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Fertility and Sterility Oct 2023To explore the dynamic transcriptional regulatory network of primordial follicle fate in obese mice to elucidate the potential mechanism of primordial follicle depletion.
OBJECTIVE
To explore the dynamic transcriptional regulatory network of primordial follicle fate in obese mice to elucidate the potential mechanism of primordial follicle depletion.
DESIGN
Experimental study and transcriptomic analysis.
ANIMALS
Healthy (n=15) and obese (n=15) female mice.
INTERVENTIONS
Six-week-old CD-1 mice were divided into healthy and high-fat diet groups and fed continuously for 12 weeks. The diet of healthy mice contained 10% fat. The diet of high-fat mice contained 60% fat.
MAIN OUTCOME MEASURES
Primordial to primary follicle transition rate, gene expression changes, enriched Kyoto Encyclopedia of Genes and Genomes pathways, and ferroptosis.
RESULTS
Primordial follicle depletion was increased in the ovaries of obese mice. We found that deposited fat around primordial and primary follicles of obese mice was higher than that for healthy mice. The proliferation of granulosa cells around primary follicles was increased in obese mice. In addition, we uncovered specific gene signatures associated with the primordial to primary follicle transition (PPT) in obese mice using laser capture microdissection RNA sequencing analysis. Gene set enrichment analysis indicated that ferroptosis, cell oxidation, vascular endothelial growth factor, and mammalian target of rapamycin signaling were increased significantly in the primordial follicles of obese mice. Notably, the ferritin, acyl CoA synthetase long-chain family member 4, and solid carrier family 7 member 11 associated proteins of the ferroptosis signaling pathway were significantly increased in the PPT phase of obese mice.
CONCLUSION
Our work suggests that ferroptosis is a key pathway activated within immature ovarian follicles in the context of obesity and that the process may be involved in the physiological regulation of the PPT as well.
Topics: Female; Animals; Mice; Transcriptome; Mice, Obese; Vascular Endothelial Growth Factor A; Ovarian Follicle; Granulosa Cells; Mammals
PubMed: 37247688
DOI: 10.1016/j.fertnstert.2023.05.165 -
Stroke and Vascular Neurology Dec 2023Extra-axial cavernous hemangiomas (ECHs) are sporadic and rare intracranial occupational lesions that usually occur within the cavernous sinus. The aetiology of ECHs...
OBJECTIVE
Extra-axial cavernous hemangiomas (ECHs) are sporadic and rare intracranial occupational lesions that usually occur within the cavernous sinus. The aetiology of ECHs remains unknown.
METHODS
Whole-exome sequencing was performed on ECH lesions from 12 patients (discovery cohort) and droplet digital polymerase-chain-reaction (ddPCR) was used to confirm the identified mutation in 46 additional cases (validation cohort). Laser capture microdissection (LCM) was carried out to capture and characterise subgroups of tissue cells. Mechanistic and functional investigations were carried out in human umbilical vein endothelial cells and a newly established mouse model.
RESULTS
We detected somatic mutation (c.121G>T, p.G41C) in 5/12 patients with ECH in the discovery cohort and confirmed the finding in the validation cohort (16/46). LCM followed by ddPCR revealed that the mutation was enriched in lesional endothelium. In vitro experiments in endothelial cells demonstrated that the mutation activated SGK-1 signalling that in turn upregulated key genes involved in cell hyperproliferation and the loss of arterial specification. Compared with wild-type littermates, mice overexpressing the mutation developed ECH-like pathological morphological characteristics (dilated venous lumen and elevated vascular density) in the retinal superficial vascular plexus at the postnatal 3 weeks, which were reversed by an SGK1 inhibitor, EMD638683.
CONCLUSIONS
We identified a somatic mutation that presents in over one-third of ECH lesions and proposed that ECHs are vascular malformations due to -induced activation of the SGK1 signalling pathway in brain endothelial cells.
Topics: Humans; Animals; Mice; Endothelial Cells; Hemangioma, Cavernous, Central Nervous System; Hemangioma, Cavernous; Mutation; Signal Transduction
PubMed: 37072338
DOI: 10.1136/svn-2022-002227 -
Journal of Dermatological Science Nov 2023Human scalp hair follicles (HFs) engage in olfactory receptor (OR)-dependent chemosensation. Activation of olfactory receptor family 2 subfamily AT member 4 (OR2AT4) by...
BACKGROUND
Human scalp hair follicles (HFs) engage in olfactory receptor (OR)-dependent chemosensation. Activation of olfactory receptor family 2 subfamily AT member 4 (OR2AT4) by the synthetic, sandalwood-like odorant Sandalore® up-regulated HF antimicrobial peptide expression of dermcidin (DCD), which had previously been thought to be produced exclusively by sweat and sebaceous glands.
OBJECTIVES
To understand if intrafollicular DCD production can be stimulated by a commonly used cosmetic odorant, thus altering human HF microbiome composition in a clinically beneficial manner.
METHODS
DCD expression was compared between fresh-frozen scalp biopsies and microdissected, full-length scalp HFs, organ-cultured in the presence/absence of the OR2AT4 agonist, Sandalore® and/or antibiotics and/or the competitive OR2AT4 antagonist, Phenirat®. Amplicon-based sequencing and microbial growth assays were performed to assess how this treatment affected the HF microbiome.
RESULTS
Synthetic odorant treatment upregulated epithelial DCD expression and exerted antimicrobial activity in human HFs ex vivo. Combined antibiotic and odorant treatment, during an ex vivo dysbiosis event, prevented HF tissue damage and favoured a more physiological microbiome composition. Sandalore®-conditioned medium, containing higher DCD content, favoured Staphylococcus epidermidis and Malassezia restricta over S. aureus and M. globosa, while exhibiting antimicrobial activity against Cutibacterium acnes. These effects were reversed by co-administration of Phenirat®.
CONCLUSIONS
We provide the first proof-of-principle that a cosmetic odorant impacts the human HF microbiome by up-regulating antimicrobial peptide production in an olfactory receptor-dependent manner. Specifically, a synthetic sandalwood-like odorant stimulates intrafollicular DCD production, likely via OR2AT4, and thereby controls microbial overgrowth. Thus, deserving further exploration as an adjuvant therapeutic principle in the management of folliculitis and dysbiosis-associated hair diseases.
Topics: Humans; Hair Follicle; Odorants; Staphylococcus aureus; Dysbiosis; Receptors, Odorant; Antimicrobial Peptides; Anti-Infective Agents
PubMed: 37858476
DOI: 10.1016/j.jdermsci.2023.09.006 -
Analytical and Bioanalytical Chemistry Apr 2024Success of mass spectrometry characterization of the proteome of single cells allows us to gain a greater understanding than afforded by transcriptomics alone but... (Review)
Review
Success of mass spectrometry characterization of the proteome of single cells allows us to gain a greater understanding than afforded by transcriptomics alone but requires clear understanding of the tradeoffs between analytical throughput and precision. Recent advances in mass spectrometry acquisition techniques, including updated instrumentation and sample preparation, have improved the quality of peptide signals obtained from single cell data. However, much of the proteome remains uncharacterized, and higher throughput techniques often come at the expense of reduced sensitivity and coverage, which diminish the ability to measure proteoform heterogeneity, including splice variants and post-translational modifications, in single cell data analysis. Here, we assess the growing body of ultrasensitive single-cell approaches and their tradeoffs as researchers try to balance throughput and precision in their experiments.
Topics: Proteome; Proteomics; Peptides; Mass Spectrometry; Protein Processing, Post-Translational
PubMed: 38358530
DOI: 10.1007/s00216-024-05171-6 -
British Journal of Pharmacology Sep 2023Complement activation may drive hypertension through its effects on immunity and tissue integrity.
BACKGROUND AND PURPOSE
Complement activation may drive hypertension through its effects on immunity and tissue integrity.
EXPERIMENTAL APPROACH
We examined expression of C3, the central protein of the complement cascade, in hypertension.
KEY RESULTS
Increased C3 expression was found in kidney biopsies and micro-dissected glomeruli of patients with hypertensive nephropathy. Renal single cell RNA sequence data from normotensive and hypertensive patients confirmed expression of C3 in different cellular compartments of the kidney. In angiotensin II (Ang II) induced hypertension renal C3 expression was up-regulated. C3 mice revealed a significant lower albuminuria in the early phase of hypertension. However, no difference was found for blood pressure, renal injury (histology, glomerular filtration rate, inflammation) and cardiac injury (fibrosis, weight, gene expression) between C3 and wildtype mice after Ang II infusion. Also, in deoxycorticosterone acetate (DOCA) salt hypertension, a significantly lower albuminuria was found in the first weeks of hypertension in C3 deficient mice but no significant difference in renal and cardiac injury. Down-regulation of C3 by C3 targeting GalNAc (n-acetylgalactosamine) small interfering RNA (siRNA) conjugate decreased C3 in the liver by 96% and lowered albuminuria in the early phase but showed no effect on blood pressure and end-organ damage. Inhibition of complement C5 by siRNA showed no effect on albuminuria.
CONCLUSION AND IMPLICATIONS
Increased C3 expression is found in the kidneys of hypertensive mice and men. Genetic and therapeutic knockdown of C3 improved albuminuria in the early phase of hypertension but did not ameliorate arterial blood pressure nor renal and cardiac injury.
Topics: Animals; Mice; Albuminuria; Hypertension, Renal; Kidney; Hypertension; Blood Pressure; Angiotensin II; RNA, Small Interfering
PubMed: 37076314
DOI: 10.1111/bph.16097 -
Cancer Research Communications Oct 2023Intraductal papillary mucinous neoplasms (IPMN) are cystic precursor lesions to pancreatic ductal adenocarcinoma (PDAC). IPMNs undergo multistep progression from...
UNLABELLED
Intraductal papillary mucinous neoplasms (IPMN) are cystic precursor lesions to pancreatic ductal adenocarcinoma (PDAC). IPMNs undergo multistep progression from low-grade (LG) to high-grade (HG) dysplasia, culminating in invasive neoplasia. While patterns of IPMN progression have been analyzed using multiregion sequencing for somatic mutations, there is no integrated assessment of molecular events, including copy-number alterations (CNA) and transcriptional changes that accompany IPMN progression. We performed laser capture microdissection on surgically resected IPMNs of varying grades of histologic dysplasia obtained from 23 patients, followed by whole-exome and whole-transcriptome sequencing. Overall, HG IPMNs displayed a significantly greater aneuploidy score than LG lesions, with chromosome 1q amplification being associated with HG progression and with cases that harbored co-occurring PDAC. Furthermore, the combined assessment of single-nucleotide variants (SNV) and CNAs identified both linear and branched evolutionary trajectories, underscoring the heterogeneity in the progression of LG lesions to HG and PDAC. At the transcriptome level, upregulation of MYC-regulated targets and downregulation of transcripts associated with the MHC class I antigen presentation machinery as well as pathways related to glycosylation were a common feature of progression to HG. In addition, the established PDAC transcriptional subtypes (basal-like and classical) were readily apparent within IPMNs. Taken together, this work emphasizes the role of 1q copy-number amplification as a putative biomarker of high-risk IPMNs, underscores the importance of immune evasion even in noninvasive precursor lesions, and reinforces that evolutionary pathways in IPMNs are heterogenous, comprised of both SNV and CNA-driven events.
SIGNIFICANCE
Integrated molecular analysis of genomic and transcriptomic alterations in the multistep progression of IPMNs, which are bona fide precursors of pancreatic cancer, identifies features associated with progression of low-risk lesions to high-risk lesions and cancer, which might enable patient stratification and cancer interception strategies.
Topics: Humans; Pilot Projects; Pancreatic Intraductal Neoplasms; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Neoplasms, Cystic, Mucinous, and Serous
PubMed: 37721516
DOI: 10.1158/2767-9764.CRC-22-0419 -
Journal of Translational Medicine Feb 2024Combined small-cell lung carcinoma (cSCLC) represents a rare subtype of SCLC, the mechanisms governing the evolution of cancer genomes and their impact on the tumor...
BACKGROUND
Combined small-cell lung carcinoma (cSCLC) represents a rare subtype of SCLC, the mechanisms governing the evolution of cancer genomes and their impact on the tumor immune microenvironment (TIME) within distinct components of cSCLC remain elusive.
METHODS
Here, we conducted whole-exome and RNA sequencing on 32 samples from 16 cSCLC cases.
RESULTS
We found striking similarities between two components of cSCLC-LCC/LCNEC (SCLC combined with large-cell carcinoma/neuroendocrine) in terms of tumor mutation burden (TMB), tumor neoantigen burden (TNB), clonality structure, chromosomal instability (CIN), and low levels of immune cell infiltration. In contrast, the two components of cSCLC-ADC/SCC (SCLC combined with adenocarcinoma/squamous-cell carcinoma) exhibited a high level of tumor heterogeneity. Our investigation revealed that cSCLC originated from a monoclonal source, with two potential transformation modes: from SCLC to SCC (mode 1) and from ADC to SCLC (mode 2). Therefore, cSCLC might represent an intermediate state, potentially evolving into another histological tumor morphology through interactions between tumor and TIME surrounding it. Intriguingly, RB1 inactivation emerged as a factor influencing TIME heterogeneity in cSCLC, possibly through neoantigen depletion.
CONCLUSIONS
Together, these findings delved into the clonal origin and TIME heterogeneity of different components in cSCLC, shedding new light on the evolutionary processes underlying this enigmatic subtype.
Topics: Humans; Lung Neoplasms; Microdissection; Small Cell Lung Carcinoma; Adenocarcinoma; Carcinoma, Large Cell; Genomics; Tumor Microenvironment
PubMed: 38383412
DOI: 10.1186/s12967-024-04968-4 -
Acta Medica Indonesiana Oct 2023Colorectal cancer (CRC) is a significant contributor to cancer-related morbidity and mortality. Biopsy remains the gold standard for CRC diagnosis, but invasive testing...
BACKGROUND
Colorectal cancer (CRC) is a significant contributor to cancer-related morbidity and mortality. Biopsy remains the gold standard for CRC diagnosis, but invasive testing may not be preferred as an initial diagnostic procedure. Therefore, alternative non-invasive approaches are needed. Circulating tumor cells (CTC) present in the bloodstream have great potential as a non-invasive diagnostic marker for CRC patients. This study aimed to assess the diagnostic potential of CTC in CRC as an adjunctive diagnostic method using a subjective manual identification method and laser capture microdissection at 40x magnification.
METHODS
A cross-sectional study was conducted on adult patients suspected to have CRC at Dr. Cipto Mangunkusumo National General Hospital, Jakarta, between November 2020 and March 2021. CTC analysis was performed using the negative selection immunomagnetic method with Easysep™ and the CD44 mesenchymal tumor marker. The identification and quantification of CTC were conducted manually and subjectively, with three repetitions of cell counting per field of view at 40x magnification.
RESULTS
Of 80 subjects, 77.5% were diagnosed with CRC, while 7.5% and 15% exhibited adenomatous polyps and inflammatory/hyperplastic polyps, respectively. The diagnostic analysis of CTC for detecting CRC (compared to polyps) using a CTC cutoff point of >1.5 cells/mL suggested sensitivity, specificity, and positive predictive value (PPV) of 50%, 88.89%, and 93.94%. Additionally, the negative predictive value (NPV), as well as the positive and negative likelihood ratio (PLR and NLR) were 34.04%, 4.5, and 0.56, respectively. The subjective manual identification and quantification of CTC were performed at 40x magnification using laser capture microdissection.
CONCLUSION
This study assessed the diagnostic potential of CTC examination in CRC as an adjunctive diagnostic method using the subjective manual identification method and laser capture microdissection at 40x magnification. Despite the limitations associated with subjective cell counting, the results showed 50% sensitivity and 88.89% specificity in diagnosing CRC. Further studies are needed to optimize the manual identification process and validate the clinical utility of CTC analysis in CRC patients.
Topics: Adult; Humans; Colonography, Computed Tomographic; Neoplastic Cells, Circulating; Cross-Sectional Studies; Colorectal Neoplasms; Predictive Value of Tests
PubMed: 38213054
DOI: No ID Found -
Frontiers in Pharmacology 2023Bitter taste receptors are involved not only in taste perception but in various physiological functions as their anatomical location is not restricted to the gustatory...
Bitter taste receptors are involved not only in taste perception but in various physiological functions as their anatomical location is not restricted to the gustatory system. We previously demonstrated expression and activity of the subtype hTAS2R46 in human airway smooth muscle and broncho-epithelial cells, and here we show its expression and functionality in human skeletal muscle cells. Three different cellular models were used: micro-dissected human skeletal tissues, human myoblasts/myotubes and human skeletal muscle cells differentiated from urine stem cells of healthy donors. We used qPCR, immunohistochemistry and immunofluorescence analysis to evaluate gene and protein hTAS2R46 expression. In order to explore receptor activity, cells were incubated with the specific bitter ligands absinthin and 3ß-hydroxydihydrocostunolide, and calcium oscillation and relaxation were evaluated by calcium imaging and collagen assay, respectively, after a cholinergic stimulus. We show, for the first time, experimentally the presence and functionality of a type 2 bitter receptor in human skeletal muscle cells. Given the tendentially protective role of the bitter receptors starting from the oral cavity and following also in the other ectopic sites, and given its expression already at the myoblast level, we hypothesize that the bitter receptor can play an important role in the development, maintenance and in the protection of muscle tissue functions.
PubMed: 37771728
DOI: 10.3389/fphar.2023.1205651 -
The Journal of Biological Chemistry Sep 2023Single-cell transcriptomics are powerful tools to define neuronal cell types based on co-expressed gene clusters. Limited RNA input in these technologies necessarily...
Single-cell transcriptomics are powerful tools to define neuronal cell types based on co-expressed gene clusters. Limited RNA input in these technologies necessarily compromises transcriptome coverage and accuracy of differential expression analysis. We propose that bulk RNA-Seq of neuronal pools defined by spatial position offers an alternative strategy to overcome these technical limitations. We report a laser-capture microdissection (LCM)-Seq method which allows deep transcriptome profiling of fluorescently tagged neuron populations isolated with LCM from histological sections of transgenic mice. Mild formaldehyde fixation of ZsGreen marker protein, LCM sampling of ∼300 pooled neurons, followed by RNA isolation, library preparation and RNA-Seq with methods optimized for nanogram amounts of moderately degraded RNA enabled us to detect ∼15,000 different transcripts in fluorescently labeled cholinergic neuron populations. The LCM-Seq approach showed excellent accuracy in quantitative studies, allowing us to detect 2891 transcripts expressed differentially between the spatially defined and clinically relevant cholinergic neuron populations of the dorsal caudate-putamen and medial septum. In summary, the LCM-Seq method we report in this study is a versatile, sensitive, and accurate bulk sequencing approach to study the transcriptome profile and differential gene expression of fluorescently tagged neuronal populations isolated from transgenic mice with high spatial precision.
PubMed: 37536628
DOI: 10.1016/j.jbc.2023.105121