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Bone marrow aspirate concentrate (BMAC) and adipose-derived stromal vascular fraction (ADSVF) are the most marketed stem cell therapies to treat a variety of conditions in the general population and elite athletes. Both tissues have been used interchangeably clinically even though their detailed composition, heterogeneity, and mechanisms of action have neither been rigorously inventoried nor compared. This lack of information has prevented investigations into ideal dosages and has facilitated anecdata and misinformation. Here, we analyzed single-cell transcriptomes, proteomes, and flow cytometry profiles from paired clinical-grade BMAC and ADSVF. This comparative transcriptional atlas challenges the prevalent notion that there is one therapeutic cell type present in both tissues. We also provide data of surface markers that may enable isolation and investigation of cell (sub)populations. Furthermore, the proteome atlas highlights intertissue and interpatient heterogeneity of injected proteins with potentially regenerative or immunomodulatory capacities. An interactive webtool is available online.
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Células-Tronco Mesenquimais , Proteoma , Proteômica , Análise de Célula Única , Humanos , Proteômica/métodos , Proteoma/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Análise de Célula Única/métodos , Tecido Adiposo/metabolismo , Transcriptoma , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Perfilação da Expressão GênicaRESUMO
BACKGROUND: Ménière's disease (MD) is a disorder of the inner ear that causes episodic bouts of severe dizziness, roaring tinnitus, and fluctuating hearing loss. To date, no targeted therapy exists. As such, we have undertaken a large whole genome sequencing study on carefully phenotyped unilateral MD patients with the goal of gene/pathway discovery and a move towards targeted intervention. This study was a retrospective review of patients with a history of Ménière's disease. Genomic DNA, acquired from saliva samples, was purified and subjected to whole genome sequencing. RESULTS: Stringent variant calling, performed on 511 samples passing quality checks, followed by gene-based filtering by recurrence and proximity in molecular interaction networks, led to 481 high priority MD genes. These high priority genes, including MPHOSPH8, MYO18A, TRIOBP, OTOGL, TNC, and MYO6, were previously implicated in hearing loss, balance, and cochlear function, and were significantly enriched in common variant studies of hearing loss. Validation in an independent MD cohort confirmed 82 recurrent genes. Pathway analysis pointed to cell-cell adhesion, extracellular matrix, and cellular energy maintenance as key mediators of MD. Furthermore, the MD-prioritized genes were highly expressed in human inner ear hair cells and dark/vestibular cells, and were differentially expressed in a mouse model of hearing loss. CONCLUSION: By enabling the development of model systems that may lead to targeted therapies and MD screening panels, the genes and variants identified in this study will inform diagnosis and treatment of MD.
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Hidropisia Endolinfática , Genômica , Doença de Meniere , Doença de Meniere/genética , Humanos , Hidropisia Endolinfática/genética , Animais , Camundongos , Masculino , Feminino , Estudos Retrospectivos , Sequenciamento Completo do Genoma , Pessoa de Meia-Idade , AdultoRESUMO
Salivary glands have essential roles in maintaining oral health, mastication, taste and speech, by secreting saliva. Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including adenoid cystic carcinoma (ACC), acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In our study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2 and myoepithelial cells/stromal cells. The localization of each cell group was verified by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Furthermore, in comparison with SGC RNA-seq data, acinar cell markers were upregulated in AciCC, but downregulated in ACC and ductal cell markers were upregulated in SDC but downregulated in MECA, suggesting that markers of origin are highly expressed in some SGC. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship.
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Carcinoma de Células Acinares , Carcinoma Adenoide Cístico , Carcinoma , Neoplasias das Glândulas Salivares , Humanos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Glândulas Salivares/patologia , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Carcinoma Adenoide Cístico/patologia , Carcinoma/patologia , Carcinoma de Células Acinares/metabolismo , RNA/metabolismoRESUMO
INTRODUCTION: Mortality from preeclampsia (PE) and PE-associated morbidities are 3-to 5-fold higher in persons of African ancestry than in those of Asian and European ancestries. METHODS: To elucidate placental contribution to worse PE outcomes in African ancestry pregnancies, we performed bulk RNA sequencing on 50 placentas from persons with severe PE (sPE) of African (n = 9), Asian (n = 18) and European (n = 23) ancestries and 73 normotensive controls of African (n = 10), Asian (n = 15) and European (n = 48) ancestries. RESULTS: Previously described canonical preeclampsia genes, involved in metabolism and hypoxia/angiogenesis including: LEP, HK2, FSTL3, FLT1, ENG, TMEM45A, ARHGEF4 and HTRA1 were upregulated sPE versus normotensive placentas across ancestries. LTF, NPR3 and PHYHIP were higher in African vs. Asian ancestry sPE placentas. Allograft rejection/adaptive immune response genes were upregulated in placentas from African but not in Asian or European ancestry sPE patients; IL3RA was of particular interest because the patient with the highest placental IL3RA expression, a person of African ancestry with sPE, developed postpartum cardiomyopathy, and was the only patient out of 123, that developed this condition. Interestingly, the sPE patients with the highest IL3RA expression among persons of Asian and European ancestries developed unexplained tachycardia peripartum, necessitating echocardiography in the European ancestry patient. The association between elevated placental IL3RA levels and unexplained tachycardia or peripartum cardiomyopathy was found to be significant in the 50 sPE patients (p = .0005). DISCUSSION: High placental upregulation of both canonical preeclampsia and allograft rejection/adaptive immune response genes may contribute to worse PE outcomes in African ancestry sPE patients.
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Placenta , Pré-Eclâmpsia , Feminino , Humanos , Gravidez , Pressão Sanguínea , Cardiomiopatias/complicações , Cardiomiopatias/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Taquicardia/complicações , Taquicardia/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Perfilação da Expressão GênicaRESUMO
OBJECTIVE: Despite great advances in obesity therapeutics in recent years, there is still a need to identify additional therapeutic targets for the treatment of this disease. We previously discovered a signature of genes, including Chloride intracellular channel 1 (Clic1), whose expression was associated with drug-induced weight gain, and in these studies, we assess the effect of Clic1 inhibition on food intake and body weight in mice. METHODS: We studied the impact of Clic1 inhibition in mouse models of binge-eating, diet-induced obese mice and genetic models of obesity (Magel2 KO mice). RESULTS: Clic1 knockout (KO) mice ate significantly less and had a lower body weight than WT littermates when either fed chow or high fat diet. Furthermore, pharmacological inhibition of Clic1 in diet-induced obese mice resulted in suppression of food intake and promoted highly efficacious weight loss. Clic1 inhibition also reduced food intake in binge-eating models and hyperphagic Magel2 KO mice. We observed that chronic obesity resulted in a significant change in subcellular localization of Clic1 with an increased ratio of Clic1 in the membrane in the obese state. These observations provide a novel therapeutic strategy to block Clic1 translocation as a potential mechanism to reduce food intake and lower body weight. CONCLUSIONS: These studies attribute a novel role of Clic1 as a driver of food intake and overconsumption. In summary, we have identified hypothalamic expression of Clic1 plays a key role in food intake, providing a novel therapeutic target to treat overconsumption that is the root cause of modern obesity.
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Obesidade , Aumento de Peso , Animais , Camundongos , Camundongos Obesos , Peso Corporal , Camundongos Knockout , Ingestão de Alimentos , Canais de Cloreto/genética , Antígenos de Neoplasias , ProteínasRESUMO
BACKGROUND: Anorexia nervosa (AN) is a complex debilitating disease characterized by intense fear of weight gain and excessive exercise. It is the deadliest of any psychiatric disorder with a high rate of recidivism, yet its pathophysiology is unclear. The Activity-Based Anorexia (ABA) paradigm is a widely accepted mouse model of AN that recapitulates hypophagia and hyperactivity despite reduced body weight, however, not the chronicity. METHODS: Here, we modified the prototypical ABA paradigm to increase the time to lose 25% of baseline body weight from less than 7 days to more than 2 weeks. We used this paradigm to identify persistently altered genes after weight restoration that represent a transcriptomic memory of under-nutrition and may contribute to AN relapse using RNA sequencing. We focused on adipose tissue as it was identified as a major location of transcriptomic memory of over-nutririon. RESULTS: We identified 300 dysregulated genes that were refractory to weight restroration after ABA, including Calm2 and Vps13d, which could be potential global regulators of transcriptomic memory in both chronic over- and under-nutrition. CONCLUSION: We demonstrated the presence of peristent changes in the adipose tissue transcriptome in the ABA mice after weight restoration. Despite being on the opposite spectrum of weight perturbations, majority of the transcriptomic memory genes of under- and over-nutrition did not overlap, suggestive of the different mechanisms involved in these extreme nutritional statuses.
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Anorexia Nervosa , Desnutrição , Camundongos , Animais , Anorexia Nervosa/genética , Transcriptoma , Peso Corporal , Tecido Adiposo , Modelos Animais de DoençasRESUMO
Introduction: Rotator cuff tears are prevalent in the population above the age of 60. The disease progression leads to muscle atrophy, fibrosis, and fatty infiltration, which is not improved upon with surgical repair, highlighting the need to better understand the underlying biology impairing more favorable outcomes. Methods: In this study, we collected supraspinatus muscle tissue from 6 month old female rabbits who had undergone unilateral tenotomy for 8 weeks at 1, 2, 4, or 8 weeks post-repair (n = 4/group). RNA sequencing and enrichment analyses were performed to identify a transcriptional timeline of rotator cuff muscle adaptations and related morphological sequelae. Results: There were differentially expressed (DE) genes at 1 (819 up/210 down), 2 (776/120), and 4 (63/27) weeks post-repair, with none at 8 week post-repair. Of the time points with DE genes, there were 1092 unique DE genes and 442 shared genes, highlighting that there are changing processes in the muscle at each time point. Broadly, 1-week post-repair differentially expressed genes were significantly enriched in pathways of metabolism and energetic activity, binding, and regulation. Many were also significantly enriched at 2 weeks, with the addition of NIF/NF-kappaB signaling, transcription in response to hypoxia, and mRNA stability alongside many additional pathways. There was also a shift in transcriptional activity at 4 weeks post-repair with significantly enriched pathways for lipids, hormones, apoptosis, and cytokine activity, despite an overall decrease in the number of differentially expressed genes. At 8 weeks post-repair there were no DE genes when compared to control. These transcriptional profiles were correlated with the histological findings of increased fat, degeneration, and fibrosis. Specifically, correlated gene sets were enriched for fatty acid metabolism, TGF-B-related, and other pathways. Discussion: This study identifies the timeline of transcriptional changes in muscle after RC repair, which by itself, does not induce a growth/regenerative response as desired. Instead, it is predominately related to metabolism/energetics changes at 1 week post-repair, unclear or asynchronous transcriptional diversity at 2 weeks post-repair, increased adipogenesis at 4 weeks post-repair, and a low transcriptional steady state or a dysregulated stress response at 8 weeks post-repair.
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BACKGROUND: Mechanisms of iron clearance from hemophilic joints are unknown. OBJECTIVES: To better understand mechanisms of iron clearance following joint bleeding in a mouse model of hemophilia. METHODS: Hemarthrosis was induced by subpatellar puncture in factor VIII (FVIII)-deficient (FVII-/-) mice, +/- periprocedural recombinant human FVIII, and hypocoagulable (HypoBALB/c) mice. HypoBALB/c mice experienced transient FVIII deficiency (anti-FVIII antibody) at the time of injury combined with warfarin-induced hypocoagulability. Synovial tissue was harvested weekly up to 6 weeks after injury for histological analysis, ferric iron and macrophage accumulation (CD68), blood and lymphatic vessel remodeling (αSMA; LYVE1). Synovial RNA sequencing was performed for FVIII-/- mice at days 0, 3, and 14 after injury to quantify expression changes of iron regulators and lymphatic markers. RESULTS: Bleed volumes were similar in FVIII-/- and HypoBALB/c mice. However, pronounced and prolonged synovial iron accumulation colocalizing with macrophages and impaired lymphangiogenesis were detected only in FVIII-/- mice and were prevented by periprocedural FVIII. Gene expression changes involved in iron handling (some genes with dual roles in inflammation) and lymphatic markers supported proinflammatory milieu with iron retention and disturbed lymphangiogenesis. CONCLUSION: Accumulation and delayed clearance of iron-laden macrophages were associated with defective lymphangiogenesis after hemarthrosis in FVIII-/- mice. The absence of such findings in HypoBALB/c mice suggests that intact lymphatics are required for removal of iron-laden macrophages and that these processes depend on FVIII availability. Studies to elucidate the biological mechanisms of disturbed lymphangiogenesis in hemophilia appear critical to develop new therapeutic targets.
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Hemofilia A , Hemostáticos , Camundongos , Humanos , Animais , Fator VIII/genética , Fator VIII/metabolismo , Hemartrose/metabolismo , Hemofilia A/terapia , Linfangiogênese , Modelos Animais de Doenças , FerroRESUMO
Background: Schools are high-risk settings for SARS-CoV-2 transmission, but necessary for children's educational and social-emotional wellbeing. Previous research suggests that wastewater monitoring can detect SARS-CoV-2 infections in controlled residential settings with high levels of accuracy. However, its effective accuracy, cost, and feasibility in non-residential community settings is unknown. Methods: The objective of this study was to determine the effectiveness and accuracy of community-based passive wastewater and surface (environmental) surveillance to detect SARS-CoV-2 infection in neighborhood schools compared to weekly diagnostic (PCR) testing. We implemented an environmental surveillance system in nine elementary schools with 1700 regularly present staff and students in southern California. The system was validated from November 2020 to March 2021. Findings: In 447 data collection days across the nine sites 89 individuals tested positive for COVID-19, and SARS-CoV-2 was detected in 374 surface samples and 133 wastewater samples. Ninety-three percent of identified cases were associated with an environmental sample (95% CI: 88%-98%); 67% were associated with a positive wastewater sample (95% CI: 57%-77%), and 40% were associated with a positive surface sample (95% CI: 29%-52%). The techniques we utilized allowed for near-complete genomic sequencing of wastewater and surface samples. Interpretation: Passive environmental surveillance can detect the presence of COVID-19 cases in non-residential community school settings with a high degree of accuracy. Funding: County of San Diego, Health and Human Services Agency, National Institutes of Health, National Science Foundation, Centers for Disease Control.
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Background: Schools are high-risk settings for SARS-CoV-2 transmission, but necessary for children's educational and social-emotional wellbeing. Previous research suggests that wastewater monitoring can detect SARS-CoV-2 infections in controlled residential settings with high levels of accuracy. However, its effective accuracy, cost, and feasibility in non-residential community settings is unknown. Methods: The objective of this study was to determine the effectiveness and accuracy of community-based passive wastewater and surface (environmental) surveillance to detect SARS-CoV-2 infection in neighborhood schools compared to weekly diagnostic (PCR) testing. We implemented an environmental surveillance system in nine elementary schools with 1700 regularly present staff and students in southern California. The system was validated from November 2020 - March 2021. Findings: In 447 data collection days across the nine sites 89 individuals tested positive for COVID-19, and SARS-CoV-2 was detected in 374 surface samples and 133 wastewater samples. Ninety-three percent of identified cases were associated with an environmental sample (95% CI: 88% - 98%); 67% were associated with a positive wastewater sample (95% CI: 57% - 77%), and 40% were associated with a positive surface sample (95% CI: 29% - 52%). The techniques we utilized allowed for near-complete genomic sequencing of wastewater and surface samples. Interpretation: Passive environmental surveillance can detect the presence of COVID-19 cases in non-residential community school settings with a high degree of accuracy. Funding: County of San Diego, Health and Human Services Agency, National Institutes of Health, National Science Foundation, Centers for Disease Control.
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BACKGROUND: Conditions affecting skeletal muscle, such as chronic rotator cuff tears, low back pain, dystrophies, and many others, often share changes in muscle phenotype: intramuscular adipose and fibrotic tissue increase while contractile tissue is lost. The underlying changes in cell populations and cell ratios observed with these phenotypic changes complicate the interpretation of tissue-level transcriptional data. Novel single-cell transcriptomics has limited capacity to address this problem because muscle fibers are too long to be engulfed in single-cell droplets and single nuclei transcriptomics are complicated by muscle fibers' multinucleation. Therefore, the goal of this project was to evaluate the potential and challenges of a spatial transcriptomics technology to add dimensionality to transcriptional data in an attempt to better understand regional cellular activity in heterogeneous skeletal muscle tissue. METHODS: The 3' Visium spatial transcriptomics technology was applied to muscle tissue of a rabbit model of rotator cuff tear. Healthy control and tissue collected at 2 and 16 weeks after tenotomy was utilized and freshly snap frozen tissue was compared with tissue stored for over 6 years to evaluate whether this technology is retrospectively useful in previously acquired tissues. Transcriptional information was overlayed with standard hematoxylin and eosin (H&E) stains of the exact same histological sections. RESULTS: Sequencing saturation and number of genes detected was not affected by sample storage duration. Unbiased clustering matched the underlying tissue type-based on H&E assessment. Connective-tissue-rich areas presented with lower unique molecular identifier counts are compared with muscle fibers even though tissue permeabilization was standardized across the section. A qualitative analysis of resulting datasets revealed heterogeneous fiber degeneration-regeneration after tenotomy based on (neonatal) myosin heavy chain 8 detection and associated differentially expressed gene analysis. CONCLUSIONS: This protocol can be used in skeletal muscle to explore spatial transcriptional patterns and confidently relate them to the underlying histology, even for tissues that have been stored for up to 6 years. Using this protocol, there is potential for novel transcriptional pathway discovery in longitudinal studies since the transcriptional information is unbiased by muscle composition and cell type changes.
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Lesões do Manguito Rotador , Animais , Amarelo de Eosina-(YS)/metabolismo , Hematoxilina/metabolismo , Músculo Esquelético/patologia , Cadeias Pesadas de Miosina/metabolismo , Coelhos , Estudos Retrospectivos , Manguito Rotador/metabolismo , Lesões do Manguito Rotador/patologia , Transcriptoma/genéticaRESUMO
Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences.
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Macrófagos Alveolares , NF-kappa B , Animais , Cromatina/genética , Cromatina/metabolismo , Pulmão/metabolismo , Camundongos , NF-kappa B/metabolismo , Transcrição GênicaRESUMO
[This corrects the article DOI: 10.3389/fphys.2021.707116.].
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Macrophage proinflammatory activation is an important etiologic component of the development of insulin resistance and metabolic dysfunction in obesity. However, the underlying mechanisms are not clearly understood. Here, we demonstrate that a mitochondrial inner membrane protein, adenine nucleotide translocase 2 (ANT2), mediates proinflammatory activation of adipose tissue macrophages (ATMs) in obesity. Ant2 expression was increased in ATMs of obese mice compared with lean mice. Myeloid-specific ANT2-knockout (ANT2-MKO) mice showed decreased adipose tissue inflammation and improved insulin sensitivity and glucose tolerance in HFD/obesity. At the molecular level, we found that ANT2 mediates free fatty acid-induced mitochondrial permeability transition, leading to increased mitochondrial reactive oxygen species production and damage. In turn, this increased HIF-1α expression and NF-κB activation, leading to proinflammatory macrophage activation. Our results provide a previously unknown mechanism for how obesity induces proinflammatory activation of macrophages with propagation of low-grade chronic inflammation (metaflammation).
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Translocador 2 do Nucleotídeo Adenina/metabolismo , Inflamação/genética , Ativação de Macrófagos/genética , Obesidade/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , CamundongosRESUMO
Rotator cuff (RC) tears are prevalent in the population above the age of 60. The disease progression leads to muscle atrophy, fibrosis, and fatty infiltration in the chronic state, which is not improved with intervention or surgical repair. This highlights the need to better understand the underlying dysfunction in muscle after RC tendon tear. Contemporary studies aimed at understanding muscle pathobiology after RC tear have considered transcriptional data in mice, rats and sheep models at 2-3 time points (1 to 16 weeks post injury). However, none of these studies observed a transition or resurgence of gene expression after the initial acute time points. In this study, we collected rabbit supraspinatus muscle tissue with high temporal resolution (1, 2, 4, 8, and 16 weeks) post-tenotomy (n = 6/group), to determine if unique, time-dependent transcriptional changes occur. RNA sequencing and analyses were performed to identify a transcriptional timeline of RC muscle changes and related morphological sequelae. At 1-week post-tenotomy, the greatest number of differentially expressed genes was observed (1,069 up/873 down) which decreases through 2 (170/133), 4 (86/41), and 8 weeks (16/18), followed by a resurgence and transition of expression at 16 weeks (1,421/293), a behavior which previously has not been captured or reported. Broadly, 1-week post-tenotomy is an acute time point with expected immune system responses, catabolism, and changes in energy metabolism, which continues into 2 weeks with less intensity and greater contribution from mitochondrial effects. Expression shifts at 4 weeks post-tenotomy to fatty acid oxidation, lipolysis, and general upregulation of adipogenesis related genes. The effects of previous weeks' transcriptional dysfunction present themselves at 8 weeks post-tenotomy with enriched DNA damage binding, aggresome activity, extracellular matrix-receptor changes, and significant expression of genes known to induce apoptosis. At 16 weeks post-tenotomy, there is a range of enriched pathways including extracellular matrix constituent binding, mitophagy, neuronal activity, immune response, and more, highlighting the chaotic nature of this time point and possibility of a chronic classification. Transcriptional activity correlated significantly with histological changes and were enriched for biologically relevant pathways such as lipid metabolism. These data provide platform for understanding the biological mechanisms of chronic muscle degeneration after RC tears.
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BACKGROUND: There is a need to identify and quantify mesenchymal stromal cells (MSCs) in human bone marrow aspirate concentrate (BMAC) source tissues, but current methods to do so were established in cultured cell populations. Given that surface marker and gene expression change in cultured cells, it is doubtful that these strategies are valid to quantify MSCs in fresh BMAC. PURPOSE: To establish the presence, quantity, and heterogeneity of BMAC-derived MSCs in minimally manipulated BMAC using currently available strategies. STUDY DESIGN: Descriptive laboratory study. METHODS: Five published strategies to identify MSCs were compared for suitability and efficiency to quantify clinical-grade BMAC-MSCs and cultured MSCs at the single cell transcriptome level on BMAC samples being used clinically from 15 orthopaedic patients and on 1 cultured MSC sample. Strategies included (1) the guidelines by the International Society for Cellular Therapy (ISCT), (2) CD271 expression, (3) the Ghazanfari et al transcriptional profile, (4) the Jia et al transcriptional profile, and (5) the Silva et al transcriptional profile. RESULTS: ISCT guidelines did not identify any MSCs in BMAC at the transcriptional level and only 1 in 9 million cells at the protein level. Of 12,850 BMAC cells, 9 expressed the CD271 gene. Only 116 of 396 Ghazanfari genes were detected in BMAC, whereas no cells expressed all of them. No cells expressed all Jia genes, but 25 cells expressed at least 13 of 22. No cells expressed all Silva genes, but 19 cells expressed at least 8 of 23. Most importantly, the liberalized strategies tended to identify different cells and most of them clustered with immune cells. CONCLUSION: Currently available methods need to be liberalized to identify any MSCs in fresh human BMAC and lack consensus at the single cell transcriptome and protein expression levels. These different cells should be isolated and challenged to establish phenotypic differences. CLINICAL RELEVANCE: This study demonstrated that improved strategies to quantify MSC concentrations in BMAC for clinical applications are urgently needed. Until then, injected minimally manipulated MSC doses should be reported as rough estimates or as unknown.
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Células-Tronco Mesenquimais , Medula Óssea , Células da Medula Óssea , Transplante de Medula Óssea , Células Cultivadas , Consenso , HumanosRESUMO
INTRODUCTION: Dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD) are characterized by cognitive alterations, visual hallucinations, and motor impairment. Diagnosis is based on type and timing of clinical manifestations; however, determination of clinical subtypes is challenging. The utility of blood DNA methylation as a biomarker for Lewy body disorders (LBD) is mostly unexplored. METHODS: We performed a cross-sectional analysis of blood methylation in 42 DLB and 50 PDD cases applying linear models to compare groups and logistic least absolute shrinkage and selection operator regression to explore the discriminant power of methylation signals. RESULTS: DLB blood shows differential methylation compared to PDD. Some methylation changes associate with core features of LBD. Sets of probes show high predictive value to discriminate between variants. DISCUSSION: Our study is the first to explore LBD blood methylation. Despite overlapping clinical presentation, we detected differential epigenetic signatures that, if confirmed in independent cohorts, could be developed into useful biomarkers.
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Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h, p53 and phc1 to specify HSPCs via modulation of Notch signalling.
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Proteínas de Ciclo Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Receptores Notch/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Células-Tronco Hematopoéticas/citologia , Mutação , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoRESUMO
Single-cell transcriptomics was used to profile cells of the normal murine middle ear. Clustering analysis of 6770 transcriptomes identified 17 cell clusters corresponding to distinct cell types: five epithelial, three stromal, three lymphocyte, two monocyte, two endothelial, one pericyte and one melanocyte cluster. Within some clusters, cell subtypes were identified. While many corresponded to those cell types known from prior studies, several novel types or subtypes were noted. The results indicate unexpected cellular diversity within the resting middle ear mucosa. The resolution of uncomplicated, acute, otitis media is too rapid for cognate immunity to play a major role. Thus innate immunity is likely responsible for normal recovery from middle ear infection. The need for rapid response to pathogens suggests that innate immune genes may be constitutively expressed by middle ear cells. We therefore assessed expression of innate immune genes across all cell types, to evaluate potential for rapid responses to middle ear infection. Resident monocytes/macrophages expressed the most such genes, including pathogen receptors, cytokines, chemokines and chemokine receptors. Other cell types displayed distinct innate immune gene profiles. Epithelial cells preferentially expressed pathogen receptors, bactericidal peptides and mucins. Stromal and endothelial cells expressed pathogen receptors. Pericytes expressed pro-inflammatory cytokines. Lymphocytes expressed chemokine receptors and antimicrobials. The results suggest that tissue monocytes, including macrophages, are the master regulators of the immediate middle ear response to infection, but that virtually all cell types act in concert to mount a defense against pathogens.