Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the common strategy is based on searching and probing set of molecular components and physical properties intended to be univocally characteristics of the target subpopulation. Pitfalls include the risk to opt for an unsuitable marker set - which may either not represent the subpopulation or also cover other unintended subpopulations - and the need to use different characterization techniques and equipment. This approach focused on specific markers may result inadequate to routinely deal with EV subpopulations that have an intrinsic high level of heterogeneity. In this paper, we show that Fourier-transform Infrared (FT-IR) spectroscopy can provide a collective fingerprint of EV subpopulations in one single experiment. FT-IR measurements were performed on large (LEVs, ~600 nm), medium (MEVs, ~200 nm) and small (SEVs ~60 nm) EVs enriched from two different cell lines medium murine prostate cancer (TRAMP-C2) and skin melanoma (B16). Spectral regions between 3100-2800 cm-1 and 1880-900 cm-1, corresponding to functional groups mainly ascribed to lipid and protein contributions, were acquired and processed by Principal Component Analysis (PCA). LEVs, MEVs and SEVs were separately grouped for both the considered cell lines. Moreover, subpopulations of the same size but from different sources were assigned (with different degrees of accuracy) to two different groups. These findings demonstrate that FT-IR has the potential to quickly fingerprint EV subpopulations as a whole, suggesting an appealing complement/alternative for their characterization and grading, extendable to healthy and pathological EVs and fully artificial nanovesicles. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.Rothia mucilaginosa has been found at high abundance on oral leukoplakia (OLK). The ability of clinical isolates to produce acetaldehyde (ACH) from ethanol has not been investigated. The objective of the current study was to determine the capacity of R. mucilaginosa isolates recovered from OLK to generate ACH. Analysis of R. mucilaginosa genomes (n = 70) shows that this species does not normally encode acetaldehyde dehydrogenase (ALDH) required for detoxification of ACH. The predicted OLK metagenome also exhibited reduced ALDH coding capacity. We analysed ACH production in 8 isolates of R. mucilaginosa and showed that this species is capable of generating ACH in the presence of ethanol. The levels of ACH produced (mean = 53 µM) were comparable to those produced by Neisseria mucosa and Candida albicans in parallel assays. These levels were demonstrated to induce oxidative stress in cultured oral keratinocytes. This study shows that R. mucilaginosa can generate ACH from ethanol in vitro at levels which can induce oxidative stress. This organism likely contributes to oral ACH levels following alcohol consumption and the significance of the increased abundance of R. mucilaginosa in patients with potentially malignant disorders requires further investigation. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.Objective Microvascular dysfunction is a feature of periodontal disease. P. gingivalis, one of the most common oral bacteria present in gingival tissue biofilms, has also been identified in the gingival capillaries of patients with chronic periodontitis. We sought to determine the effect of P. gingivalis W83 infection on microvascular endothelium in vivo and in vitro. Methods and Results Interdental papillae of rats with P. gingivalis-induced alveolar bone loss had a more dilated and denser subepithelial capillary network than uninfected controls. P. gingivalis W83 was detected in the epithelial layers, the subepithelial connective tissue matrix, and subgingival capillaries. P. gingivalis invaded human dermal microvascular endothelial cells (HD-MVECS) and persisted up termination (24 h). Colocalization analysis at 2.5, 6, and 24 h post-inoculation showed that 79-88% of internalized bacteria were in ICAM-1 positive endosomes, and 10-39% were in Rab5, Rab7, or LAMP1 positive compartments, but never in autophagosomes. Antibody-based blockade of ICAM-1 significantly reduced W83 invasion in HD-MVECS. P. gingivalis infected HD-MVECS were unable to form vascular networks in Matrigel. Conclusions P. https://www.selleckchem.com/products/bromelain.html gingivalis perturbs microvascular endothelial function and invasion of these cells via ICAM-1 may be important for microbial persistence within tissues. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.Background The oral microbiome, which consists of various habitats, has been shown to be influenced by smoking. However, differences in the tongue microbiomes of current and former smokers, as well as their resultant functional consequences, have rarely been investigated in East Asian populations. Methods We used 16S rRNA amplicon sequencing of tongue-coating samples obtained from East Asian subjects who were current, former, or never smokers to identify differences in their tongue microbiomes and related metagenomic functions. Two sets of participants from 2016 to 2017 (n = 657 and n = 187, respectively) were analyzed separately. Results We found significant differences between the overall microbiome compositions of current versus never smokers (p = 0.0015), but not between former versus never smokers (p = 0.43) based on the weighted UniFrac distance. Twenty-nine of 43 investigated genera showed significantly different expression levels in current versus never smokers. Neisseria and Capnocytophaga were less abundant, and Streptococcus and Megasphaera were more abundant in current smokers. Moreover, the abundances of metagenomic pathways, including those related to nitrate reduction and the tricarboxylic acid cycle, were significantly different between current and never smokers. Conclusions The tongue microbiomes and related metagenomic pathways of current smokers differ from those of never smokers among East Asians. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the common strategy is based on searching and probing set of molecular components and physical properties intended to be univocally characteristics of the target subpopulation. Pitfalls include the risk to opt for an unsuitable marker set - which may either not represent the subpopulation or also cover other unintended subpopulations - and the need to use different characterization techniques and equipment. This approach focused on specific markers may result inadequate to routinely deal with EV subpopulations that have an intrinsic high level of heterogeneity. In this paper, we show that Fourier-transform Infrared (FT-IR) spectroscopy can provide a collective fingerprint of EV subpopulations in one single experiment. FT-IR measurements were performed on large (LEVs, ~600 nm), medium (MEVs, ~200 nm) and small (SEVs ~60 nm) EVs enriched from two different cell lines medium murine prostate cancer (TRAMP-C2) and skin melanoma (B16). Spectral regions between 3100-2800 cm-1 and 1880-900 cm-1, corresponding to functional groups mainly ascribed to lipid and protein contributions, were acquired and processed by Principal Component Analysis (PCA). LEVs, MEVs and SEVs were separately grouped for both the considered cell lines. Moreover, subpopulations of the same size but from different sources were assigned (with different degrees of accuracy) to two different groups. These findings demonstrate that FT-IR has the potential to quickly fingerprint EV subpopulations as a whole, suggesting an appealing complement/alternative for their characterization and grading, extendable to healthy and pathological EVs and fully artificial nanovesicles. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.Rothia mucilaginosa has been found at high abundance on oral leukoplakia (OLK). The ability of clinical isolates to produce acetaldehyde (ACH) from ethanol has not been investigated. The objective of the current study was to determine the capacity of R. mucilaginosa isolates recovered from OLK to generate ACH. Analysis of R. mucilaginosa genomes (n = 70) shows that this species does not normally encode acetaldehyde dehydrogenase (ALDH) required for detoxification of ACH. The predicted OLK metagenome also exhibited reduced ALDH coding capacity. We analysed ACH production in 8 isolates of R. mucilaginosa and showed that this species is capable of generating ACH in the presence of ethanol. The levels of ACH produced (mean = 53 µM) were comparable to those produced by Neisseria mucosa and Candida albicans in parallel assays. These levels were demonstrated to induce oxidative stress in cultured oral keratinocytes. This study shows that R. mucilaginosa can generate ACH from ethanol in vitro at levels which can induce oxidative stress. This organism likely contributes to oral ACH levels following alcohol consumption and the significance of the increased abundance of R. mucilaginosa in patients with potentially malignant disorders requires further investigation. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.Objective Microvascular dysfunction is a feature of periodontal disease. P. gingivalis, one of the most common oral bacteria present in gingival tissue biofilms, has also been identified in the gingival capillaries of patients with chronic periodontitis. We sought to determine the effect of P. gingivalis W83 infection on microvascular endothelium in vivo and in vitro. Methods and Results Interdental papillae of rats with P. gingivalis-induced alveolar bone loss had a more dilated and denser subepithelial capillary network than uninfected controls. P. gingivalis W83 was detected in the epithelial layers, the subepithelial connective tissue matrix, and subgingival capillaries. P. gingivalis invaded human dermal microvascular endothelial cells (HD-MVECS) and persisted up termination (24 h). Colocalization analysis at 2.5, 6, and 24 h post-inoculation showed that 79-88% of internalized bacteria were in ICAM-1 positive endosomes, and 10-39% were in Rab5, Rab7, or LAMP1 positive compartments, but never in autophagosomes. Antibody-based blockade of ICAM-1 significantly reduced W83 invasion in HD-MVECS. P. gingivalis infected HD-MVECS were unable to form vascular networks in Matrigel. Conclusions P. https://www.selleckchem.com/products/bromelain.html gingivalis perturbs microvascular endothelial function and invasion of these cells via ICAM-1 may be important for microbial persistence within tissues. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.Background The oral microbiome, which consists of various habitats, has been shown to be influenced by smoking. However, differences in the tongue microbiomes of current and former smokers, as well as their resultant functional consequences, have rarely been investigated in East Asian populations. Methods We used 16S rRNA amplicon sequencing of tongue-coating samples obtained from East Asian subjects who were current, former, or never smokers to identify differences in their tongue microbiomes and related metagenomic functions. Two sets of participants from 2016 to 2017 (n = 657 and n = 187, respectively) were analyzed separately. Results We found significant differences between the overall microbiome compositions of current versus never smokers (p = 0.0015), but not between former versus never smokers (p = 0.43) based on the weighted UniFrac distance. Twenty-nine of 43 investigated genera showed significantly different expression levels in current versus never smokers. Neisseria and Capnocytophaga were less abundant, and Streptococcus and Megasphaera were more abundant in current smokers. Moreover, the abundances of metagenomic pathways, including those related to nitrate reduction and the tricarboxylic acid cycle, were significantly different between current and never smokers. Conclusions The tongue microbiomes and related metagenomic pathways of current smokers differ from those of never smokers among East Asians. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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