Circulating endometrial cells (CECs) have emerged as a new biomarker of advanced disease in women with endometriosis. The identification of several subtypes of CECs (e.g., stem cell-like, epithelial, glandular, stromal) has opened the way for characterization of endometriosis-associated CECs. This study focused on the isolation and characterization of CECs and disseminated endometrial cells (DECs) in patients with spontaneous pneumothorax (SP). The primary objective was to differentiate between cancer and non-cancer cells in patients with no previous cancer diagnosis. The MetaCell® size-based separation protocol was used to enrich CECs/DECs. Evaluation of the captured cells by 3D microscopy was performed using a NANOLIVETM microscope using a holographic approach. Based on gene expression analysis (GEA), we can conclude that mitochondria are much more active in primary tumors compared to endometriosis tissue (e.g. MT-ND1, MT-ATP6 genes). The culture of DECs is made of stromal, stem and immune cells. In vitro culture of DECs is characterized by an increase in the epithelial marker KRT18. Similarly, NFE2L2, a proerythroid factor, is also elevated. Further, a significant decrease in the amount of stem and immune cells was observed in the cell culture of DECs. The data presented here show how morphologically plastic the changes in the mitochondrial network can be and how cells can reflect them at the level of gene expression. The markers identified could help in the accompanying diagnostic process of the spontaneous pneumothorax in women of reproductive age.
- MeSH
- Adult MeSH
- Endometriosis * pathology diagnosis genetics MeSH
- Endometrium pathology metabolism MeSH
- Humans MeSH
- Mitochondria * metabolism pathology MeSH
- Pneumothorax * pathology diagnosis MeSH
- Gene Expression Profiling methods MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is the most common repeat-mediated disease in humans. It exclusively affects corneal endothelial cells (CECs), with ≤81% of cases associated with an intronic TCF4 triplet repeat (CTG18.1). Here, we utilise optical genome mapping (OGM) to investigate CTG18.1 tissue-specific instability to gain mechanistic insights. METHODS: We applied OGM to a diverse range of genomic DNAs (gDNAs) from patients with FECD and controls (n = 43); CECs, leukocytes and fibroblasts. A bioinformatics pipeline was developed to robustly interrogate CTG18.1-spanning DNA molecules. All results were compared with conventional polymerase chain reaction-based fragment analysis. FINDINGS: Analysis of bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin. However, clusters of CTG18.1 molecules, encompassing ∼1800-11,900 repeats, were exclusively detected in diseased CECs from expansion-positive cases. Additionally, both progenitor allele size and age were found to influence the level of leukocyte-specific CTG18.1 instability. INTERPRETATION: OGM is a powerful tool for analysing somatic instability of repeat loci and reveals here the extreme levels of CTG18.1 instability occurring within diseased CECs underpinning FECD pathophysiology, opening up new therapeutic avenues for FECD. Furthermore, these findings highlight the broader translational utility of FECD as a model for developing therapeutic strategies for rarer diseases similarly attributed to somatically unstable repeats. FUNDING: UK Research and Innovation, Moorfields Eye Charity, Fight for Sight, Medical Research Council, NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Grantová Agentura České Republiky, Univerzita Karlova v Praze, the National Brain Appeal's Innovation Fund and Rosetrees Trust.
- MeSH
- Alleles MeSH
- Trinucleotide Repeat Expansion MeSH
- Fuchs' Endothelial Dystrophy * genetics pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Chromosome Mapping MeSH
- Genomic Instability MeSH
- Organ Specificity genetics MeSH
- Aged MeSH
- Transcription Factor 4 * genetics metabolism MeSH
- Trinucleotide Repeats genetics MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.
- MeSH
- Alternative Splicing genetics MeSH
- Endothelial Cells metabolism MeSH
- Trinucleotide Repeat Expansion * genetics MeSH
- Fuchs' Endothelial Dystrophy * genetics MeSH
- Humans MeSH
- Endothelium, Corneal metabolism pathology MeSH
- Transcription Factor 4 * genetics metabolism MeSH
- Transcriptome genetics MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
Východiska: Aferéza kmenových buněk je v procesu autologní transplantace kmenových buněk klíčovým krokem. Dostupné separátory krevních buněk (blood cell separators – BCS) mají díky svým technickým vlastnostem a vlivem obsluhy různou účinnost. Materiál a metody: Retrospektivě byla shromažďována data z aferéz periferních kmenových buněk provedených pomocí dostupných BCS od výrobce Fresenius (ComTec a Amicus) v Ukrajinském národním onkologickém institutu (National Cancer Institute Ukraine) v letech 2017–2020. Byl vypočítán koeficient účinnosti odběru (collection efficiency coefficient – CEC) a pro každý separátor byl upraven vzorec pro predikci celkového objemu zpracované krve (total volume of processed blood – TVPB). Výsledky: Analýza dat 60 pacientů (celkem 92 provedených aferéz). Průměrné hodnoty CEC byly (53,8 ± 36,6) % pro přístroj Amicus a (44,2 ± 37,3) % pro přístroj ComTec; p = 0,22. Při použití přístroje Amicus byl získán nižší objem produktu v porovnání s přístrojem ComTec; p = 2×10–7. Množství odebraných kmenových buněk bylo v obou skupinách srovnatelné: (5,8 ± 5,7) ×106/kg pro Amicus a (4,1 ± 3,1) ×106/kg pro ComTech; p = 0,064. Aby bylo dosaženo optimálního množství kmenových buněk, byla provedena úprava vzorce pro odhad TVPB. Závěr: Hodnoty CEC pro oba přístroje byly v obecně akceptovaném rozmezí 30–50 % a výrazně se nelišily. Nicméně při použití přístroje Amicus bylo dosaženo nižších objemů produktu, přičemž ostatní charakteristiky byly srovnatelné.
Background: Stem cells apheresis is a key step in the process of the autologous stem cell transplantation. Available blood cell separators (BCS) have different efficiency due to the technical characteristics and influence of the operator. Materials and methods: Retrospectively, data were collected of the peripheral blood stem cells apheresis performed using available BCS manufactured by Fresenius (ComTec and Amicus) in the National Cancer Institute Ukraine from 2017 to 2020. The collection efficiency coefficient (CEC) was calculated, the formula for predicting the total volume of processed blood (TVPB) was adapted for each separator. Results: The analysis included data from 60 patients (total of 92 apheresis procedures). The mean CEC was established at the level of (53.8 ± 36.6) % for the Amicus device and (44.2 ± 37.3) % for the ComTec device; P = 0.22. The lower product volume was obtained using the Amicus device compared to the ComTec device; P = 2×10–7. The amount of collected stem cells was comparable in both groups (5.8 ± 5.7) ×106/kg and (4.1 ± 3.1) ×106/kg, respectively; P = 0.064. The adaptation of the formula for predicting the TVPB to achieve the optimum amount of stem cells was performed. Conclusion: The CEC for each device was within the generally accepted limits of 30–50%, and did not differ significantly. Nevertheless, using of the Amicus BCS allowed to collect lower volumes of the product, maintaining other characteristics of the product competitive.
BACKGROUND: The etiopathogenesis of abdominal aortic aneurysm (AAA) is still unclarified, but vascular inflammation and matrix metalloproteases activation have a recognized role in AAA development and progression. Circulating lipoproteins are involved in tissue inflammation and repair, particularly through the regulation of intracellular cholesterol, whose excess is associated to cell damage and proinflammatory activation. We analyzed lipoprotein metabolism and function in AAA and in control vasculopathic patients, to highlight possible non-atherosclerosis-related, specific abnormalities. METHODS: We measured fluorometrically serum esterified/total cholesterol ratio, as an index of lecithin-cholesterol acyltransferase (LCAT) activity, and cholesteryl ester transfer protein (CETP) activity in patients referred to vascular surgery either for AAA (n=30) or stenotic aortic/peripheral atherosclerosis (n=21) having similar burden of cardiovascular risk factors and disease. We measured high-density lipoprotein (HDL)-cholesterol efflux capacity (CEC), through the ATP-binding cassette G1 (ABCG1) and A1 (ABCA1) pathways and serum cell cholesterol loading capacity (CLC), by radioisotopic and fluorimetric methods, respectively. RESULTS: We found higher LCAT (+23%; p < 0.0001) and CETP (+49%; p < 0.0001) activity in AAA sera. HDL ABCG1-CEC was lower (-16%; p < 0.001) and ABCA1-CEC was higher (+31.7%; p < 0.0001) in AAA. Stratification suggests that smoking may partly contribute to these modifications. CEC and CETP activity correlated with CLC only in AAA. CONCLUSIONS: We demonstrated that compared to patients with stenotic atherosclerosis, patients with AAA had altered HDL metabolism and functions involved in their anti-inflammatory and tissue repair activity, particularly through the ABCG1-related intracellular signaling. Clarifying the relevance of this mechanism for AAA evolution might help in developing new diagnostic parameters and therapeutic targets for the early management of this condition.
- MeSH
- Adenosine Triphosphate MeSH
- Aortic Aneurysm, Abdominal * MeSH
- Anti-Inflammatory Agents MeSH
- Atherosclerosis * MeSH
- Cholesterol metabolism MeSH
- Sterol O-Acyltransferase metabolism MeSH
- Cholesterol, HDL MeSH
- Homeostasis MeSH
- Lecithins MeSH
- Humans MeSH
- Lipoproteins metabolism MeSH
- Metalloproteases metabolism MeSH
- Cholesterol Ester Transfer Proteins MeSH
- Inflammation metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Antimicrobial peptides (AMPs) are produced to control bacteria, fungi, protozoa, and other infectious agents. Sand fly larvae develop and feed on a microbe-rich substrate, and the hematophagous females are exposed to additional pathogens. We focused on understanding the role of the AMPs attacin (Att), cecropin (Cec), and four defensins (Def1, Def2, Def3, and Def4) in Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. Larvae and adults were collected under different feeding regimens, in addition to females artificially infected by Leishmania infantum. AMPs' gene expression was assessed by qPCR, and gene function of Att and Def2 was investigated by gene silencing. The gene knockdown effect on bacteria and parasite abundance was evaluated by qPCR, and parasite development was verified by light microscopy. We demonstrate that L. longipalpis larvae and adults trigger AMPs expression during feeding, which corresponds to an abundant presence of bacteria. Att and Def2 expression were significantly increased in Leishmania-infected females, while Att suppression favored bacteria growth. In conclusion, L. longipalpis AMPs' expression is tuned in response to bacteria and parasites but does not seem to interfere with the Leishmania cycle.
- Publication type
- Journal Article MeSH
The review presents a comprehensive survey of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, micropreparation, and physicochemical and biochemical characterization of peptides since 2017 up to about the middle of 2019. Progress in the study of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression, EOF control, and detection) are described. Advances in CE and CEC methods are demonstrated and their applications in the following areas are presented: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. In addition, micropreparative separations and determinations of important physicochemical characteristics of peptides by CE and CEC methods are reported.
- MeSH
- Electrophoresis, Capillary * MeSH
- Capillary Electrochromatography * MeSH
- Peptide Mapping MeSH
- Peptides analysis chemistry isolation & purification MeSH
- Proteins analysis chemistry isolation & purification MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
BACKGROUND: The occurrence of catamenial pneumothorax (CP) is rare, and the awareness of this diagnosis among physicians is insufficient. CP is highly correlated with pelvic endometriosis and remains the most common form of thoracic endometriosis syndrome. Circulating endometrial cells (CECs) have been previously detected in patients with pelvic endometriosis. Could CECs bring new insights into pneumothorax management? METHODS: This study aims to describe the occurrence and molecular characteristics of CECs in women with spontaneous pneumothorax (SP) (N = 20) with high suspicion of its catamenial character. CECs were enriched from peripheral blood by size-based separation (MetaCell). In addition to cytomorphology, gene expression profiling of captured cells was performed for 24 endometriosis-associated genes. RESULTS: CECs were present in all 20 patients with SP. Enriched CECs exhibited four character features: epithelial, stem cell-like, stroma-like, and glandular. However, not all of them were present in every sampling. Gene expression profiling revealed two distinct phenotypes of CECs in SP and/or CP: one of them refers to the diaphragm openings syndrome and the other to endometrial tissue pleural implantations. Comparisons of the gene expression profiles of CECs in pneumothorax (CECs-SP group) with CECs in pelvic endometriosis (CECs-non-SP group) have revealed significantly higher expression of HER2 in the CECs-SP group compared with the CECs-non-SP group. CONCLUSIONS: This proof-of-concept study demonstrates successful isolation and characterization of CECs in patients with SP. Identification of CECs in SP could alert endometriosis involvement and help early referral to gynecologic consultation for further examination and treatment.
- MeSH
- CA-125 Antigen genetics MeSH
- Adult MeSH
- Endometriosis blood genetics MeSH
- Endometrium cytology MeSH
- Keratin-18 genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Membrane Proteins genetics MeSH
- Young Adult MeSH
- Mucin-1 genetics MeSH
- Pleural Diseases blood genetics MeSH
- Pneumothorax blood diagnosis genetics MeSH
- Receptor, ErbB-2 genetics MeSH
- Case-Control Studies MeSH
- Liquid Biopsy MeSH
- Transcriptome MeSH
- Vimentin genetics MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Conazole fungicides are currently used pesticides with considerable chronic toxicity and ecotoxicity that are also on EU list for substitution. They enter the soil forming short- or long-term residues. In this study two of their representatives, epoxiconazole (EPC) and tebuconazole (TBC), have been tested with 20 soils from the Czech Republic for their adsorption. Adsorption, by means of Kd coefficients, was compared to "basic" (TOC, pH, clay …) and "advanced" (surface area, minerals ..) soil properties. After doing multivariate analysis of the variables it was apparent that adsorption of both pesticides was highly associated with pH (negatively correlated), and less associated with soil organo-mineral complex (TOC, clay and surface area) and C and N in soil organic matter (OM). Particle sizes or cation exchange capacity (CEC) did not show correlation with adsorption, but showed an association in multidimensional space in factor analysis (FA). Some correlations were revealed between EPC adsorption and soil organic matter parameters. Recalculating Kd to Koc and to Gibb's free energy (ΔG) and its values indicated that the adsorption of EPC and TBC is mainly weak physical adsorption - partitioning. Also, ΔG values gave better correlation with pH(H2O) than Kd. Surface area impacted EPC adsorption. From the several soil minerals, kaolinite showed EPC and TBC adsorption. EPC adsorption was not highly influenced with pH changes compared to TBC. The number and types of H-bonds with molecular geometry govern the sorption, which might crucially affect leachibility in soil, and this may indicate that TBC is more leachable than EPC for the same soil.
- MeSH
- Adsorption MeSH
- Epoxy Compounds chemistry MeSH
- Clay MeSH
- Cations MeSH
- Soil Pollutants chemistry MeSH
- Minerals chemistry MeSH
- Pesticides chemistry MeSH
- Soil chemistry MeSH
- Triazoles chemistry MeSH
- Particle Size MeSH
- Agriculture MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
Large-scale suspect and non-targeted screening approaches based on high-resolution mass spectrometry (HRMS) are today available for chemical profiling and holistic characterisation of biological samples. These advanced techniques allow the simultaneous detection of a large number of chemical features, including markers of human chemical exposure. Such markers are of interest for biomonitoring, environmental health studies and support to risk assessment. Furthermore, these screening approaches have the promising capability to detect chemicals of emerging concern (CECs), document the extent of human chemical exposure, generate new research hypotheses and provide early warning support to policy. Whilst of growing importance in the environment and food safety areas, respectively, CECs remain poorly addressed in the field of human biomonitoring. This shortfall is due to several scientific and methodological reasons, including a global lack of harmonisation. In this context, the main aim of this paper is to present an overview of the basic principles, promises and challenges of suspect and non-targeted screening approaches applied to human samples as this specific field introduce major specificities compared to other fields. Focused on liquid chromatography coupled to HRMS-based data acquisition methods, this overview addresses all steps of these new analytical workflows. Beyond this general picture, the main activities carried out on this topic within the particular framework of the European Human Biomonitoring initiative (project HBM4EU, 2017-2021) are described, with an emphasis on harmonisation measures.
- MeSH
- Biological Monitoring * MeSH
- Chromatography, Liquid MeSH
- Environmental Health MeSH
- Environmental Pollutants * analysis toxicity MeSH
- Humans MeSH
- Environmental Monitoring MeSH
- Environmental Exposure analysis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
