: 10.1016/j.healun.2024.06.001 -
Neoplasia (New York, N.Y.) Aug 2024In invasive lung adenocarcinoma (LUAD), patients with micropapillary (MIP) or solid (SOL) components had a significantly poorer prognosis than those with only lepidic...
In invasive lung adenocarcinoma (LUAD), patients with micropapillary (MIP) or solid (SOL) components had a significantly poorer prognosis than those with only lepidic (LEP), acinar (ACI) or papillary (PAP) components. It is interesting to explore the genetic features of different histologic subtypes, especially the highly aggressive components. Based on a cohort of 5,933 patients, this study observed that in different tumor size groups, LUAD with MIP/SOL components showed a different prevalence, and patients with ALK alteration or TP53 mutations had a higher probability of developing MIP/SOL components. To control individual differences, this research used spatial whole-exome sequencing (WES) via laser-capture microdissection of five patients harboring these five coexistent components and identified genetic features among different histologic components of the same tumor. In tracing the evolution of components, we found that titin (TTN) mutation might serve as a crucial intratumor potential driver for MIP/SOL components, which was validated by a cohort of 146 LUAD patients undergoing bulk WES. Functional analysis revealed that TTN mutations enriched the complement and coagulation cascades, which correlated with the pathway of cell adhesion, migration, and proliferation. Collectively, the histologic subtypes of invasive LUAD were genetically different, and certain trunk genotypes might synergize with branching TTN mutation to develop highly aggressive components.
Topics: Humans; Exome Sequencing; Adenocarcinoma of Lung; Mutation; Lung Neoplasms; Biomarkers, Tumor; Male; Female; Connectin; Prognosis; Middle Aged
PubMed: 38850835
DOI: 10.1016/j.neo.2024.101013 -
ENeuro Jun 2024Social behavior is important for our well-being, and its dysfunctions impact several pathological conditions. Although the involvement of glutamate is undeniable, the...
Social behavior is important for our well-being, and its dysfunctions impact several pathological conditions. Although the involvement of glutamate is undeniable, the relevance of vesicular glutamate transporter type 3 (VGluT3), a specific vesicular transporter, in the control of social behavior is not sufficiently explored. Since midbrain median raphe region (MRR) is implicated in social behavior and the nucleus contains high amount of VGluT3+ neurons, we compared the behavior of male VGluT3 knock-out (KO) and VGluT3-Cre mice, the latter after chemogenetic MRR-VGluT3 manipulation. Appropriate control groups were included. Behavioral test battery was used for social behavior (sociability, social discrimination, social interaction, resident intruder test) and possible confounding factors (open field, elevated plus maze, Y-maze tests). Neuronal activation was studied by c-Fos immunohistochemistry. Human relevance was confirmed by VGluT3 gene expression in relevant human brainstem areas. VGluT3 KO mice exhibited increased anxiety, social interest, but also aggressive behavior in anxiogenic environment and impaired social memory. For KO animals, social interaction induced lower cell activation in the anterior cingulate, infralimbic cortex, and medial septum. In turn, excitation of MRR-VGluT3+ neurons was anxiolytic. Inhibition increased social interest 24 h later but decreased mobility and social behavior in aggressive context. Chemogenetic activation increased the number of c-Fos+ neurons only in the MRR. We confirmed the increased anxiety-like behavior and impaired memory of VGluT3 KO strain and revealed increased, but inadequate, social behavior. MRR-VGluT3 neurons regulated mobility and social and anxiety-like behavior in a context-dependent manner. The presence of VGluT3 mRNA on corresponding human brain areas suggests clinical relevance.
Topics: Animals; Male; Social Behavior; Mice, Knockout; Humans; Anxiety; Raphe Nuclei; Mice; Neurons; Mice, Inbred C57BL; Behavior, Animal; Mice, Transgenic; Amino Acid Transport Systems, Acidic; Proto-Oncogene Proteins c-fos; Aggression
PubMed: 38839305
DOI: 10.1523/ENEURO.0332-23.2024 -
BioRxiv : the Preprint Server For... May 2024Somatic mosaicism is a hallmark of malignancy that is also pervasively observed in human physiological aging, with clonal expansions of cells harboring mutations in...
Somatic mosaicism is a hallmark of malignancy that is also pervasively observed in human physiological aging, with clonal expansions of cells harboring mutations in recurrently mutated driver genes. Bulk sequencing of tissue microdissection captures mutation frequencies, but cannot distinguish which mutations co-occur in the same clones to reconstruct clonal architectures, nor phenotypically profile clonal populations to delineate how driver mutations impact cellular behavior. To address these challenges, we developed single-cell Genotype-to-Phenotype sequencing (scG2P) for high-throughput, highly-multiplexed, single-cell joint capture of recurrently mutated genomic regions and mRNA phenotypic markers in cells or nuclei isolated from solid tissues. We applied scG2P to aged esophagus samples from five individuals with high alcohol and tobacco exposure and observed a clonal landscape dominated by a large number of clones with a single driver event, but only rare clones with two driver mutations. mutants dominate the clonal landscape and are linked to stunted epithelial differentiation, while mutants and double-driver mutants promote clonal expansion through both differentiation biases and increased cell cycling. Thus, joint single-cell highly multiplexed capture of somatic mutations and mRNA transcripts enables high resolution reconstruction of clonal architecture and associated phenotypes in solid tissue somatic mosaicism.
PubMed: 38826366
DOI: 10.1101/2024.05.22.595241 -
BioRxiv : the Preprint Server For... May 2024TRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and...
RATIONALE
TRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and tissue fibrosis. These are key features in the pathophysiology of lymphatic system diseases, including lymphedema and lipedema; however, the role of TRPV4 channels in the lymphatic system remains largely unexplored. TRPV4 channels are calcium permeable, non-selective cation channels that are activated by diverse stimuli, including shear stress, stretch, temperature, and cell metabolites, which may regulate lymphatic contractile function.
OBJECTIVE
To characterize the expression of TRPV4 channels in collecting lymphatic vessels and to determine the extent to which these channels regulate the contractile function of lymphatics.
METHODS AND RESULTS
Pressure myography on intact, isolated, and cannulated lymphatic vessels showed that pharmacological activation of TRPV4 channels with GSK1016790A (GSK101) led to contractile dysregulation. The response to GSK101 was multiphasic and included, 1) initial robust constriction that was sustained for ≥1 minute and in some instances remained for ≥4 minutes; and 2) subsequent vasodilation and partial or complete inhibition of lymphatic contractions associated with release of nitric oxide. The functional response to activation of TRPV4 channels displayed differences across lymphatics from four anatomical regions, but these differences were consistent across different species (mouse, rat, and non-human primate). Importantly, similar responses were observed following activation of TRPV4 channels in arterioles. The initial and sustained constriction was prevented with the COX inhibitor, indomethacin. We generated a and single-cell RNA sequencing (scRNAseq) dataset from intact and microdissected collecting lymphatic vessels. Our data uncovered a subset of macrophages displaying the highest expression of compared to other cell types within and surrounding the lymphatic vessel wall. These macrophages displayed a transcriptomic profile consistent with that of tissue-resident macrophages (TRMs), including differential expression of , , , , , , , , and ; and at least half of these macrophages also expressed This subset of macrophages also highly expressed , which encodes the thromboxane A2 (TXA2) synthase. Inhibition of TXA2 receptors (TXA2Rs) prevented TRPV4-mediated contractile dysregulation. TXA2R activation on LMCs caused an increase in mobilization of calcium from intracellular stores through Ip3 receptors which promoted store operated calcium entry and vasoconstriction.
CONCLUSIONS
Clinical studies have linked cancer-related lymphedema with an increased infiltration of macrophages. While these macrophages have known anti-inflammatory and pro-lymphangiogenic roles, as well as promote tissue repair, our results point to detrimental effects to the pumping capacity of collecting lymphatic vessels mediated by activation of TRPV4 channels in macrophages. Pharmacological targeting of TRPV4 channels in LYVE1-expressing macrophages or pharmacological targeting of TXA2Rs may offer novel therapeutic strategies to improve lymphatic pumping function and lymph transport in lymphedema.
PubMed: 38826322
DOI: 10.1101/2024.05.21.595189 -
The American Journal of Case Reports May 2024BACKGROUND Aside from the rarity of mobile spinal schwannomas, the coexistence of these tumors with herniated intervertebral disc is also scarce. Furthermore, cauda...
BACKGROUND Aside from the rarity of mobile spinal schwannomas, the coexistence of these tumors with herniated intervertebral disc is also scarce. Furthermore, cauda equina syndrome (CES), as a manifestation of intraspinal schwannomas has been reported rarely. Described here is a case of simultaneous lumbar disc bulge and mobile spinal schwannoma presented with intermittent symptoms of CES. CASE REPORT A 62-year-old man presented with severe but intermittent leg pain for 2 weeks, which later progressed to an episode of lower extremity weakness and difficulty in urination. Magnetic resonance imaging revealed an intraspinal tumor that moved in position relative to the L1-2 disc bulge on scans 6 h apart, with associated spontaneous regression in symptoms. The tumor was found to be a mobile spinal schwannoma, originated from a nerve root. A standard microdissection technique was used to remove the tumor through a spinous process-sparing unilateral approach, with complete laminectomy of L1. Use of intraoperative ultrasound facilitated the accurate tumor localization. Postoperatively, the patient no longer had symptoms. CONCLUSIONS This report presents a combination of a common spinal pathology, intervertebral disc herniation, alongside a rare condition, mobile spinal schwannoma, whose uncommon clinical manifestations, such as CES can cause irreversible neurological deficits. Surgeons need to remain vigilant of potential atypical scenarios when treating patients. Surgical treatment challenges regarding the mobility of tumors, such as accurate localization, should be addressed using intraoperative imaging to avoid wrong-level surgery. To mitigate the irreversible neurological complications, patients should receive comprehensive information for alarming signs of CES.
Topics: Humans; Male; Neurilemmoma; Middle Aged; Cauda Equina Syndrome; Intervertebral Disc Displacement; Lumbar Vertebrae; Magnetic Resonance Imaging; Spinal Neoplasms; Spinal Cord Neoplasms
PubMed: 38794785
DOI: 10.12659/AJCR.942717 -
Cells May 2024The oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is...
The oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is crucial to maintaining them in a healthy and stable condition. In this study, for the first time, bulk RNA-seq analysis was performed to explore the gene expression of laser microdissected epithelium and lamina propria from mice, aiming to investigate the differences between keratinized and non-keratinized oral mucosa. Based on the differentially expressed genes (DEGs) and Gene Ontology (GO) Enrichment Analysis, bone morphogenetic protein 2 (BMP-2) was identified to be a potential regulator of oral mucosal keratinization. Monoculture and epithelial-mesenchymal cell co-culture models in the air-liquid interface (ALI) indicated that BMP-2 has direct and positive effects on epithelial keratinization and proliferation. We further performed bulk RNA-seq of the ALI monoculture stimulated with BMP-2 in an attempt to identify the downstream factors promoting epithelial keratinization and proliferation. Analysis of the DEGs identified, among others, , , , , , , and as key factors. In summary, these results revealed the involvement of a well-known growth factor responsible for bone development, BMP-2, in the mechanism of oral mucosal keratinization and proliferation, and pointed out the possible downstream genes involved in this mechanism.
Topics: Bone Morphogenetic Protein 2; Mouth Mucosa; Animals; Mice; Keratins; Cell Proliferation; Gene Expression Regulation; Humans; Gene Ontology
PubMed: 38786031
DOI: 10.3390/cells13100807