The detection of HPV infection and microbial colonization in cervical lesions is currently done through PCR-based viral or bacterial DNA amplification. Our objective was to develop a methodology to expand the metaproteomic landscape of cervical disease and determine if protein biomarkers from both human and microbes could be detected in distinct cervical samples. This would lead to the development of multi-species proteomics, which includes protein-based lateral flow diagnostics that can define patterns of microbes and/or human proteins relevant to disease status. In this study, we collected both non-frozen tissue biopsy and exfoliative non-fixed cytology samples to assess the consistency of detecting human proteomic signatures between the cytology and biopsy samples. Our results show that proteomics using biopsies or cytologies can detect both human and microbial organisms. Across patients, Lumican and Galectin-1 were most highly expressed human proteins in the tissue biopsy, whilst IL-36 and IL-1RA were most highly expressed human proteins in the cytology. We also used mass spectrometry to assess microbial proteomes known to reside based on prior 16S rRNA gene signatures. Lactobacillus spp. was the most highly expressed proteome in patient samples and specific abundant Lactobacillus proteins were identified. These methodological approaches can be used in future metaproteomic clinical studies to interrogate the vaginal human and microbiome structure and metabolic diversity in cytologies or biopsies from the same patients who have pre-invasive cervical intraepithelial neoplasia, invasive cervical cancer, as well as in healthy controls to assess how human and pathogenic proteins may correlate with disease presence and severity.
- MeSH
- biologické markery * analýza metabolismus MeSH
- biopsie MeSH
- cervix uteri * mikrobiologie patologie MeSH
- dospělí MeSH
- galektin 1 metabolismus analýza genetika MeSH
- Lactobacillus MeSH
- lidé MeSH
- lumican MeSH
- mikrobiota MeSH
- nádory děložního čípku patologie mikrobiologie MeSH
- proteomika * metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologické markery * MeSH
- galektin 1 MeSH
- lumican MeSH
N-Acetyllactosamine (LacNAc; Galβ4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and metastatic formation. In this study, we synthesized a series of fifteen N-(2-hydroxypropyl)methacrylamide (HPMA)-based glycopolymers with varying LacNAc amounts and presentations and evaluated the impact of their architecture on the binding affinity to Gal-1 and Gal-3. The controlled radical reversible addition-fragmentation chain transfer copolymerization technique afforded linear polymer precursors with comparable molecular weight (Mn ≈ 22,000 g mol-1) and narrow dispersity (D̵ ≈ 1.1). The precursors were conjugated with the functionalized LacNAc disaccharide (4-22 mol % content in glycopolymer) prepared by enzymatic synthesis under catalysis by β-galactosidase from Bacillus circulans. The structure-affinity relationship study based on the enzyme-linked immunosorbent assay revealed that the type of LacNAc presentation, individual or clustered on bi- or trivalent linkers, brings a clear discrimination (almost 300-fold) between Gal-1 and Gal-3, reaching avidity to Gal-1 in the nanomolar range. Whereas Gal-1 strongly preferred a dense presentation of individually distributed LacNAc epitopes, Gal-3 preferred a clustered LacNAc presentation. Such a strong galectin preference based just on the structure of a multivalent glycopolymer type is exceptional. The prepared nontoxic, nonimmunogenic, and biocompatible glycopolymers are prospective for therapeutic applications requiring selectivity for one particular galectin.
- MeSH
- akrylamidy chemie MeSH
- aminocukry chemie MeSH
- Bacillus enzymologie MeSH
- beta-galaktosidasa metabolismus MeSH
- disacharidy chemická syntéza MeSH
- ELISA MeSH
- epitopy MeSH
- galektin 1 analýza metabolismus MeSH
- galektiny analýza metabolismus MeSH
- katalýza MeSH
- krevní proteiny analýza metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- polymerizace MeSH
- polymery chemie metabolismus farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- aminocukry MeSH
- beta-galaktosidasa MeSH
- disacharidy MeSH
- epitopy MeSH
- galektin 1 MeSH
- galektiny MeSH
- krevní proteiny MeSH
- LGALS1 protein, human MeSH Prohlížeč
- LGALS3 protein, human MeSH Prohlížeč
- N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
- N-acetyllactosamine MeSH Prohlížeč
- polymery MeSH
Keratin 19 and nuclear reactivity to an endogenous lectin, galectin-1, represent a potential marker of epidermal stem cells. We detected expression of keratin 19 and nuclear binding sites for galectin-1 in adult cells migrating from the hair follicle, where cells expressing keratin 19 are located in the bulge region. The results were compared with the expression of both markers in cells adhering from suspension prepared from the interfollicular epidermis without keratin-19-positive cells and with nuclear binding sites for galectin-1. The results were compared with data from basal cell carcinomas. All cells were analyzed concerning size, as it is known that cell diameter influences the clonogenic potential of keratinocytes. The major result of this study is the observation of transient expression of keratin 19 and nuclear galectin-1 binding sites in originally negative interfollicular epidermal cells induced by adhesion. These cells were very small in size, similar to basal cells of the interfollicular epidermis or the bulge region of the hair follicle. The influence of the suspension regimen on beta1-integrin expression, cell diameter and growth was also monitored. A population of cells highly positive for beta1 integrin of the same diameter as keratin-19-positive cells insensitive to induction of terminal differentiation by lack of anchorage was characterized. Cells of the same size were also observed in the keratin-19-positive cells of basal cell carcinomas. In conclusion, the expression of poor levels of differentiation induced by cell adhesion is transient. Also, keratin 19 expression should not be exclusively regarded as a marker of stem cell activity.
- MeSH
- antigeny CD29 analýza MeSH
- bazocelulární karcinom metabolismus patologie MeSH
- buněčná adheze MeSH
- buněčné kultury MeSH
- časové faktory MeSH
- epidermální buňky MeSH
- epidermis chemie MeSH
- galektin 1 analýza MeSH
- galektin 3 analýza MeSH
- keratin-10 MeSH
- keratin-20 MeSH
- keratinocyty chemie cytologie MeSH
- keratiny analýza MeSH
- kultivované buňky MeSH
- lidé MeSH
- nádory kůže metabolismus patologie MeSH
- pohyb buněk MeSH
- proteiny intermediálních filament analýza MeSH
- vlasový folikul chemie cytologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- antigeny CD29 MeSH
- galektin 1 MeSH
- galektin 3 MeSH
- keratin-10 MeSH
- keratin-20 MeSH
- keratiny MeSH
- KRT10 protein, human MeSH Prohlížeč
- KRT20 protein, human MeSH Prohlížeč
- proteiny intermediálních filament MeSH
